Dissertations / Theses: 'RNA biomarkers'

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Yazbek, Hanna Marcelino. "Exosomal RNA as a source of urine biomarkers for prostate cancer." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/66563/.

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Introduction: In this study we exploited the recent development of methods that have enabled the analysis of RNA present in urine exosomes of prostate cancer patients. We report RNA expression patterns that contain diagnostic and prognostic information for prostate cancer, and association with response to hormone treatment. Methods: First catch urine following digital rectal examination were collected from 662 men. 3 groups of patients were used: Low, Intermediate, and High-risk according to NICE stratification criteria, and two control groups: benign and advanced disease. 50-gene transcript expression analysis using NanoString technology was performed on 192 samples. Exosomal RNA Next-Generation Sequencing was performed on 18 samples for novel biomarker discovery. Results: Expression analysis showed that PCa-specific transcripts such as TMPRSS2/ERG fusion transcripts were identifiable in exosomes from PCa urine samples. LPD analysis highlighted expression levels of 15 transcripts with diagnostic potential (significantly up-regulated in cancer samples in comparison to benign control) and 17 transcripts with prognostic potential (differentialy expressed in high risk and advanced disease in comparison to lower grade disease). I also report two gene transcripts (SERPINB5/Maspin, HPRT) that were significantly differentially expressed in patients who failed to respond to hormone deprivation therapy for high risk/metastatic disease. Three genes (STEAP4, ARexons4_8 and NAALADL2) were significantly differentially expressed in patients who relapsed within 12 months of hormone treatment initiation. Next-Generation Sequencing of twenty samples identified 45 genes to be significantly differentially expressed between non-cancer and cancer samples (28 were up regulated and 17 down regulated). 33 out of the 45 genes showed a significant linear trend in association with cancer risk. Conclusions: Urine Exosomal RNA contains PCa specific transcripts. Gene expression analysis and Next Generation Sequencing identified genes that are significantly differentially expressed between cancer and non-cancer cases as well as prognostic genes and genes that can predict response to hormone treatment.

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Islam, Nazmul. "Engineering translational approaches for molecular diagnostics of cancer: Multifunctional nanomaterials and electrochemical sensors for clinically relevant RNA biomarker detection." Thesis, Griffith University, 2018. http://hdl.handle.net/10072/374754.

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Over the past several years, the human transcriptome has been repeatedly interrogated, and with the recent breakthrough in sequencing technologies, ribonucleic acids (RNAs) comprising different coding and noncoding transcripts, such as messenger RNA (mRNA), microRNA (miRNA), and long-noncoding RNA (lncRNA), are becoming progressively crucial for revolutionising personalised cancer management. Improved diagnostics, prognostics and streamlined therapeutic potentiality makes them an excellent choice as biomarkers. Non-coding RNAs are key regulators of the gene expression network and are involved in the control of a range of important cellular pathways, such as cell cycle, cell proliferation, differentiation, apoptosis, and post-transcriptional regulation. Abnormalities in the expression of these RNAs can affect one or several of these cellular pathways, which might contribute towards initiation and progression of cancer. Despite recent advances in RNA-based fundamental research, their detection approaches are largely confined to laboratory-based molecular biology techniques, such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarrays, and RNA sequencing. Although the analytical performance and reliability of these methods are excellent, most of these methods require enzymatic amplification, cumbersome sample pre-treatment, multi-step assay protocol, high maintenance cost, and technical expertise. The development of a simple, sensitive, and low cost method that can be used for rapid detection of RNA biomarkers for a meaningful clinical application at the time and place of patient care (i.e., point-of-care) is of great importance to clinical and translational research. This PhD project endeavours to engineer such translational approaches to circumvent the aforementioned challenges for developing an inexpensive, sensitive, specific, and portable biosensor platform. This thesis initially studies the biogenesis, diagnostic, and prognostic potential of RNA biomarkers followed by a comprehensive appraisal of recent progress in the development of RNA biosensors with a special emphasis on electrochemical-detection approaches. We then report on the development of a biosensing platform consisting of four novel readout schemes for the simple, rapid, and inexpensive analysis of various RNA biomarkers (i.e., mRNAs, miRNAs and lncRNAs). First, employing the nucleotides’ affinity towards gold, we developed an amplification-free electrochemical assay for the detection of tumour-specific mRNAs. This straightforward sensor adopted differential pulse voltammetry to enable the readout using simple direct adsorption of magnetically isolated analytes on unmodified disposable electrodes. Subsequent to the development of this proof of concept sensor, we attempted to address the increasing demand for detecting the ultralow levels of RNAs from the complex biological sample via introducing two novel readout strategies for detecting miRNAs. Utilising the coupling of electrocatalytic strength of two in-house synthesised porous graphene oxide-loaded iron oxide (GO/IO hybrid material), gold-loaded nanoporous ferric oxide nanocubes (Au-NPFe2O3NC), and [Ru(NH3)6]3+/[Fe(CN)6]3- electrocatalytic cycle, two ultrasensitive assays were reported, where the detection was achieved by chronocoulometric (CC) charge measurement of surface bound cationic [Ru(NH3)6]3+, which was electrostatically attached to the anionic phosphate backbone of target RNAs. In our final readout strategies, we extended our approach towards a translational- focused assay platform which enabled naked-eye, colorimetric and electrochemical interrogation of lncRNA via 3,3′,5,5′-tetramethylbenzidine (TMB)/Horseradish peroxidase (HRP)-based colorimetric assay. All of the readout platforms reported herein have shown excellent analytical performance with high sensitivity (LOD for mRNA and miRNA = picomolar to attomolar level, LOD for lncRNA = single cell approaching) and specificity. The applicability of the assays was also demonstrated in complex biological samples (a cohort of cancer cell lines and patient samples) with high reproducibility. The analytical performance of the assays was also validated with the standard RT-qPCR approach. We believe that our research efforts will lead to the development of a translational-focused point-of-care platform for RNA analysis, which in turn will not only hold the potential to improve patient care and outcomes but might also prove to be a venture of immense commercial significance.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Liu, Ming-lai, and 廖明麗. "Roles of microRNAs in hepatocellular carcinoma: biomarkers, matabolisms and pathway regulators." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46918929.

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Götschke, Jeremias [Verfasser], and Susanne [Akademischer Betreuer] Krauss-Etschmann. "Beschreibung eines potentiellen micro-RNA-basierten Biomarkers für allergisches Asthma / Jeremias Götschke ; Betreuer: Susanne Krauss-Etschmann." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/115653352X/34.

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Franklin, Oskar. "Stromal components and micro-RNAs as biomarkers in pancreatic cancer." Doctoral thesis, Umeå universitet, Kirurgi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128000.

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Background Pancreatic ductal adenocarcinoma (PDAC) patients have the poorest 5-year survival rates of all cancer forms. It is difficult to diagnose at early disease stages, tumour relapse after surgery is common, and current chemotherapies are ineffective. Carbohydrate antigen 19-9 (Ca 19-9), the only clinically implemented PDAC biomarker, is insufficient for diagnostic and screening purposes. PDAC tumours are characterised by a voluminous stroma that is rich in extracellular matrix (ECM) molecules such as collagens, hyaluronan (HA) and matricellular proteins. These stromal components have been suggested to promote PDAC cell migration, proliferation, evasion of apoptosis and chemotherapy resistance. Those events are mediated via interactions with adhesion receptors, such as integrins and CD44 receptors expressed on cancer cell surfaces. Micro-RNAs (miRNA) post-transcriptionally regulate gene expression in health and disease. At the time of PDAC diagnosis, miRNA levels are altered both in plasma and tumour tissue. Before PDAC diagnosis, tissue miRNA levels are altered in precursor lesions, raising the possibility that plasma miRNAs might aid in early detection. In this thesis, it is hypothesised that stromal components and miRNAs can serve as tissue or blood based biomarkers in PDAC. The aims are: (1) to characterise the expression of stromal components and their receptors in normal and cancerous tissue; (2) to find potential stroma-associated tissue and blood-based biomarkers for diagnosis and prognosis estimates; (3) to determine the cellular effects of type IV collagen (Col IV) in PDAC; (4) to determine if plasma miRNAs that are altered in manifest PDAC can be used to diagnose PDAC earlier. Methods The expression patterns of Col IV, Col IV-binding integrin subunits (α1, α2, β1), Endostatin, Osteopontin (OPN) and Tenascin C (TNC) were analysed in frozen PDAC and normal pancreatic tissue. A tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded primary tumours and lymph node metastases. The TMA was used to study the expression levels and associations with survival of the standard CD44 receptor (CD44s), its variant isoform 6 (CD44v6), HA, OPN and Col IV. Circulating levels of HA, Col IV, Endostatin, OPN and TNC were measured in PDAC patients and healthy individuals, and compared with conventional tumour markers (Ca 19-9, CEA, Ca 125 and TPS). The functional roles of Col IV were studied in PDAC cell lines by: (1) growth on different matrices (2) blocking Col IV binding integrin subunits, (3) blocking the Col IV domains 7s, CB3 and NC1, and (4) by down regulation of PDAC cell synthesis of Col IV using siRNA transfection. Plasma miRNAs alterations were screened for in samples from patients with manifest disease, using real-time quantitative PCR (RT-qPCR). To find early miRNA alterations, levels of those miRNAs that were altered at diagnosis were measured in prediagnostic plasma samples. Results High tissue expression of both the standard CD44 receptor (CD44s) and its variant isoform CD44v6 as well as low expression of stromal OPN were associated with poor survival. In addition, high CD44s and low OPN predicted poor survival independent of established prognostic factors. Circulating Col IV, Endostatin, OPN, TNC and HA were increased in preoperative samples from PDAC patients. Preoperatively, higher levels of serum-HA and plasma-Endostatin were associated with shorter survival. Postoperatively, higher levels of Col IV, Endostatin and OPN were associated with shorter survival. On the contrary, only one of the conventional tumour markers was associated with survival (Ca 125). Col IV stimulated PDAC cell proliferation and migration and inhibited apoptosis in vitro, dependent on the collagenous domain (CB3) of Col IV and the Col IV binding integrin subunit β1. Reduced endogenous Col IV synthesis inhibited these effects, suggesting that PDAC cells synthesise Col IV to stimulate tumour-promoting events via a newly discovered autocrine loop. 15 miRNAs were altered in early stage PDAC patients and the combination of these markers outperformed Ca 19-9 in discriminating patients from healthy individuals. However, none of the miRNAs were altered in prediagnostic samples, suggesting that plasma miRNA alterations appear late in the disease course. Conclusions Up regulated stromal components in PDAC tumours are detectable in blood samples and are potential diagnostic and prognostic biomarkers in PDAC. High circulating levels of Col IV, Endostatin, OPN and HA predict poor survival, as well as high expression of CD44s and CD44v6 and low expression of OPN in tumour tissue. PDAC cells synthesise Col IV, which forms BM-like structures close to cancer cells and promote tumour progression in vitro via an autocrine loop. Several plasma-miRNAs are altered in PDAC, but are not useful for early discovery.

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Sohiya, Yotsukura. "Computational Framework for the Dissection of Cancer Genomic Architecture and its Association in Different Biomarkers." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/217149.

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Sundaramurthy, Gopinath. "A Probabilistic Approach for Automated Discovery of Biomarkers using Expression Data from Microarray or RNA-Seq Datasets." University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1459528594.

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Bajak, Edyta Zofia. "Genotoxic stress: novel biomarkers and detection methods : uncovering RNAs role in epigenetics of carcinogenesis /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-415-5/.

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Tong, Chiu-hung, and 唐朝虹. "MiR-143 and its downstream targets: possible biomarkers for cervical cancer and precursors." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46579436.

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Alvarez, Michelle. "PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION." Doctoral diss., University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4198.

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It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.
Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomolecular Sciences PhD

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Chapman, M. H. "Whole genome RNA expression profiling for the identification of novel biomarkers in the diagnosis and prognosis of biliary tract cancer." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1310148/.

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Biliary tract cancer (BTC) is difficult to diagnose, in part related to the lack of reliable tumour markers. The aim of this project was to use whole genome RNA expression profiling in order to identify novel biomarkers for diagnosis and prognosis in biliary tract cancer. Chapter 1 summarises clinical aspects of BTC as well as current diagnostic and prognostic tests. Chapter 2 addresses the identification of circulating tumour cells for the diagnosis of BTC. It includes details of a study investigating measurement of circulating cytokeratin 19 fragments (CYFRA 21-1), demonstrating that CYFRA 21-1 is a more specific, but less sensitive diagnostic marker than CA19-9, and predicts a poor prognosis in BTC. Chapter 3 investigates the potential for using RNA isolated from archived formalin fixed, paraffin embedded (FFPE) surgical and explanted liver tissues from patients with primary sclerosing cholangitis (PSC) with and without cholangiocarcinoma, for use in whole genome RNA expression analysis. We demonstrate that, although technically possible, the rarity of samples and RNA degradation that occurs as a result of the tissue processing, are such that further evaluation using these materials is not feasible at this time. Chapter 4 addresses and validates methodology for isolating RNA from samples of biliary brushings taken at the time of endoscopic retrograde cholangiopancreatography (ERCP). We demonstrate that RNA isolated from biliary brushings is of low quantity and degraded, and that this degradation occurs in vivo. However, we demonstrate that such RNA is still useful for downstream applications such as quantitative real time PCR and is therefore suitable for whole genome RNA expression analysis using microarray technology. Chapter 5 describes the methods and results obtained from using whole genome RNA expression analysis using microarray of RNA isolated from ERCP biliary brushings. The results are presented as a shortlist of candidate genes requiring further validation. Chapter 6 provides results of qPCR studies performed in order to validate the gene expression profile identified by microarray. A selection of candidate genes are investigated using TaqMan Array and SYBR Green qPCR and demonstrate a high correlation with the pattern of expression shown by microarray. Chapter 7 investigates whether a selection of the genes identified in malignant biliary brushings are similarly upregulated in fresh frozen surgical resection material from patients with benign and malignant biliary diseases. In addition, we provide evidence for gene translation and upregulation at the protein level by immunohistochemistry for a selection of the protein products. Chapter 8 discusses the main conclusions drawn from the work as well as potential future studies.

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Daniel, Rhonda W. "Dysregulation of microRNAs in Blood as Biomarkers for Diagnosing Prostate Cancer." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3975.

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Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers. Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease. It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.

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Guillemette, Shawna S. "Investigating Tumor Suppressors in the DNA Damage Response: Caretakers of the Genome and Biomarkers to Predict Therapeutic Response: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/712.

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Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

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Guillemette, Shawna S. "Investigating Tumor Suppressors in the DNA Damage Response: Caretakers of the Genome and Biomarkers to Predict Therapeutic Response: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/712.

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Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

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Ghanipour, Lana. "Colorectal Cancer : Aspects of Heredity, Prognosis and Tumour Markers." Doctoral thesis, Uppsala universitet, Kolorektalkirurgi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-224624.

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Colorectal cancer (CRC) is one of the most common cancer types and leading causes of cancer death worldwide. Since CRC is a heterogenic disease, there is a demand for increased knowledge of the underlying genetic and epigenetic mechanisms. The aim of this thesis was to investigate heredity and potential tumour markers in relation to prognosis. In paper I, survival of patients with CRC and a positive family history of CRC in first-degree relatives was analysed. Patients with colon cancer and positive family history of CRC had improved survival compared to patients with negative family history. This improvement in survival could not be explained by known clinico-pathological factors. In paper II, we investigated the prognostic value of Tryptophanyl t-RNA synthetase (TrpRS) in tissues from patients operated for CRC. Low protein expression of TrpRS in primary tumour tissues correlated with increased risk of recurrence and poorer survival. In paper III, the prognostic value of microsatellite instability (MSI) and the correlation to heredity for CRC in first-degree relatives was investigated. Patients with proximal colon cancer and MSI had improved cancer specific survival. There were no correlation between MSI and heredity. In paper IV, we evaluated the potential use of proximity ligation assay (SP-PLA) in patients with CRC, by simultaneous analysis of 35 proteins in only 5 μl plasma. SP-PLA is a suitable method for protein detection and might give valuable guidance in pursuing new prognostic and predictive tumour markers. However, none of the markers selected for present SP-PLA analyses gave better prognostic information than CEA. In conclusion, heredity is related to better survival independent of MSI in patients with CRC and MSI is associated with better prognosis in proximal colon cancer. Detection and increased knowledge of molecular mechanism in CRC is important, however it needs to be further investigated and validated in clinical use.

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Lopes, Patrícia Ferreira. "Diversidade taxonômica e potencial de biodegradação de bactérias isoladas de reservatórios de petróleo da Bacia de Campos (RJ)." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-22122010-095359/.

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O presente trabalho teve como objetivos caracterizar uma coleção de 98 bactérias isoladas de amostras de petróleo e água de formação de reservatórios da Bacia de Campos (RJ) utilizando técnicas de taxonomia molecular e avaliar o potencial de degradação de biomarcadores do petróleo. O sequenciamento e análise filogenética do gene RNAr 16S revelaram Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/B. thuringiensis Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/ H.campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis /S. moderatus, Staphylococcus hominis e Staphylococcus pasteuri/S. warneri. Os resultados evidenciaram a preferência pela biotransformação do ácido nonadecanóico e esqualano. A caracterização da microbiota presente nos reservatórios e avaliação do potencial de biodegradação pode contribuir para fornecer subsídios para estudos futuros sobre os mecanismos biológicos responsáveis pela biodegradação do petróleo.
This study is aimed to characterize a collection of 98 bacteria isolated from oil and formation water samples derived from reservoirs of the Campos Basin (RJ) using molecular biology-based techniques and to evaluate the degradation potential of petroleum biomarkers. Further sequencing and phylogenetic analysis of 16S rRNA genes revealed species of Bacillus firmus, megaterium, pumilus, sphaericus, simplex, cereus/thuringiensis, Marinobacter lutaoensis, Halomonas shengliensis/H. alimentaria/H. campisalis, Citreicella thiooxidans, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Micrococcus luteus, Kocuria rosea, Streptomyces alboniger/S. chartreusis/S. moderatus, Staphylococcus hominis and Staphylococcus pasteuri/S. warneri. The results showed the preference of bacteria for the biotransformation of nonadecanoic acid and squalane. The characterization of the microbiota associated to reservoirs and the evaluation of their biodegradation potential may provide subsidies for future studies about the biological mechanisms responsible for petroleum biodegradation.

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Bou, Samra Elias. "Recherche et caractérisation de biomarqueurs pronostiques dans les leucémies myélomonocytaires chroniques et aiguës myéloïdes par exploration des transcriptomes." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20057/document.

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Un défi de la transcriptomique est d'explorer l'intégralité du répertoire des transcrits normaux et anormaux. Les analyses de GEP (Gene Expression Profiling) basées sur la technologie des puces à ADN sont largement exploitées en cancérologie depuis plusieurs années. Parallèlement, les nouvelles méthodes basées sur le séquençage à haut débit offrent désormais la possibilité de réaliser des analyses précises et sensibles nécessaires à l'étude des cellules normales et cancéreuses. Quelle que soit la méthode, la caractérisation de l'ensemble des transcrits codants et non-codants représente un réel défi biologique pour la recherche de nouveaux marqueurs de diagnostic et de pronostic, et pour la bonne prise en charge des patients. Dans ce travail, j'ai eu l'occasion de traiter deux aspects différents de la biologie qui convergent vers l'identification de transcrits exprimés de manière aberrante dans les leucémies myéloïdes. Le premier aspect a consisté à proposer une sélection de biomarqueurs moléculaires pour la caractérisation de la leucémie myélomonocytaire chronique (LMMC). A partir de l'expression de ces gènes, nous avons développé un score de pronostic qui a permis de définir deux groupes de patients cliniquement distincts. Nous avons ensuite complété notre étude par une caractérisation phénotypique par cytométrie en flux des sous-populations cellulaires aberrantes constituant les lignages mono- et granulocytaires. Une partie de ce travail a été étendue aux leucémies aiguës myéloïdes (LAM) à caryotype normal (CN). L'autre aspect a consisté à participer à la mise en place d'une approche computationnelle intégrée pour caractériser de nouveaux ARNs non annotés et fort probablement non-codants. En explorant des données de Digital Gene Expression (DGE), nous avons quantifié et caractérisé la fraction de ces transcrits dans les régions intergéniques. Nous avons vérifié l'expression de ces nouveaux transcrits dans les tissus normaux et cancéreux en croisant avec d'autres données d'expression, telles que le RNA-Sequencing, et regarder leur conservation et leur expression dans d'autres espèces
A challenge of transcriptomics is to explore the full repertoire of normal and abnormal transcripts. Gene expression profiling analyses based on microarray technology are widely used in cancer research since many years. Meanwhile, new methods based on high-throughput sequencing methods offers henceforth the possibility to undergo accurate and sensitive analyses necessary for studying normal and cancer cells. Despite the method, the characterization of all coding and non-coding transcripts is a real biological challenge in identifying novel diagnostic and prognostic markers, and for the proper care of patients. In the present work, I had the opportunity to address two different aspects of biology, both convergent toward the identification of aberrantly expressed transcripts in myeloid leukemia. The first aspect was to provide a selection of molecular biomarkers for the characterization of chronic myelomonocytic leukemia (CMML). We developed a gene expression-based prognostic score which identified two clinically distinct groups of patients. We then completed our study with a phenotypic characterization by flow cytometry of aberrant subpopulations constituting the myeloid and granulocytic lineages. A part of this work has been extended to acute myeloid leukemia (AML) patients with normal karyotype. The other aspect was to participate in the implementation of an integrated computational approach in order to characterize novel non annotated RNAs, more likely non-coding. We quantified and characterized the proportion of these transcripts in intergenic regions by exploring Digital Gene Expression (DGE) data. We checked their expression in normal and cancer tissues by crossing with RNA-Seq data, and their conservation and expression in other species

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Corral, Vázquez Celia. "Human sperm transcriptome: characterization, biological relevance, and biomarker functionality." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669365.

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Se ha demostrado que la contribución del espermatozoide al embrión va más allá de la transmisión del genoma paterno. Diversos estudios han mostrado que el espermatozoide humano contiene una compleja población de RNAs implicados en funciones relacionadas con la fertilidad. Por tanto, la visión de estas moléculas como meros restos de eventos previos ha quedado atrás. Este nuevo paradigma abre las puertas a nuevas aplicaciones del RNA en el ámbito de los biomarcadores de fertilidad. Sin embargo, los análisis transcriptómicos en espermatozoides presentan diversas limitaciones debidas a la heterogeneidad y delicada naturaleza de estas moléculas, además de la poca cantidad de RNA contenida en dichas células. En este contexto, el objetivo de esta Tesis Doctoral es caracterizar el transcriptoma del espermatozoide humano y establecer las bases para desarrollar nuevos biomarcadores de fertilidad masculina. Dentro de este objetivo, se plantearon las siguientes metas: 1) optimizar metodologías específicas para analizar el RNA espermático mediante qRT-PCR y RNA-seq; 2) proporcionar un perfil integrado y una caracterización funcional de los mRNAs y lncRNAs espermáticos mediante RNA-seq; y 3) establecer nuevos biomarcadores de fertilidad a partir de la carga transcriptómica del espermatozoide. Con este propósito, se adaptaron tanto el protocolo experimental como el análisis de datos a las limitaciones propias del RNA espermático y a la tecnología transcriptómica usada. Por tanto, se implementaron métodos de eliminación de células no espermáticas de las muestras seminales, así como controles de calidad para asegurar la ausencia de DNA y RNA no espermático. Además, se usó un método basado en solventes orgánicos para los estudios qRT-PCR, y kits de solventes no orgánicos para RNA-seq. Los datos obtenidos se normalizaron usando métodos específicos de la técnica empleada. En concreto, para la normalización de los datos de expresión de miRNAs espermáticos en estudios singleplex qRT-PCR era necesario establecer miRNAs normalizadores. Esto se consiguió comparando los resultados derivados de unos datos que se normalizaron mediante: i) el método Mean-Centering Restricted (MCR); y ii) el nivel de expresión de diferentes miRNAs. Los miRNAs hsa-miR-100-5p y hsa-miR-30a-5p mostraron una expresión estable y ubicua, y su uso derivó en resultados con una calidad semejante a los conseguidos mediante la normalización por MCR. Por tanto, se sugirió esta combinación de miRNAs como la mejor opción para la normalización de futuros estudios singleplex qRT-PCR de miRNAs espermáticos. Por otro lado, se empleó RNA-seq, para caracterizar el transcriptoma espermático de individuos fértiles. Los resultados revelaron una red de mRNAs y lncRNAs en alto estado de fragmentación, pero que contenían un grupo de transcritos ubicuos. Los análisis de Ontología Génica de todos los mRNAs expresados mostraron una implicación en procesos de espermatogénesis y reproducción, la cual era más significativa en los análisis de los mRNAs altamente expresados, ubicuos y altamente estables. Aparte, los potenciales genes dianas en cis de los lncRNAs mostraron relación con procesos de desarrollo embrionario y adhesión celular, la cual prevalecía en los genes dianas que no estaban expresados en espermatozoides. Finalmente, el hecho de hallar transcritos ubicuos y de expresión correlacionada indicó un posible uso de estas moléculas como biomarcadores de fertilidad. Por tanto, se evaluó y se validó la presencia de pares de miRNAs espermáticos con una expresión correlacionada en individuos fértiles y no correlacionada en pacientes infértiles de diferentes etiologías (astenozoospermia, teratozoospermia, oligozoospermia e infertilidad inexplicable [UMI]). El par hsa-miR-942-5p/hsa-miR-1208 permitió clasificar correctamente el 85.71% de los casos de infertilidad, alcanzando el mayor potencial de diagnóstico de pacientes con alteraciones seminales. El par hsa-miR-34b-3p/hsa-miR-93-3p destacó por su potencial para discernir pacientes UMI. Aparte, varios pares de mRNAs y lncRNAs también mostraron expresiones correlacionadas en individuos fértiles, constituyendo unos candidatos potenciales para futuros estudios.
The biological relevance of sperm contribution to the embryo has been shown to go beyond a mere transmission of the paternal genome. Several findings revealed that human spermatozoa carry a complex population of coding and non-coding RNAs with potential implications in multiple fertility-related pathways. Accordingly, the consideration of these molecules as simple residual pools of earlier processes has been left behind. This new paradigm also opens the possibility for potential applications in the field of male fertility biomarkers. However, sperm transcriptomic analysis has several limitations due to the heterogeneity and delicate nature of these molecules, besides the small amount of RNA contained in spermatozoa. In this context, the objective of this Doctoral Thesis is to characterize the human sperm transcriptome to set up the basis for developing new biomarkers of male fertility. Within this goal, the following aims were undertaken: 1) To optimize specific methodologies of sperm RNA analysis using qRT-PCR and RNA-seq strategies; 2) To provide an integrative profiling and functional characterization of sperm mRNAs and lncRNAs by RNA-seq technologies; and 3) To establish new fertility biomarkers among the transcriptomic cargo of the human spermatozoa. For this purpose, the experimental protocols and data analysis were adapted to the inherent limitations of sperm RNA and to the used transcriptomic technology. Therefore, methods for the elimination of non-sperm cells from semen samples were implemented, together with strict quality controls for ensuring the absence of DNA and non-sperm RNA. Besides, an organic solvent-based method was used for qRT-PCR studies, and non-organic solvent kits were employed for RNA-seq. The obtained data were normalized by specific methods depending on the used technique. In particular, the normalization of sperm miRNA qRT-PCR singleplex studies required the determination of a suitable set of normalizing miRNAs molecules. This was achieved by comparing the results derived from a sperm miRNA expression dataset normalized by: i) the reference Mean Centering Restricted (MCR) method; and ii) the expression level of different miRNAs. The miRNAs hsa-miR-100-5p and hsa-miR-30a-5p showed ubiquitous and stable expressions, and data normalized by their mean expression led to results with an appropriate quality when compared to MCR. Therefore, this miRNA combination was suggested as the most suitable choice for data normalization in further sperm singleplex studies. RNA-seq analysis was used to characterize the sperm transcriptome cargo of fertile individuals. Results revealed a complex network of mRNAs and lncRNAs with a high fragmentation status, but containing a host of ubiquitous transcripts. Gene ontology analyses of the whole set of expressed mRNAs showed an enrichment of spermatogenesis and reproduction processes, which was more significant in the sets of highly expressed, ubiquitous, and highly stable mRNAs. Additionally, the functional profiling of potential cis-target genes of the observed lncRNAs showed a significant involvement in embryo development and cell adhesion. This implication became more evident in those cis-target genes that were not present among the sperm mRNA cargo. Finally, the detection of ubiquitous transcripts and pairs of RNAs with correlated expressions suggested a potential use of these molecules as fertility biomarkers. Accordingly, the presence of sperm miRNA pairs with a correlated expression in fertile individuals that was disrupted in infertile patients of different ethiologies (asthenozoospermia, teratozoospermia, oligozoospermia, and Unexplained Male Infertility or UMI) was evaluated and validated by qRT-PCR. The hsa-miR-942-5p/hsa-miR-1208 pair allowed correctly classifying the 85.71% of infertile individuals, thus achieving the highest potential for discerning infertility cases with seminal alterations. Additionally, the pair hsa-miR-34b-3p/hsa-miR-93-3p was highlighted due to its high potential for discerning UMI patients. Besides, several pairs of ubiquitous lncRNAs and mRNAs were also observed to display a correlated expression in fertile individuals, becoming potential candidates for further biomarker studies.

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19

Zhang, Zhouwei. "Investigation of DNA and RNA markers by novel technologies demonstrates DNA content intratumoral heterogeneity and long non-coding RNA aberrations in breast tumors." ScholarWorks @ UVM, 2014. https://scholarworks.uvm.edu/graddis/323.

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BACKGROUND: Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50Âµm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH). METHODS: Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests. RESULTS: Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (p CONCLUSION: Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.

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Crabtree, Nathaniel Mark. "Multi-Class Computational Evolution| Development, Benchmark Comparison, and Application to RNA-Seq Biomarker Discovery." Thesis, University of Arkansas at Little Rock, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10620232.

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A computational evolution system (CES) is a knowledge-discovery engine that constructs and evolves classifiers with a small number of features to identify subtle, synergistic relationships among features and to discriminate groups in high-dimensional data analysis. CESs have previously been designed to only analyze binary datasets. In this work, the CES method has been expanded to accommodate multi-class data.

The multi-class CES was compared to three common classification and feature selection methods: random forest, random k-nearest neighbor, and support vector machines. The four classifiers were evaluated on three real RNA sequencing datasets. Performance was evaluated via cross validation to assess classification accuracy, number of features selected, stability of the selected feature sets, and run-time.

The three common classification and feature selection methods were originally designed for microarray data, which is fundamentally different from RNA-Seq data. In order to preprocess RNA-Seq count data for classification, the data was normalized and transformed via a variance stabilizing transformation to remove the variance-mean relationship that is commonly observed in RNA-Seq count data.

Compared to the three competing methods, the multi-class CES selected far fewer features. The identified features are potential biomarkers that may be more relevant than the longer lists of features identified by the competing methods. The CES performed best on the dataset with the smallest sample size, indicating that it has a unique advantage in these situations since most classification algorithms suffer in terms of accuracy when the sample size is small.

The CES identified numerous potentially-important biomarkers in each of the three real datasets that are validated by previous research and worthy of additional investigation. CES was especially helpful at identifying important features in the rat blood RNA-Seq data set. Subsequent ontological analysis of these selected features revealed protein folding as an important process in that dataset. The other contribution of this research to science was to extend the applicability of CES to biomarker discovery in multi-class settings. New software algorithms based on CES have already been developed, and the multi-class modifications presented here are directly applicable and would also benefit the newer software.

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21

Hopkins, Tom. "The RNA-binding protein LARP1 as potential biomarker and therapeutic target in ovarian cancer." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/32144.

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Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3' untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer.

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22

Dinh, Tru-Khang T. "Circulating Micro-RNAs as Biomarkers for Thoracic Radiation Therapy in Lung Cancer." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:27007756.

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Risk of normal tissues toxicity limits the amount of thoracic radiation therapy that can be routinely prescribed for the treatment of non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic radiation may provide a way to predict eventual toxicities during the multi-week treatment regimen. This enables dose adjustment before the symptomatic onset of late effects, such as radiation pneumonitis and esophagitis. Micro-RNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by decreasing the translation of messenger RNAs. miRNAs constitute a major fraction of small RNAs reproducibly found in circulation, in part due to their protective encapsulation within exosomes. They are therefore attractive candidates as serological biomarkers. In this study, we performed miRNA profiling of the blood of 5 NSCLC patients at 5 dose-points during thoracic RT and found 10 miRNAs that correlated well with total radiation dose as well as other common dosimetric parameters. We then assessed these 10 miRNAs in samples from a separate cohort of 21 NSCLC patients receiving RT and identified miR-29a-3p and miR-150-5p as potential, reproducible biomarkers that decreased in circulation with increasing radiation dose. We also conducted in-vitro experiments to measure the expression levels of these miRNAs intracellularly and within exosomes in three NSCLC cell lines and two lung bronchoepithelial and fibroblast lines. The exosomal expression of miR-29a-3p and miR-150-5p decreased with radiation. However, this was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in NSCLC and stromal cells as a response to radiation. One may therefore hypothesize that outlier trends in levels of circulating miR-29a-3p and miR-150-5p may predict unexpected responses to radiation therapy, such as toxicity.

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Pokharel, Rounak. "Label Free Micro-RNA Biomarker Detection in Serum Samples for Potential Diagnosis Application at Point-of-Care Settings." Thesis, North Dakota State University, 2020. https://hdl.handle.net/10365/31880.

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The number of new cancer cases is projected to rise to 23.6 million by 2030 according to the National Cancer Institute. Obesity & cardiovascular diseases are among the leading causes of death worldwide according to recent reports. Biomarkers— any molecules found within a human body that can be used to monitor an individual's health — have been shown to play a significant role in the detection of cancer, obesity, and cardiovascular diseases. Recent studies have shown that in the diagnosis and screening of various human diseases, including cancer, obesity and cardiovascular diseases, circulating microRNAs (miRNAs) are important biomarkers. A crucial roadblock to using microRNA in screening applications is the lack of effective and low-cost microRNA detection. To address this issue, in this study, we have developed a viable method that combines the dielectrophoresis and electrical impedance. Results show this approach can measure very small concentrations of label-free microRNAs (1pM).

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Fadhil, Rushdi S. "Salivary micro RNAs as Potential Biomarkers for Oral and Head and Neck Cancers." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/396154.

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Head and neck cancer (HNC) could include several squamous cell carcinoma (SCC) cancers that are considered a serious health problem worldwide today. The incidence rate of HNC has seen a sustained increase in Australia. For example, in Queensland, there is a 10.6 % increase of oral squamous cell carcinoma (OSCC) in male annually. Head and neck squamous cell carcinomas (HNSCC) patients may be asymptomatic, therefore, poor prognosis and metastasis are usually common in the patients. Subsequently, late diagnosis has a low rate of survival which most likely results in extreme severity of tumours at the time of diagnosis. The survival rate of patients with HNC is very poor, currently assessed at not more than five years with a high probability of local recurrence (40%). Therefore, a delay in diagnosis is still valid while early diagnostic methods are still needed. Traditional diagnostic methods such as radiology and endoscopic biopsy are major techniques performed for this type of cancer. However, these clinical methods are stressful, relatively invasive in nature and extremely expensive. Thus, there is an urgent need for more specific, sensitive and straight forward early diagnostic procedures for investigating HNC. Furthermore, there is a necessity to discover novel biomarkers for early HNC diagnosis. MicroRNAs which are small noncoding molecules can be tested in different bodily fluids for e.g. blood, urine, saliva and serum. These have been verified as significant regulators of gene expression in cancer biology and may play a significant role in the early diagnosis of HNC. Several studies have revealed the importance of miRNAs in the diagnosis of different types of cancer. However, few studies have investigated the expression of salivary profile miRNAs in HNC patients before treatment, and the possible utility of this easily accessible biological fluid as a diagnostic biomarker for HNC. Salivary biomarkers for HNC are still relatively limited compared with other types of cancers such as breast, lung and colon. Regarding existing knowledge, the literature review is inadequate for Australian HNC researchers. Furthermore, conflicting results have been reported in miRNAs expression levels in different types of cancer, including HNC and these might be as a result of contrasting experiment design, validation methods or sample variations. To address this literature gap, this study was designed to profile the expression of different salivary miRNA in HNSSC patients in Australian as compared to healthy individuals; as well as comparing grades and stages of cancer, cancer sites and habit status. Significant variation in salivary miRNAs expression has been hypothesised between HNC patients and healthy individuals which is invaluable in the diagnosis of HNC. To test the hypothesis, several studies have been carried out. The aims of these studies are: i) To analyse the profile miRNAs expressions and their significance in HNC Australian patients and healthy adults using oral rinse samples; and ii) to test the possible utility of saliva sample as an easily accessible biological fluid and stress free for diagnostic biomarker isolation for HNC. Chapter one explores the scientific problem and aims of this study. Chapter two provides a comprehensive literature review of the background in this field of work and the methodology used to conduct our research. Chapter three detected the potential identification of the significant variation of salivary miRNAs in OSCC. Five different miRNAs (miR-21, miR-10b, miR-125a, miR-31 and miR-200a) were selected from the literature review to optimise the conditions of RNA extraction and thermocycles of real time PCR. Besides, these miRNAs have been highlighted in conflicting results both oncogenic and suppressor genic in different types of cancer. However, very little is known about the role and expression of these miRNAs in saliva samples of oral cancer. Thus, the study of this chapter documented the role of these miRNAs that is specifically associated with HNSCC. In the above study, the result revealed that the overall expression of salivary miR-125a was significantly lower in OSCC patients compared with healthy individuals, while salivary miR-21 in OSCC patients was much higher than healthy individuals. Interestingly, upregulated salivary miR-21 was associated with tumour’s stage of OSCC (p≤0.05). A Receiver Operator Curve (ROC) was also constructed to estimate the discriminatory power of miR- 21 and miR- 125a for their potential to distinguish between healthy adults and the OSCC groups. These two miRNAs were shown to have good discriminatory ability with AUC values of 0.95 and 1, respectively. In chapter four, next generation sequencing was used to investigate the variation of miRNAs profile expression levels between OSCC patients and healthy individuals, as a more efficient and precise technique. This study aimed to detect potentially significant miRNAs in OSCC. The salivary miRNA expression profiling identified 2565 miRNAs differentially co-expressed. However, 1927 miRNAs expressions are nonsignificantly differentiated between OSCC and the healthy individuals’ group. There are 638 miRNAs significantly co-expressed in the supernatant saliva of OSCC patients compared with healthy individuals group where p-value ≤0.05; 114 miRNAs with a pvalue ≤ 0.001; while 27 miRNAs were significantly different with a p-value ≤ 0.0001. This study contributes to knowledge of the association between 27 dysregulated salivary miRNAs in OSCC and the potential to use them for OSCC diagnosis (p = 0.0001). 15 of these miRNAs were up-regulated (miR-7703, miR-3928-5p, miR-889- 5p, miR-3147, miR-4474-3p, miR-3170, miR-6895-5p, miR-1238-5p, miR-4521, miR- 548ac, miR-3158-3p, miR-1343-3p, miR-7152-5p, miR-3148, miR-3124-3p, ) and 12 miRNAs were down-regulated (miR-let-7f-5p, miR-let-7a-5p, miR-1247-5p, miR-574- 3p, miR-194-5p, miR-200c-3p, miR-32-5p, miR-6126, miR-99a-5p, miR-345-5p, miR- 301a-3p, miR-101-3p). Also, this study pointed out for the first time that overexpression of miR-7703 is a great and significant biomarker that could be used for cancer diagnosis. Hence, to confirm these results, it is reasonable to validate these miRNAs as signature diagnostic biomarkers in a large majority of patients. Chapter five was aimed to validate the previous findings of chapter four in relation to the five dysregulated miRNAs in 150 patients with head and neck cancer (HNC) along with 80 healthy individuals. Thus, five dysregulated miRNAs (miR-7703, miR- let-7a- 5p, miR- 345-5p, miR- 3928 and miR- 1470) were selected for the validation study using a real time PCR technique. In this study, significant expression differences of miR-let-7a-5p and miR-3928 (p≤0.05) in HNC patients compared with healthy individuals were confirmed. These miRNAs were revealed to provide a good discriminative ability with AUC values of 0.85 and 0.74, respectively. Taken together, the data reveals that the availability of significant miRNA could play a great role in HNC early diagnosis. Those studies contribute to knowledge on the correlation of miRNAs expression with OSCC and HNC and improve the availability of current diagnostic methods of cancer. In addition, the literature findings of miRNA studies supported our results and reported consistent results in the role of miRNA in cancer. However, this significance of the dysregulated expressions of miRNAs requires further investigation and additional research.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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25

ARIAS, JESICA PAOLA RADA. "NATURAL AND ANTHROPOGENIC ORGANIC MATTER IN SEDIMENTS IN RIA DE AVEIRO, PORTUGAL: CHARACTERIZATION BY LIPID BIOMARKERS." PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2015. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=33663@1.

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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
FUNDAÇÃO DE APOIO À PESQUISA DO ESTADO DO RIO DE JANEIRO
BOLSA NOTA 10
A matéria orgânica (MO) sedimentar de origem natural na Ria de Aveiro e no estuário Mondego foi caracterizada usando a composição elementar (C e N) e lipídios biomarcadores (esteróis, n-álcoois e triterpenoides). A contribuição por esgotos foi avaliada através de coprostanol e outros esteróis fecais. Amostras de sedimento superficial foram coletadas em 22 estações ao longo das regiões. Os lipídios biomarcadores foram extraídos, purificação com sílica-gel e analisados por cromatografia em fase gasosa acoplada a espectrometria de massas. O carbono orgânico total (9,94 e 43,00 mg g(-1)) teve uma correlação com o nitrogênio total (r(2) = 0,86). A razão C/N molar (14,34 mais ou menos 1,75) foi um valor típico de MO continental. Existe uma contribuição maior de MO terrestre nas amostras, como mostra as evidencias: a predominância de n-álcoois de cadeia longa (maior ou igual à C22), maior abundancia no somatório de sitosterol, estigmasterol e campesterol sobre outros fitoesterois, como o colesterol e o brassicasterol; e a alta concentração de triterpenoides de plantas superiores, como Beta-amirina e a Alfa amirina. A concentração de coprostanol (0,05 a 16,6 microgramas g(-1)) indicou a presença de esgotos em sete estações localizadas no largo do Laranjo (S4, S5, S6, S7, S10), porto de Aveiro (S14) e no canal de Ílhavo (S16). Os aportes mais significativos foram no largo do Laranjo (S8 e S9) e no porto do Mondego (S19).
The sedimentary organic matter (OM) of natural origin in the Ria de Aveiro and the estuary of Mondego River was characterized using elemental composition (C and N) and lipid biomarkers (sterols, alcohols and triterpenoids). In addition, the contribution by sewage was evaluated by coprostanol and other fecal sterols. Surface sediment samples were collected at 22 stations along the two regions. The lipid biomarkers was extracted, separated with silica-gel and analyzed by gas chromatography coupled to mass spectrometry. Concentrations of total organic carbon (9.94 and 43.00 mg g(-1)) was highly correlated with total nitrogen (r(2) = 0.86). The C/N molar ratio: 14,34 plus-minus 1,75 are typical values of OM of continental origin. There is a major contribution of terrestrially OM to the sediments, as suggested by proxies, including: predominance of long-chain (greater-than or equal to C22) alcohols, higher abundance of sitosterol, estigmasterol and campesterol over other phytosterols, like cholesterol and brassicasterol, and elevated concentrations of triterpenoids from higher plants, as Beta-amyrin and Alpha-amyrin. The concentrations of coprostanol (0.05 to 16.6 micrograms g(-1)) indicated the presence of sewage in seven stations located in the Largo do Laranjo (S4, S5, S6, S7, S10), Port of Aveiro (S14) and in Ílhavo Channel (S16). The more significant contribution of sewage was identified in the Largo do Laranjo (S8 and S9) and the Port of Mondego (S19).

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26

Matson, Cole Wesley. "Combining environmental chemistry, somatic biomarkers, and population genetics: an innovative approach in wildlife ecotoxicology." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/72.

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The Caspian region and specifically the Apsheron peninsula of Azerbaijan is known to be polluted with a variety of environmental contaminants, making risk assessment difficult. The wetlands of Sumgayit contain particularly complex mixtures of contaminants. Flow cytometry and the micronucleus assay were used to assess chromosomal damage in aquatic turtles and frogs inhabiting contaminated wetlands in Azerbaijan. By evaluating biomarkers that are indicative of somatic effects, elevated chromosomal damage was documented at several sites in Azerbaijan relative to reference sites. Sediment samples were analyzed for polycyclic aromatic hydrocarbons (PAHs), organochlorines (OCs), and mercury to evaluate contaminant associations with genetic damage. Sediment samples revealed heterogeneous patterns of PAH and mercury concentrations throughout Sumgayit. Significant positive correlations were documented between both PAH and mercury sediment concentrations and chromosomal damage. Population genetic methods were employed to study the effects of long-term chronic contaminant exposure in marsh frogs from Sumgayit. The Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. One of the most contaminated sites in Sumgayit, WTP, appears to be a source of new mutations as a result of an increased mutation rate. Finally, the Sumgayit region seems to act as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. This study provides not only exposure and biomarker data, but also an integrated method for assessing the cumulative population impacts of contaminant exposure by studying both population genetic and evolutionary effects. The results presented here will be used in conjunction with those of ongoing research involving both wildlife and humans to develop comprehensive ecological and human risk assessments.

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27

Ferraioli, Domenico. "Assessment and relevance of the putative DNA/RNA helicase Schlafen-11 in ovarian and breast cancer." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1324/document.

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Schlafen 11 (SLFN11) est une ADN/ARN hélicase décrite pour la première fois pour son rôle dans le développement et la différenciation des thymocytes chez la souris. Elle fait partie d'une famille de protéines présentant divers degrés d'homologie entre les espèces, mais qu’est présente de façon constante chez les mammifères. Le rôle de cette ADN/ARN hélicase, SLFN11, a été associé de façon causale à la sensibilité de réponse aux différents agents alkylants (agents endommageant l'ADN, les inhibiteurs de topo-isomérase I et II) dans le NCI-60. Dans la première étude, nous avons développé un protocole d’immunohistochimie (IHC) sur des biopsies paraffinées de carcinome séreux de l'ovaire de haut grade (HGSOC), afin de valider un anticorps (Ab) anti-SLFN11 et d’en déterminer l'expression. En IHC, nous avons testé et validé un Ab anti-SLFN11, en choisissant entre deux anti-SLFN11 Ab utilisés normalement pour le Western Blot. Premièrement, il a été développé dans une culture cellulaire (CCB) de HGSOC et, successivement, dans une série indépendante de micro-array (TMA) de HGSOC. Pour chaque cas, nous avons évalué soit le score d'intensité (IS) que le score de distribution (DS) en évaluant au moins 300 cellules. Un score histologique (HS) a été obtenu comme suit : HS=IS x DS. Successivement, nous avons appliqué notre protocole à une plus large série d'échantillons de HGSOC pour confirmer nos résultats préliminaires. Nous avons trouvé un anticorps fiable dans les séries CCB et TMA permettant de déterminer l'expression IHC de SLFN11. Ces résultats ont été confirmés dans notre plus large série de HGSOC. Brièvement, comme pour les séries indépendantes de TMA, nous avons constaté que la HS de l'expression de SLFN11 est présente dans environ 60%. En parallèle, le SLFN11 n'a pas été exprimé dans 40 % des cas qui, cliniquement, correspondent, dans environ 60 % de ces cas (16/27), aux patients résistant aux sels de platine. Une faible expression de SLFN11 en IHC pourrait être corrélée à la réponse à la chimiothérapie(CT) à base de platine. Dans la deuxième étude, nous étudions l’état transcriptionnel du SLFN11 dans le cancer du sein en effectuant une méta-analyse de plus de 7000 cas à partir de 35 étudies publiquement disponibles. Par l’analyse de corrélation, nous avons identifié 537 transcrits qui corrèle, au-delà du 95e percentile selon le coefficient de Pearson, avec l’expression de SLFN11. En particulier, voie l’analyse par “Gene Ontology” SLFN11 est lié au transcrits impliqués dans le système immunitaire : "réponse immunitaire", "l’activation lymphocytaire" et "l’activation des lymphocytes T". En outre, voie le “likehood lasso regression ”, nous avons signalé une très forte association entre le SLFN11 et les signatures immunitaires dans le cancer du sein. Enfin, grâce à la “multiple corresponded analysis ”, nous avons découvert un sous-groupe de patients, défini "SLFN11-Hot cluster", caractérisé par une expression élevé de SLFN11, récepteurs d'œstrogènes(ER) négatives, un phénotype basal, un jeune âge, une signature élevée de CD3D et de STAT1. En utilisant la "Cox proportional hazard regression", l’expression élevé de SLFN11, l’indice de prolifération élevé et le ER négative sont des paramètres indépendants lié à la survie sans maladie chez les patients soumises à la CT. Notre deuxième travail decrit un rôle spécifique pour le SLFN11 dans le cancer du sein probablement en relation avec la modulation du système immunitaire et une forte corrélation entre l’expression de SFLN11 et un sous-type moléculaire spécifique de cancer du sein (récepteurs négatifs aux œstrogènes, phénotype de type basal). Autres études devront être réalisées afin de: 1) mieux comprendre la fonction du SLFN11 dans les cellules cancéreuses, 2) valider un protocole IHC fiable et standardisé pour évaluer l’expression de SLFN11, 3) utiliser SFLN11 comme biomarqueur prédictif de réponse aux DDA et PARP inhibiteurs et 4) établir sa relation avec le système immunitaire
Schlafen 11 (SLFN11) is a putative DNA/RNA helicase, first described for its role in thymocyte development and differentiation in mouse models. SLFN11 is part of a family of proteins with various degree of homology across species, but intriguingly being consistently present only in vertebrates and especially in mammals. Recently, the role of this putative DNA/RNA helicase, SLFN11, was causally associated with sensitivity to DNA damaging agents, such as platinum salts, topoisomerase I and II inhibitors, and other alkylators in the NCI-60 panel of cancer cell lines. In the first study, we validate an anti-SLFN11 antibody in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples, developing an immunohistochemistry (IHC) protocol in order to determinate the expression of SLFN11 in our series of HGSOC. Indeed, we tested and validated a reliable SLFN 11 antibody (Ab) in IHC choosing between two anti-SLFN11 Ab used normally for Western Blot (WB) in culture cell block (CCB) of ovarian carcinoma and in an independent series of HGSOCs tissue micro-array (TMA). For each case, we evaluated both the Intensity Score (IS) and the Distribution Score (DS) evaluating at least 300 cells. A Histological Score (HS)was obtained as follow: HS=IS x DS. Successively, we applied our protocol to a large case series of HGSOC samples to confirm our preliminary results. We found one antibody to be reliable in CCB and TMA series allowing to determinate clearly IHC expression of SLFN11. These results were confirmed in our large case series of FFPE HGSOC samples. Briefly, as for TMA independent series, we found that the HS for SLFN11 expression presents a normal distribution with a prevalent (≈ 60%) intermediate expression. Parallel SLFN11 was not expressed in practically 40% of cases that clinically corresponded to the platinum resistant patients in about 60% of cases (16/27). So, we believe that low IHC expression of SLFN 11 should be correlated to response to the platinum-based chemotherapy. In the second study, we investigate the transcriptional landscape of SLFN11 in breast cancer performing a gene expression microarray meta-analysis of more than 7000 cases from 35 publicly available data sets. By correlation analysis, we identified 537 transcripts in the top 95th percentile of Pearson’s coefficients with SLFN11 identifying “immune response”, “lymphocyte activation” and “T cell activation” as top Gene Ontology enriched processes. Furthermore, we reported very strong association of SLFN11 with immune signatures in breast cancer through penalized maximum likelihood lasso regression. Finally, through multiple corresponded analysis we discovered a subgroup of patients, defined “SLF11-hot cluster”, characterized by high SLFN11 levels, estrogen receptor(ER) negativity, basal-like phenotype, elevated CD3D, STAT1 signature, and young age. Using Cox proportional hazard regression, we characterized that SLFN11 high levels, high proliferation index, and ER negativity are independent parameters for longer disease-free interval in patients undergoing chemotherapy. We believe that our second work supports proof of concept that: i) A clear and specific role for SLFN11 in breast cancer, in likely connection with the immune system modulation in such disease entity, ii) a strong correlation between high SFLN 11 and specific molecular subtype of breast cancer (estrogen receptor negativity, basal-like phenotype). Further studies will be performed to confirm our hypothesis in order to: 1) better understand the function of SLFN 11 in cancer cell, 2) validate an easy, reliable and standardized IHC protocol to assessment SLFN11, 3) use SFLN11expression as a predictive biomarker of response to DDA and PARP inhibitors and 4) determinate the relationship with immune system

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28

Liu, Yu. "Better understanding of canine telomerase and its potential applications in canine oncology." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6499.

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Telomerase, discovered in 1985, is considered a near-universal marker of malignancy and therefore has a potential use in cancer therapeutics and diagnostics. In this study, I used several approaches to gain a better understanding of telomerase and its potential applications in the canine context, for both cancer therapeutics and diagnosis. Having already developed an effective siRNA viral vector in vitro, the challenge still remained to deliver it efficiently in vivo. Thus, I initially investigated two possible approaches for in vivo delivery. First, I investigated a cell-based system for direct delivery to the tumours. Specifically I optimised a system for efficient gene-transfer to endothelial cells using a green fluorescent protein plasmid vector, and monitored systemic delivery by ex vivo imaging of dye-labelled cells in a canine xenograft tumour mouse model. In parallel, in vitro I investigated the gene transfer mediated by a novel dendrimer vector that can form nanoparticles with DNA and accumulate in tumour sites in vivo after i.v. administration. In order to utilize these delivery systems, I developed a DNA plasmid-based siRNA vector and tested its efficacy on canine tumour cells. To investigate telomerase as a cancer biomarker, I conducted a study that aimed to detect circulating telomerase reverse transcriptase (TERT) mRNA in serum taken from canine cancer patients. For this I developed several systems for effective RNA isolation from serum and used both conventional and quantitative PCR assays to detect TERT expression. Although for the first time I can confirm the existence of mRNA in serum of canine cancer patients, in this clinical study, I could only detect telomerase transcripts in a very small proportion of canine cancer patients. In a final pilot study to investigate anti-ageing technologies, I looked at the potential for drug-dependant telomerase induction rather than inhibition. For this I investigated the ability of three candidate drugs to induce TERT mRNA activation in canine embryonic fibroblasts. In this study, telomerase induction was measured using the quantitative PCR method that I had developed for serum detection. In summary, I have demonstrated that a cell-based delivery vehicle has a potential application in cancer therapy, but that more development is required before it can be applied clinically. I have also reported here that PPIG3 dendrimer-based gene transfer in vitro is low in canine cancer cells and thus require more optimisation and development before it can be utilised as an efficient systemic delivery vehicle. For the siRNA experiment, unfortunately, I did not observe any telomerase genesilencing in canine cancer cells using the plasmid-based siRNA expression vector, and therefore the gene sequence of cTR that we were targeting as well as the siRNA plasmid-vector that we used needs further validation in canine cells. I also suggest that TERT mRNA may not be a good serum biomarker for canine cancer diagnostics as I did not find TERT transcript in most of our serum samples from canine cancer patients, although circulating mRNA of a housekeeping gene was detected. Finally, in a pilot study, I have demonstrated that telomerase can be induced in normal canine somatic cells using small molecules. However, the long-term effects of telomerase induction on ageing must be determined in future studies.

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29

Schulz, Elena [Verfasser], Alexander [Gutachter] Baraniskin, and Claus [Gutachter] Doberauer. "Zirkulierende Fragmente der small nuclear RNA U2 als neuer diagnostischer Biomarker für das primäre ZNS-Lymphom / Elena Schulz ; Gutachter: Alexander Baraniskin, Claus Doberauer ; Medizinische Fakultät." Bochum : Ruhr-Universität Bochum, 2018. http://d-nb.info/1163451320/34.

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30

Zimmer, Christian [Verfasser], Jürgen [Akademischer Betreuer] Brockmöller, Volker [Akademischer Betreuer] Ellenrieder, and Margarete [Akademischer Betreuer] Schön. "Identifizierung von Biomarkern für die Prognose der Gemcitabin-Therapie beim Pankreaskarzinom: RNA-, DNA- und Immunhistochemische- Analysen / Christian Zimmer. Gutachter: Volker Ellenrieder ; Margarete Schön. Betreuer: Jürgen Brockmöller." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1075372852/34.

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31

Voß, Madeleine [Verfasser], Wolfgang A. [Gutachter] Schulz, and Michael [Gutachter] Sabel. "Lange nicht-kodierende RNAs als Biomarker in Plattenepithelkarzinomen von Kopf und Hals / Madeleine Voß ; Gutachter: Wolfgang A. Schulz, Michael Sabel." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1211554511/34.

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32

Chambers, David L. "Abiotic Factors Underlying Stress Hormone Level Variation Among Larval Amphibians." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/27817.

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Anthropogenic disturbances can alter the abiotic composition of freshwater systems. These compositional changes can act as physiological stressors towards system inhabitants. However, little is known about how these altered abiotic factors influence stress hormones (corticosterone) in larval amphibians. Throughout the following chapters, I examined the effects of several abiotic factors on baseline and stress-induced corticosterone levels in the larvae of four amphibian species: Jefferson salamander (Ambystoma jeffersonianum), spotted salamander (A. maculatum), wood frog (Rana sylvatica), and grey treefrog (Hyla versicolor). Chapter II examined corticosterone level differences throughout development in A. jeffersonianum and R. sylvatica larvae under field, mesocosm, and laboratory venues. Baseline corticosterone levels in R. sylvatica increased near metamorphic climax in all venues, but not in A. jeffersonianum. Rather, baseline corticosterone levels differed with respect to venue throughout development in A. jeffersonianum. Chapter III examined corticosterone level differences among free-living A. jeffersonianum populations and possible abiotic factors underlying these hormone differences. Corticosterone levels significantly differed across populations. Increased baseline corticosterone levels significantly correlated to low pH. There was also a trend for increased baseline corticosterone levels to be positively correlated with chloride levels and negatively correlated with conductivity. Chapter IV examined the effects of laboratory manipulated pH on corticosterone levels in A. jeffersonianum, A. maculatum, R. sylvatica, and H. versicolor. There was a significant correlation between increased baseline corticosterone levels to low pH in all four species. Prey consumption (in both Ambystoma species) and survival (in A. jeffersonianum, A. maculatum, and R. sylvatica) were also negatively correlated to low pH. Chapter V examined the effects of increased conductivity on corticosterone levels in A. jeffersonianum, R. sylvatica, and H. versicolor. Increased conductivity exposure significantly correlated to increased baseline corticosterone levels in A. jeffersonianum and R. sylvatica. Prey consumption in A. jeffersonianum was also negatively correlated to increased conductivity. My dissertation shows that abiotic factors, such as pH and conductivity, can influence corticosterone levels in larval amphibians. These results suggest that corticosterone levels in larval amphibians may be a suitable biomarker reflective of altered freshwater habitat quality. However, my results also suggest that one should use a high degree of caution when using corticosterone levels in larval amphibians as a means to infer the health status of a population.
Ph. D.

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33

Carregosa, Vanessa Silva. "Tolerance and response of clams in Ria de Aveiro to salinity changes." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/13424.

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Mestrado em Biologia Marinha
Unlike the concern that has been growing in relation to the impacts of contamination in marine benthic populations, the responses of aquatic organisms to natural alterations, namely changes in salinity, have received little attention. In fact, salinity is one of the dominant environmental factors that most affect marine bivalves, limiting their spatial distribution in the environment. Tide combined with fresh water inputs, from rivers or heavy rainy periods, and extreme dry seasons can dramatically alter the salinity of water, causing alterations in the benthic populations, namely intertidal bivalves. Furthermore, salinity of a given environment will restrict the spatial distribution of the species, which is especially important when assessing the spread of an invasive species into a new environment. In order to understand how native (Venerupis decussata and Venerupis corrugata) and invasive (Venerupis philippinarum) clam species cope with salinity changes, physiological, biochemical and metabolomic patterns were investigated. The results obtained showed that V. decussata and V. philippinarum presented high mortality at low (0 and 7) but tolerate high (35 and 42) salinities. On the other hand, V. corrugata presented high mortality rates both at low (0 and 7) and high salinities (35 and 42). The quantification of Na and K content revealed that, along the salinity gradient, V. decussata was the species with higher ability to maintain the ionic homeostasis. The biochemical parameters also showed that V. decussata was the clam that best cope with salinity changes and V. corrugata was the most sensitive. Furthermore, the results obtained showed that clams under salinity stressful conditions can alter their biochemical mechanisms, such as increasing their antioxidant defences, to cope with the higher oxidative stress resulting from hypo and hypersaline conditions. Among the physiological and biochemical parameters analysed (glycogen, glucose and protein content; lipid peroxidation (LPO) levels, antioxidant enzymes activity; total, reduced and oxidized glutathione), superoxide dismutase (SOD), LPO and glutathione S-transferase (GST) showed to be useful biomarkers to assess salinity impacts in clams. The effects of salinity changes in the metabolic profile of the three species were also studied using 1H Nuclear Magnetic Resonance (NMR) spectroscopy of clam extracts. Multivariate analysis of the NMR spectra enabled metabolite changes to be observed in relation to clams exposure to different salinity concentrations. When exposed to low salinities, energy reserves of clams may be exhausted, increasing the osmotic imbalance, affecting the metabolic performance and increasing the oxidative stress. V. corrugata showed to be the most sensitive clam to salinity changes. The optimal salinity for V. decussata and V. philippinarum was between 21 and 28 and for V. corrugata was salinity 21. This study showed that changes in salinity have different impacts in native and invasive species
As respostas dos organismos aquáticos a alterações naturais, nomeadamente, alterações de salinidade, têm recebido pouca atenção, inversamente à preocupação que tem vindo a crescer em relação aos impactos da contaminação em populações marinhas bentónicas. De facto, a salinidade é um dos factores ambientais dominantes que mais afetam os bivalves marinhos, o que limita a sua distribuição espacial no ecossistema. As marés combinadas com entradas de água doce, de rios ou períodos de chuva longos e estações secas extremas, podem alterar drasticamente a salinidade da água, provocando alterações nas populações de bivalves bentónicos, nomeadamente intertidais. Além disso, a salinidade de um determinado ambiente irá restringir a distribuição espacial das espécies, o que é especialmente importante quando se avalia a propagação de uma espécie invasora num ambiente novo. A fim de entender como espécies nativas (Venerupis decussata e Venerupis corrugata) e invasoras (Venerupis phiippinarum) de molluscos lidam com as mudanças de salinidade, foram investigados parâmetros fisiológicos, bioquímicos e metablómicos. Os resultados obtidos mostraram que V. decussata e V. philippinarum apresentaram elevada mortalidade em salinidades baixas (0 e 7), mas toleram as salinidades mais altas (35 e 42). Por outro lado, V. corrugata apresentou elevadas taxas de mortalidade tanto em salinidades baixas (0 e 7) como em salinidades altas (35 e 42). A quantificação do teor de Na e K, revelou que ao longo do gradiente de salinidade, a V. decussata foi a espécie com maior capacidade de manter a homeostasia iónica. Os parâmetros bioquímicos também mostraram que V. decussata foi a espécie que melhor lidou com as mudanças de salinidade enquanto a V. corrugata foi a mais sensível. Além disso, os resultados obtidos mostraram que as ameijoas, sob condições adversas de salinidade, podem alterar os seus mecanismos bioquímicos, nomeadamente aumentando as suas defesas antioxidantes, para lidar com um maior stress oxidativo resultante das condições de hipo e hipersalinidade. Entre os parâmetros fisiológicos e bioquímicos analisados (glicogénio, glucose, proteinas, níveis de peroxidação lípidica (LPO), atividade de enzimas antioxidantes; glutationa total, reduzida e oxidada), LPO, superoxide dismutase (SOD) e glutathiona S-transferase (GST) mostraram ser biomarcadores úteis para avaliar os impactos de salinidade em bivalves. Os efeitos das alterações de salinidade no perfil metabólico das três espécies foram também estudados através de Ressonância Magnética Nuclear de 1H (RMN). A análise multivariada dos espectros de RMN permitiu a observação de alterações em relação à exposição de ameijoas a diferentes concentrações de salinidade. Quando expostos a baixas salinidades, as reservas energéticas destes organismos podem ser esgotadas, aumentando o desequilíbrio osmótico, afetando o desempenho metabólico e aumentando o stress oxidativo. V. corrugata mostrou ser a amêijoa mais sensível a mudanças de salinidade. O intervalo de salinidades entre 21 e 28 foi o ideal para V. decussata e V. philippinarum e a salinidade 21 foi a ideal para V. corrugata. Este estudo mostrou que as mudanças de salinidade têm impactos diferentes em espécies nativas e invasoras.

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34

Sengal, Asmerom Tesfamariam. "Prognostic, predictive, and therapeutic role of FGFR2 isoforms and cognate FGF ligands in endometrial cancer." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/205851/1/Asmerom%20Tesfamariam_Sengal_Thesis.pdf.

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This project investigated the role of FGFR2 isoforms (FGFR2b/FGFR2c) and their cognate FGF ligands in endometrial cancer development, prognosis, and treatment response via designing and validating an innovative BaseScope RNA in-situ hybridization assay and generating patient tumour-derived organoids. FGFR2c and high FGF18 expression were significantly associated with aggressive tumour characteristics and poor survival outcome. It was also noted FGFR2c expression is associated with progestin treatment failure in atypical hyperplasia and well-differentiated endometrial cancers. Overall, FGFR2c and FGF18 are independent prognostic biomarkers that could improve our ability to predict patient prognosis and predict response to FGFR inhibitor treatment in endometrial cancer.

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35

Muys, Bruna Rodrigues. "Caracterização da Estrutura e Regulação dos Genes MGC16121 e CR596471." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-29082013-142331/.

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Os genes MGC16121 e CR596471 localizam-se no cromossomo X (Xq26) entre os loci HPRT1 e PLAC1, uma região rica em genes associados com a reprodução humana. A importância de tais genes reside na possibilidade de estarem envolvidos no desenvolvimento placentário e fetal e de serem expressos em poucos tecidos normais. Camundongos portadores de deleções próximas do gene ortólogo HPRT1 de humanos apresentam cerca de um terço do tamanho dos camundongos selvagens ou em alguns casos são natimortos. No entanto, este fenótipo não é observado quando o gene está mutado. Assim, pode-se supor que o fenótipo anormal das cobaias não é resultado da deficiência do HPRT1, mas sim de genes e/ou microRNAs (miRNAs) próximos a ele. Estes resultados abrem perspectivas em relação ao estudo dos genes MGC16121, CR596471 e miRNAs das vizinhanças. O objetivo deste trabalho foi caracterizar a estrutura, a expressão e o mecanismo de regulação por metilação dos genes MGC16121 e CR596471. Adicionalmente foram analisados quanto ao perfil de expressão e regulação por metilação os miRNAs das vizinhanças (miR-424, 503, 450a, 450b-5p e 542-3p). O gene MGC16121 mostrou-se específico de placenta e também expresso em 50% das 18 linhagens tumorais analisadas. Já CR596471 e os miRNAs das vizinhanças foram mais expressos em placenta do que qualquer outro tecido normal analisado, sendo o primeiro expresso também em 100% das linhagens tumorais avaliadas. Houve correlação positiva e significativa entre todos os genes e miRNAs em relação à expressão em tecidos normais, porém o mesmo não foi observado para linhagens tumorais. A respeito da regulação, os genes CR596471 e MGC16121 e os miRNAs miR-424, 503 e 450a foram regulados negativamente por metilação do DNA em pelo menos uma das três linhagens tratadas com o agente demetilante 5-aza-2-deoxicitidina. Apoiando este fato, os dinucleotídeos CpG das ilhas CpGs situadas próximas às regiões 5 dos genes CR596471 e MGC16121 foram pelo menos em parte desmetilados após o mesmo tratamento.Os dados relativos à estrutura primária dos genes indicam que os transcritos, apesar de serem lncRNAs apresentaram características de mRNAs. Para MGC16121 foi determinado um transcrito composto de 3 éxons e, para CR596471, um transcrito composto de 3 éxons e outro composto de 2 éxons. Os transcritos aqui determinados são relativamente conservados quando comparados a sequências de RNA encontradas em outros mamíferos, principalmente em primatas. Adicionalmente, o transcrito de MGC16121 possui subestruturas secundárias visivelmente semelhantes com aquelas dos transcritos homólogos encontrados em alguns primatas. De acordo com os resultados, o gene MGC16121 pode ser considerado um possível bom marcador para diagnóstico, prognóstico e talvez para terapias contra cânceres. Todavia, mais experimentos devem ser realizados para verificar a função dos genes MGC16212 e CR5976471, além de avaliar mais robustamente a capacidade do gene MGC16121 ser utilizado como ferramenta na medicina contra o câncer.
CR596471 and MGC16121 genes lie on chromosome X (Xq26) between the HPRT1 and PLAC1 loci, a region rich in genes associated with human reproduction. The importance of such genes is the possibility that they might be involved in placental and fetal development, aware that they are expressed in few normal tissues. Deletions in mice around the orthologous gene of human HPRT1 affect their development or lead to stillbirth. However, this phenotype is not observed when this gene is mutated. So we can assume that the abnormal phenotype of mice cannot be due to HPRT1 deficiency, but to genes and/or microRNAs (miRNAs) nearby. These results support the idea of investigating the mechanisms involved in the regulation of the MGC16121 and CR596471 genes, and their neighbor miRNAs. This study aimed to characterize the structure, expression and regulation mechanism by methylation of genes MGC16121 and CR596471. In addition, the expression profile and methylation regulation of the neighbor miRNAs (miR-424, 503, 450a, 450b-5p and 542-3p) were analyzed. MGC16121 was demonstrated to be placenta specific and expressed in 50% of 18 tumor cell lines analyzed. CR596471 and the neighbor miRNAs were more expressed in placenta than in any other normal tissue analyzed. The former was also expressed in all tumor cell lines evaluated. There was significant and positive correlation between all genes and miRNAs regarding normal tissue expression. However, the same was not observed for the tumor cell lines. With respect to regulation, the genes CR596471 and MGC16121, and miRNAs miR-424, 503 and 450a were negatively regulated by DNA methylation at least in one of the three cell lines treated with the demethylating agent 5- aza-2-deoxycytidine. Supporting these results, the CpG dinucleotides from CpG islands located near the CR596471 and MGC16121 5 regions were at least partially demethylated after the same treatment. The data concerning to genes primary structures indicate that the transcripts, despite of being considered lncRNAs, presented mRNAs characteristics. It was determined one transcript for MGC16121 gene which consisted of three exons, and for CR596471 gene, two transcripts were found, one with three exons and other composed of two exons. The transcripts herein determined are relatively conserved when compared to RNAs sequences found in other mammals, mostly in primates. Besides, the MGC16121 transcript presents similar secondary substructures to those found in homologous transcripts from other primate species. According to the results, MGC16121 gene could be considered a possible good biomarker to diagnosis, prognosis and perhaps to therapies against cancers. Nevertheless, more experiments must be accomplished in order to verify the functions of MGC16121 and CR596471 genes, in addition to evaluate more robustly the competence of MGC16121 gene to be used as a tool in medicine against cancer.

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36

Sousa, Rodrigo Guarischi Mattos Amaral de. "O transcritoma da retinopatia induzida por oxigênio e uma assinatura gênica prognóstica baseada em angiogênese para predição de recidiva de cancer de mama." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-28092017-112917/.

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Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras.
Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

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Dybos, Sandra Amalie. "Verdien av miRNA som biomarkør for kreft i prostata : Optimalisering av metode for RNA-isolering av formalinfiksert parafininnstøpt vev, samt undersøkelse av miRNA-uttrykk i ulik grad av prostatakreft." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for biologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-19434.

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Prostatakreft er den kreftformen som hyppigst rammer norske menn og dødelighet på grunn av avansert kreft i prostata er et klinisk problem. For å forstå molekylære ulikheter som skiller progressiv sykdom fra ikke-progressiv sykdom, vil identifisering av nye biomarkører som kan bidra til å gi en mer korrekt prognose være med på å forenkle behandling av pasienter med prostatakreft, og det er svært ønskelig med bedre behandlingsmetoder enn det som finnes i dag.miRNA er viktig under regulering av genuttrykk og det har vist seg at miRNA har en betydelig rolle under utvikling av kreft. Så langt er det bare et lite antall studier som har undersøkt uttrykk av miRNA relatert til prostatakreft. Målet med denne oppgaven var å utarbeide en optimal metode for isolering av total-RNA fra formalinfiksert parafininnstøpt vevsmateriale til bruk i undersøkelse av miRNA i vev fra prostata. Videre ville det være interessant å undersøke hvilken miRNA-profil som uttrykkes i Gleason grad 3, 4 og 5 for å finne ut om det er forskjell i miRNA-profil knyttet til alvorlighetsgrad av prostatakreft.Det ble tatt utgangspunkt i metode for isolering av total-RNA med RecoverAllTM Nucleic Isolation Acid Kit for formalinfiksert parafininnstøpt materiale, for å bedre utbytte total-RNA i vevsprøver fra prostata. Utprøving av ulike parametere som temperatur, inkubasjonstid med enzym, oppkutting av vevssylinder og homogenisering av vevssylinder ble gjennomført for å undersøke hvorvidt endringer kunne bidra til et bedre utbytte av RNA fra vevsmateriale som er formalinfiksert. Resultatene viser at optimal metode for isolering av RNA krever inkubasjon med enzymbehandling over natt (18 timer) for å få best utbytte RNA og høyest RIN-score. Ved isolering av RNA til analyse av miRNA er det tilstrekkelig med 3 timers inkubasjon med enzymbehandling, da mengde av kvantitert miRNA ser ut til å påvirkes i liten grad av inkubasjonstid over 3 timer. Mengde kvantitert miRNA i formalinfiksert materiale og ferskt materiale er tilnærmet likt, som betyr at formalinfiksert vevsmateriale kan benyttes på lik linje som ferskt materiale med hensyn på undersøkelse av miRNA.Det kommer tydelig frem av resultatene at det er gjennomgående lave konsentrasjoner RNA til stede i vevsmaterialet, og det er tydelig at RNAet er svært degradert, noe som gjenspeiles i den generelt lave RIN-scoren. Til tross for dårlig kvalitet på isolert RNA ble det laget cDNA-bibliotek til bruk i sekvensering av miRNA i formalinfiksert vevsmateriale. cDNA-bibliotekene viste mye adapterdimer som er forstyrrende for sekvensering av miRNA, og på bakgrunn av den dårlige kvaliteten på RNAet og forstyrrende DNA-sekvenser i prøvene, var det ikke mulig å gjennomføre sekvensering.

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Moralles, Patricia Regina Manzine. "Biomarcadores sanguíneos para a doença de Alzheimer : avaliação da expressão gênica da ADAM10 e de micro- RNAs." Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7759.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
ADAM10 is an-secretase that cleaves APP in the non-amyloidogenic pathway, thereby inhibiting -amyloid peptide (A ) production in Alzheimer´s disease (AD). Studies have shown decreased ADAM10 platelet levels in AD patients as well as the deregulation of microRNAs (miRNAs) related to molecules involved in the pathophysiology of this disease. The objective was to verify and compare ADAM10 gene expression and micro-RNAs (miRNAs) between AD patients and controls without cognitive impairment. It is a comparative study, based on the assumptions of quantitative research. Biological samples were collected, analyzed and stored in a biorepository. The ADAM10 gene expression in whole blood was studied in 47 AD, 32 healthy controls and 21 mild cognitive impairment (MCI) subjects by RT-qPCR techniques and analyzed by relative expression by 2- Ct. For miRNAs analyses, using MegaplexTM and MirWalk 2.0 database, were analyzed by RT-qPCR ~700 miRNAs in total blood and 21 miRNAs of them were validated in a sample of 21 AD subjects and 17 healthy controls. Statistical association tests, regression and diagnostic accuracy were performed. No significant differences in ADAM10 gene expression were observed between AD and control groups. Therefore, the decrease of ADAM10 protein in platelets of AD patients was not caused by a reduction in mRNA encoding for ADAM10. Mir-144-5p, miR-374 and miR-221 were downregulated in AD subjects, with moderate accuracy diagnosis. However, the association of selected miRNAs expression and Mini Mental State Examination (MMSE) was significantly better as a diagnostic tool compared to their expression separately. The validated miRNAs are involved in the regulation of pathways related to neurodegenerative diseases (beta-amyloid cascade, ubiquitination, transcriptional regulator, synaptic transmission, vesicle trafficking). Specifically, miR-144-5p, miR-374 and miR-221 are relevant for AD, as regulators of APP, BACE1 and ADAM10 translation. To the best of our knowledge, this is the first study to demonstrate a downregulation of these specific miRNAs in total blood of Alzheimer’s disease patients, compared to healthy cognitive controls. These findings are in agreement with AD protein outcomes and place the miRNAs evaluated as potential biomarkers that can be used to improve AD diagnosis.
ADAM10 é uma -secretase que cliva a APP através do caminho não amiloidogênico, inibindo desta forma a produção do peptídeo -amiloide (A ) na doença de Alzheimer (DA). Estudos apresentam a diminuição plaquetária da proteína ADAM10 em idosos com DA, assim como a desregulação de microRNAs (miRNAs) relacionados com moléculas envolvidas com a fisiopatologia desta doença. O objetivo geral foi verificar e comparar a expressão gênica da ADAM10 e de miRNAs entre idosos com DA e controles sem alterações cognitivas. Trata-se de um estudo de comparação, baseado nos pressupostos da pesquisa quantitativa. Amostras biológicas foram coletadas, analisadas e armazenadas em um biorrepositório. A expressão gênica da ADAM10 em sangue total foi estudada em 47 sujeitos com DA, 32 controles saudáveis e 21 sujeitos com transtorno neurocognitivo leve (TNCL), através de técnicas de RT-qPCR e analisada pela expressão relativa por 2- Ct. Para análises dos miRNAs, utilizando Megaplex e a base de dados MiRWalk 2.0, foram analisados por RTqPCR ~700 miRNAs no sangue total e 21 deles foram validados em uma amostra de 21 sujeitos com DA e 17 controles. Testes estatísticos de associação, regressão e acurácia diagnóstica foram realizados. Não foi observada diferença significante na expressão gênica da ADAM10 entre sujeitos com DA e controles. Assim, a diminuição dos níveis proteicos da ADAM10 plaquetária em pacientes com DA não foi devido a redução do mRNA codificante para ADAM10. Mir-144-5p, miR-374 e miR-221 estavam menos expressos em indivíduos com DA, com moderada acurácia diagnóstica. Entretanto, a associação da expressão dos miRNAs selecionados com o Mini Exame do Estado Mental (MEEM) foi significativamente melhor como uma ferramenta de diagnóstico em comparação com as análises individuais. Os miRNAs validados estão envolvidos na regulação de vias relacionadas a doenças neurodegenerativas (cascata beta-amiloide, ubiquitinação, reguladores de transcrição, transmissão sináptica, tráfego de vesículas). Especificamente, o miR-144-5p, miR-374 e miR- 221 são relevantes para a DA, como reguladores da tradução da APP, BACE1 e da ADAM10. Segundo nosso conhecimento, este é o primeiro estudo a demonstrar a expressão reduzida desses miRNAs no sangue total de pacientes com DA, em comparação com controles cognitivamente saudáveis. Estes resultados estão de acordo com os resultados proteicos da DA e destacam os miRNAs avaliados como potenciais biomarcadores que podem ser utilizados para o aperfeiçoamento do diagnóstico da DA.

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Ipas, Hélène. "Contingent microARN des exosomes, diagnostic et physiopathologie des gliomes." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00986111.

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Les tumeurs gliales du cerveau et en particulier les glioblastomes sont des tumeurs de très mauvais pronostic. Les paramètres qui contrôlent des phénotypes comme l'agressivité, la migration, ou la chimio-résistance de ces tumeurs sont mal connus. Dans ce contexte tumoral, il est envisagé que les microARN (ARN non-codants d'une vingtaine de bases) soient des acteurs essentiels des phénomènes de modification phénotypique parce qu'ils sont capables d'orchestrer l'expression de nombreux gènes. Nous avons montré que les microARN sont des marqueurs tissulaires précieux pour le diagnostic permettant de différencier les deux types principaux de gliomes à partir de prélèvements tumoraux. Nous avons aussi observé que plusieurs microARN sont, en outre, sécrétés par les cellules gliales saines ou cancéreuses au sein de microvésicules appelées exosomes. Le contenu en ARN de ces exosomes a été caractérisé par analyse moléculaire transcriptomique (ARN messagers et microARN) par techniques d'hybridation sur puces à ADN Affymetrix. Les profils ARN exosomaux sains et cancéreux sont distincts, mais ils ne reflètent pas intégralement le profil ARN des cellules dont ils sont issus. Des conditions de stress hypoxique ou l'utilisation de composés pharmacologiques (GW4869 et 5-aza-2'-désoxycitidine) n'affectent pas la quantité d'exosomes produite par la lignée de glioblastome (U87) en culture. Les profils ARN sont cependant modifiés, et le contenu des exosomes produits semble donc être un mécanisme actif et régulé. Enfin, des exosomes cancéreux incubés avec des cellules saines ont très peu d'effet sur le phénotype de celles-ci. Les microARN tissulaires et exosomaux seraient donc des acteurs importants de la physiopathologie du gliome et de sa progression, dont les rôles restent encore à préciser.

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Perez, Lopez Raquel. "A study of multi-parametric MRI as a prognostic and response biomarker in patients with castration resistant prostate carcinoma and bone metastases." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405715.

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Múltiples drogas han sido aprobadas para el tratamiento de cáncer de próstata avanzado en las últimas décadas, incluyendo innovadoras terapias hormonales (abiraterona y enzalutamida), quimioterapias basadas en taxanos (docetaxel y cabazitaxel) y radiofármacos (Rad 223). Además, se han desarrollado muchos otros tratamientos como inhibidores de PARP e inmunoterapias. Aunque, a pesar del fascinante progreso conseguido en el manejo del cáncer de próstata, su forma metastásica sigue siendo una condición fatídica, causando elevada morbilidad y mortalidad en todo el mundo. El desarrollo de todos estos tratamientos ha aportado nuevas oportunidades para los pacientes con cáncer de próstata avanzado pero, también significa un reto para los médicos a la hora de optimizar la selección de tratamientos y llevar a cabo una secuencia de tratamientos eficiente. Es necesario, por tanto, desarrollar biomarcadores predictivos y pronósticos que ayuden en la toma de decisiones terapéuticas. La valoración de respuesta en pacientes con cáncer de próstata avanzado representa un reto. El esqueleto es el órgano en el que ocurren con mayor frecuencia las metástasis en pacientes con cáncer de próstata, representando en muchas ocasiones la única localización de enfermedad metastásica. Las técnicas de imagen estándares utilizadas en la actualidad, tomografía computarizada y gammagrafía ósea, no muestran la verdadera extensión de las metástasis óseas y presentan de forma subóptima cambios biológicos como respuesta a tratamientos. El cambio de terapia se basa muchas veces en cambios de PSA, el cual no es ni un biomarcador pronóstico ni de respuesta en tratamientos no hormonales. Por ello, hay una necesidad médica imperiosa de desarrollar biomarcadores de respuesta precisos. 5 La RNM incluyendo secuencias funcionales permite el estudio de características anatómicas así como moleculares y metabólicas. Los estudios de difusión son una secuencia funcional de RNM que evalúa el movimiento de las moléculas de agua en los tejidos e informa de la microestructura y celularidad tisular. En mi programa de doctorado he analizado el papel de la RNM multiparamétrica, incluyendo imagen de difusión, como biomarcador pronóstico y de respuesta en pacientes con cáncer de próstata resistente a castración y metástasis óseas. El estudio presentado en el manuscrito “El volumen de metástasis óseas evaluado mediante estudio de difusión de cuerpo entero se asocia con supervivencia en el cáncer de próstata resistente a castración” muestra que la carga de metástasis óseas cuantificada por imagen de difusión de cuerpo entero es un biomarcador pronóstico en pacientes con cáncer de próstata. El estudio presentado en el manuscrito “Imagen de difusión como biomarcador de respuesta evaluando metástasis óseas en cáncer de próstata” representa el primer estudio de difusión de cuerpo entero dentro de un ensayo clínico prospectivo en pacientes con cáncer de próstata metastásico. Cambios en parámetros cuantificables derivados de las imágenes de difusión de las metástasis óseas son un indicador de respuesta al inhibidor de PARP olaparib. También he estudiado la correlación de las características de RNM multiparamétrica con parámetros histológicos de biopsias óseas. Esto es crucial para la validación de la imagen de difusión como biomarcador de metástasis óseas en cáncer de próstata. Los positivos resultados de estos estudios contribuyen a la eventual puesta en práctica de la imagen de difusión en el tratamiento del cáncer de próstata.
Multiple new drugs have been approved for advanced prostate cancer treatment over the last decade including novel endocrine therapies (abiraterone acetate and enzalutamide), taxane-‐based chemotherapies (docetaxel and cabazitaxel) and radiopharmaceuticals (Rad 223). Moreover, many other treatments such as PARP-‐ inhibitors and immunotherapy are currently been developed. However, despite the exciting progress achieved in the management of prostate cancer, metastatic prostate cancer remains a fatal condition, causing marked morbidity and mortality worldwide. The development of all these treatments has brought a wide range of opportunities for patients with advanced prostate cancer, but also challenges physicians to optimize treatment selection and pursue a rational and efficient sequence of drugs for each patient. Therefore, there is a need for predictive and prognostic biomarkers that help in treatment decision-‐making. Assessment of response to anticancer therapy in patients with advanced prostate cancer represents a challenge. The skeleton is the most frequent organ of distal metastases in prostate cancer patients, representing very often the only site of metastatic disease. The currently used standard imaging techniques, computed tomography and bone scan, do not depict the true extent of bone metastases and are suboptimal in capturing biological changes occurring in response to treatment. Treatment switch decisions are too often being made based on PSA changes, which are neither a survival surrogate biomarker nor a good response biomarker for non-‐ hormonal agents. Therefore, the development of accurate response biomarkers for bone metastases remains an unmet medical need. K MRI including functional sequences allows the study of anatomical and as well molecular and metabolic features. Diffusion-‐weighted imaging is a functional MRI technique that studies the movement of water molecules within a tissue and informs of tissue microstructure and cellularity. In my PhD studies I evaluated multiparametric MRI including diffusion-‐weighted imaging as a prognostic and response biomarker in patients with castration resistant prostate cancer and bone metastases. The study presented in the manuscript “Volume of bone metastasis assessed with whole-‐ body diffusion-‐weighted imaging is associated with overall survival in metastatic castration-‐resistant prostate cancer” showed that the burden of bone metastases assessed with whole body DWI is a prognostic biomarker in patients with advanced prostate cancer. The study presented in the manuscript “Diffusion-‐weighted imaging as a treatment response biomarker evaluating bone metastases in prostate cancer” represents the first study of whole-‐body diffusion-‐weighted imaging in the setting of a prospective clinical trial in patients with metastatic prostate cancer. Changes in quantitative variables derived from diffusion-‐weighted imaging of bone metastases are indicators of response to the PARP inhibitor olaparib. I also studied the correlation of multiparametric MRI features with histological findings in bone biopsies. This is crucial towards the validation of DWI as a biomarker for bone metastases in prostate cancer. The positive results of these studies contribute towards the eventual implementation of DWI in prostate cancer care.

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Kitow, Janina [Verfasser], and Thomas [Akademischer Betreuer] Thum. "Die Rolle der mitochondrialen long-non-coding RNA als blutbasierte Biomarker für myokardiales Remodeling bei Patienten mit hypertropher Kardiomyopathie / Janina Kitow ; Akademischer Betreuer: Thomas Thum ; Institut für Molekulare und Translationale Therapiestrategie der Medizinischen Hochschule Hannover." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2019. http://d-nb.info/1192515684/34.

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Spornraft, Melanie [Verfasser], Michael W. [Akademischer Betreuer] Pfaffl, and Ralph P. [Akademischer Betreuer] Kühn. "Profiling of circulating small RNAs and their potential as transcriptomic biomarkers to detect anabolic drug abuse in bovines / Melanie Spornraft. Betreuer: Michael W. Pfaffl. Gutachter: Michael W. Pfaffl ; Ralph P. Kühn." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/108203424X/34.

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Vladimir, Stojšić. "Učestalost i prognostički značaj genskih alteracija u tumorskim ćelijama i njihova povezanost sa kliničko-patološkim karakteristikama bolesnika sa ranim stadijumom adenokarcinoma bronha." Phd thesis, Univerzitet u Novom Sadu, Medicinski fakultet u Novom Sadu, 2018. https://www.cris.uns.ac.rs/record.jsf?recordId=105379&source=NDLTD&language=en.

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Napredak na polju molekularne biologije omogućio je identifikaciju molekularnih markera za karcinom bronha sa vrednim prognostičkim i prediktivnim značajem i njihova uloga kod uznapredovalog, metastatskog oblika bolesti je u velikoj meri istražena, dok kod ranih stadijuma bolesti još uvek nije sasvim jasna. Cilj ovog istraživnja bio je da se utvrdi učestalost najčešćih genskih alteracija u tumorskim ćelijama bolesnika sa ranim stadijumom adenokarcinoma bronha, da se utvrdi pojedinačna zavisnost ispitivanih genskih alteracija u tumorskim ćelijama sa određenim kliničko-patološkim karakteristikama i da se utvrdi potencijalni prognostički značaj pojedinačne genske alteracije u tumorskim ćelijama na vreme preživljavanja bez povratka bolesti i ukupno vreme preživljavanja. Istraživanje je obuhvatilo 161 bolesnika sa adenokarcinomom bronha, stadijuma bolesti od I do IIIA, kod kojih je sprovedena radikalna hirurška resekcija u Institutu za plućne bolesti Vojvodine u periodu izmedju 2007 i 2014 godine. U tumorskim uzorcima fiksiranim u parafinu odredjivane su mutacije EGFR, KRAS i PIK3CA gena, ALK i ROS1 rearanžman i PD1 i PD-L1 ekspresija. Kliničkopatološke karakteristike su preuzete iz registra za karcinom bronha Instituta za plućne bolesti Vojvodine. Ukupno preživljavanje je računato od dana operacije do dana smrti, a preživljavanje bez povratka bolesti je računato od dana operacije do momenta ponovne pojave bolesti. Od 161 testiranog tumorskog uzorka, prisustvo mutacija detektovano je kod 96 uzoraka (59.6%). Prisustvo mutacije KRAS gena detektovano je kod 69 (42.9%), mutacije EGFR gena kod 10 (6.2%), a mutacije PIK3CA gena kod 7 (4.3%) tumorskih uzoraka. ALK rearanžman je detektovan kod 3 (1.9%), a ROS1 rearanžman kod 7 (4.3%) tumorskih uzoraka. PD-1 ekspresija detektovana je u 71 tumorskom uzorku (45%), dok je PD-L1 ekspresija detektovana u 59 tumorskih uzoraka (36.6%). PD-1 ekspresija nije bila značajno povezana ni sa jednim od klinčko-patoloških karakteristika (uključujući KRAS, EGFR, ALK, ROS1 i PI3KCA status). PD-L1 ekspresija je bila značajno povezana sa tipom hirurgije (P = 0.01) i sa prisustvom KRAS mutacije (P = 0.02). Mutacioni status u domenu KRAS gena je bio značajno povezan sa godinama starosti (P = 0.004), polom (P = 0.006) i pušačkim statusom (P = 0.004). Mutacioni status u domenu EGFR gena je bio značajno povezan sa pušenjem (P < 0.001) i sa godinama starosti (P = 0.013). Mutacioni statusi u domenu gena za ALK, ROS1 i PI3KCA nisu bili značajno povezani ni sa jednom od ispitivanih kliničko-patoloških karakteristika. Prisustvo PD-1 ekspresije je bilo značajno povezano sa preživljavanjem bez povratka bolesti (P = 0.03) i ukupnim preživljavanjem (P = 0.01). PD-L1 ekspresija, KRAS, EGFR, ALK, ROS1 i PIK3CA mutacioni status nisu bili značajno opvezani sa preživljavanjem bez povratka bolesti i ukupnim preživljavanjem. Najčešće detektovane genske alteracije su mutacije u domenu KRAS i EGFR gena. Prisustvo KRAS mutacije je značajno povezano sa godinama starosti ispitanika, polom i pušačkim statusom dok je prisustvo EGFR mutacije značajno povezano sa godinama starosti ispitanika i pušačkim statusom. Prisustvo PD-L1 ekspresije je značajno povezano sa vrstom hirurškog lečenja i sa prisustvom KRAS mutacija. Jedino prisustvo PD-1 ekspresije u tumorskim ćelijama predstavlja nezavistan prognostički faktor za preživljavanje bez povratka bolesti i ukupno preživljavanje bolesnika sa ranim stadijumom adenokarcinoma bronha.
Advances in the field of molecular biology gave us insight into biomarkers for lung cancer with great prognostic and predictive value and their role in advanced stage disease is well known while in early stage disease is yet to be proven. The aim of this study was to determine the frequencies of the most common gene alterations in patients with early stage lung adenocarcinoma, to determine the relationship between gene alterations in tumor cells and clinicopathologial characteristics and to determine prognostic value of each gene alteration regarding overall survival and disease free survival. One hundred sixty-one patients diagnosed with lung adenocarcinoma clinical stage I-IIIA who underwent radical surgical resection at the Institute for Pulmonary Diseases of Vojvodina between 2007 and 2014 were included in this study. Mutations in EGFR, KRAS and PIK3CA gene, ALK and ROS1 rearrangement and PD-1 and PD-L1 expression were determined in representative formalin-fixed, paraffin-embedded (FFPE) tumor block from each patient. Clinical data were extracted from the institutional lung cancer registry of the Institute for Pulmonary Diseases. Overall survival was calculated as time from the day of surgery to the day of death. Disease free survival was calculated as time from the day of surgery to the day of disease relapse. Among 161 tested tumor tissue, presence of mutation was found in 96 (59.6%) of them. There were 69 (42.9%) mutations in KRAS gene, 10 (6.2%) in EGFR gene and 7 (4.3%) in PIK3CA gene. ALK and ROS1 rearrangement were present in 3 (1.9%) and 7 (4.3%), respectively. PD-1 expression was determined in 71 (45.0%) tumor sample while PD-L1 expression was determined in 59 (36.6%). PD-1 expression was not correlated with any of the clinicopathologial characteristics (including KRAS, EGFR, ALK, ROS1 and PIK3CA mutational status). PD-L1 expression correlated with type of surgery (P = 0.01) and KRAS positivity (P = 0.02). KRAS mutation status correlated with age (P = 0.004), sex (P = 0.006) and smoking status (P = 0.004). EGFR status correlated with smoking status (P < 0.001) and age (P = 0.013). ALK, ROS1 and PIK3CA status were not correlated with any of the clinicopathologial characteristics. PD-1expression was significantly associated with disease free survival (P = 0.03) and overall survival (P = 0.01). PD-L1 expression, KRAS, EGFR, ALK, ROS1 and PIK3CA status were not associated with disease free survival and overall survival. The most frequent gene alteration are mutations in KRAS and EGFR gene. Presence of KRAS mutation is in correlation with patients age, sex and smoking status while presence of EGFR mutation is in correlation with patients age and smoking status. PD-L1 expression is in correlation with type of surgery and KRAS mutational status. Only presence of PD-1 expression represent an independent prognostic factor for disease free survival and overall survival.

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44

Gourvest, Morgane. "Etude des longs ARNs non codants dans les leucémies aiguës myéloïdes : relevance clinique et caractérisation fonctionnelle." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30117.

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Les longs ARNs non codants (lncRNAs) sont définis comme des transcrits ayant une taille supérieure à 200 nucléotides et dépourvus de potentiel codant. Longtemps considérés comme inutiles, leur étude récente a démontré qu’ils jouent un rôle important dans l’expression de nos gènes. On compte d’ailleurs de plus en plus d’exemples de ces lncRNAs dérégulés dans les cancers. Notre étude visait à évaluer l’existence de profils d’expression particuliers de lncRNAs au sein des leucémies aiguës myéloïdes à caryotype normal (LAM-CN), dont l’implication dans cette pathologie n’est que peu décrite. Le séquençage des ARNs que l’on a effectué sur une cohorte de 40 patients atteints de LAM-CN nous a permis de faire ressortir une signature minimale de 12 lncRNAs différentiellement exprimés chez les patients porteurs de la mutation dans le gène de la nucléophosmine (NPM1). Ces résultats ont été validés par RT-qPCR (Fluidigm) sur une cohorte indépendante composée de 134 nouveaux patients atteints de LAM-CN. Parmi cette signature, nous avons identifié un biomarqueur potentiel, le XLOC_109948, dont la faible expression est associée à un bon pronostic, particulièrement chez les patients NPM1 mutés. De plus, l’inhibition de ce lncRNA par transfection transitoire de gapmeRs dans une lignée cellulaire de LAM NPM1 muté augmente l’apoptose de ces cellules traitées à l’aracytine, suggérant un rôle du XLOC_109948 dans la sensibilité au traitement. Nous avons également caractérisé un autre lncRNA de la signature NPM1, baptisé LONA (LncRNA Overexpressed in NPM1-Mutated AML patients). Nous avons remarqué d’une part, que la mutation NPM1 induit une délocalisation nucléaire du lncRNA LONA, ce qui impacte ses fonctions cellulaires. Des stratégies perte et gain de fonctions ont montré que LONA aurait un rôle oncogénique en contexte de LAM NPM1 muté où il est impliqué in vitro et in vivo dans les processus de différentiation myéloïde et de croissance cellulaire en régulant l’expression de gènes cruciaux tels que THBS1, ASB2 ou MAFB. A l’inverse, la dérégulation de LONA en contexte de LAM NPM1 sauvage induit des effets opposés et suppresseurs de tumeurs, suggérant des régulations différentes en fonction du statut mutationnel de NPM1. D’autre part, le locus du lncRNA LONA est situé sur le chromosome 6 humain, au sein du cluster HIST1 codant des gènes des histones canoniques. Chez les patients NPM1 muté, l’expression du lncRNA LONA est inversement corrélée à celle de gènes voisins codant des histones. De manière cohérente, la diminution du lncRNA LONA dans notre lignée de LAM NPM1 muté est associée à une augmentation de l’expression de certaines des histones proximales du cluster. Par immunoprécipitation d’ARN, nous avons montré que LONA interagit avec le complexe de répression Polycomb (PRC2), suggérant sa contribution dans les régulations épigénétiques de la transcription des histones. De manière plus préliminaire, le lncRNA LONA pourrait également réguler l’étape de maturation des messagers des histones canoniques, en séquestrant telle une éponge moléculaire le snRNA U7, un petit ARN régulateur impliqué dans la maturation des extrémités 3’ des ARNs messagers des histones. L’ensemble de ces données suggère que les lncRNAs pourraient être considérés comme des biomarqueurs potentiels robustes, et apparaissent comme des acteurs clés dans le développement des leucémies aiguës myéloïdes
Long noncoding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides without protein-coding potential. Long considered as useless, their recent study has demonstrated that lncRNAs have important roles in gene expression regulation. Cumulative evidence points toward the implication for lncRNAs deregulation in tumorigenesis. In this study, we sought to evaluate specific lncRNAs expression profiles among cytogenetically normal AML patients (CN-AML), their involvement in this pathology being barely referenced. The RNA sequencing that we performed on forty CN-AML patients allowed us to highlight a minimal set of 12 differentially expressed lncRNAs in AML patients bearing the mutation in the Nucleophosmin gene (NPM1). These results were confirmed by RT-qPCR (Fluidigm) on a validation set of 134 CN-AML patients. Among these, we identified one putative biomarker, the lncRNA XLOC_109948, whose low expression indicates a good prognosis, especially for NPM1-mutated patients. Consistently, the downregulation of XLOC_109948 using GapmeRs in a NPM1-mutated AML cell line enhances apoptosis of these cells treated with aracytine, suggesting the role of XLOC_109948 in drug sensitivity. We also functionally characterized another lncRNA of the NPM1 signature, that we named LONA (lncRNA overexpressed in NPM1-mutated AML patients). On one hand, we observed that the mutation of NPM1 leads to a nuclear delocalization of LONA lncRNA, which consequently modulates its cellular functions. Loss and gain of functions strategies allowed us to show that LONA seems to have oncogenic effects in a NPM1 mutated AML context, where it is implicated in vitro in myeloid differentiation and in vivo cellular growth processes by regulating the expression of master genes such as THSB1, ASB2, and MAFB. At the contrary, the deregulation of LONA lncRNA in a NPM1 wild type AML context leads to opposite and tumor suppressor effects, suggesting a different regulation depending on the mutational status of NPM1. On the other hand, the LONA’s genomic locus is located on chromosome 6, within a cluster of histone coding genes. In NPM1 mutated AML patients, we observed that the expression of LONA inversely correlates with the expression of some neighboring histones genes. Consistently, the downregulation of LONA lncRNA by using GapmeRs in a NPM1 mutated AML cell line leads to the upregulation of some proximal histones genes of the cluster. By RNA immunoprecipitation, we showed that LONA interacts with the Polycomb Repressive Complex 2 (PRC2), suggesting its contribution to epigenetic regulation of histone genes transcription and chromatin remodeling. More preliminary, we also think that LONA could regulate the maturation step of histone messengers by sequestrating, as a molecular sponge, the snRNA U7, a small regulatory RNA implicated in the maturation of histone messengers 3’ ends. Altogether, these data suggest that lncRNAs could be considered as strong prognostic biomarkers and emerged as key players in the pathogenesis of Acute Myeloid Leukemia

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Strassner, James P. "The Role of Interferon Gamma in Melanocyte Clearance During Vitiligo." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1023.

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Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

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46

Souza, AlethÃia CarÃzia Baracho de Lima. "Estudos sobre as interaÃÃes das proteÃnas seminais com as cÃlulas espermÃticas e componentes dos diluidores usados na criopreservaÃÃo do sÃmen e sobre marcadores moleculares de parÃmetros do sÃmen em animais de produÃÃo." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=12911.

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FundaÃÃo de Amparo Ã Pesquisa do Estado do CearÃ
A tese Ã composta por dois capÃtulos. O primeiro capÃtulo inclui o trabalho cujo objetivo foi investigar o potencial uso de pares de transcritos correlativos baseados em microarranjos como marcadores de fertilidade masculina usando a displasia da bainha fibrosa (DFS) como modelo afetado. Atualmente Ã bastante reconhecido que a tecnologia de microarranjos pode ser limitada pelos custos e que a qualidade dos transcritos permanece relativamente desconhecida. Para responder essas questÃes, nÃs analizamos pares de transcritos estÃveis por qPCR com um processo sistemÃtico de desenho de primers sistemÃtico. Nesse estudo experimental, nÃs utilizamos amostras de homens com fertilidade comprovada e de homens com diagnÃtico de DFS. Nossa abordagem foi baseada nas sequÃncias de primers dos seis genes de interesse, os quais foram desenhados utilizando os programas Oligo7 e Primer3Plus. A especificidade do primer foi inicialmente analisada in silico atravÃs de pesquisas nos bancos de dados ENSEMBL, University of California Santa Cruz (UCSC), e National Center for Biotechnology Information (NCBI) para uso de sequÃncias especÃficas aos genes alvos. A habilidade dos pares de transcritos em classificar as amostras de homens de fertilidade comprovada das amostras de DFS foi avaliada. Nossos resultados mostraram que em conjunÃÃo com a identificaÃÃo de quatro novos pares estÃveis, a comparaÃÃo dos coeficientes de correlaÃÃo dos valores de C(t) dos DSF revelou a interrupÃÃo de quatro pares estÃveis identificados nas amostras de homens normais. Esta seleÃÃo de pares estÃveis resolve a questÃo sobre a DSF. Em conclusÃo, os resultados mostram efetivamente que o desenho de primers e qPCR podem fornecer um ensaio molecular de baixo custo para avaliar a fertilidade masculina. O segundo capÃtulo divide-se em dois estudos e avalia em carneiros os efeitos de uma dieta suplementada com farelo de castanha de caju. No estudo 1, nosso objetivo foi detectar a presenÃa de transcritos para Heat Shock Protein (HSP70), clusterina (CLU), proteÃna semelhante Ã subunidade alfa do complexo T (TCP1) e proteÃna do complexo T subunidade 8 (CCT8) no espermatozÃide de ovinos, seguindo a mesma metodologia para qPCR utilizada no capitulo 1. As sequÃncias de primers foram desenhadas utilizando os programas Primer3Plus e Oligo Analyzer. Gene para protamina 2 (PRM2) foi usado como controle interno de reaÃÃo. O sÃmen foi coletado de machos pÃberes Morada Nova utlizando eletroejaculador. As amostras selecionadas para extraÃÃo de RNA espermÃtico seguiram as recomendaÃÃes do ColÃgio Brasileiro de ReproduÃÃo Animal quanto aos parÃmetros de motilidade, vigor e concentraÃÃo. Nossos resultados mostraram a presenÃa de mRNA para a HSP70 nos espermatozÃides de ovinos. Maiores estudos sÃo necessÃrios a fim de confirmar ou refutar a presenÃa das chaperonas TCP1 e CCT8 no espermatozÃide ovino. A presenÃa do transcrito da HSP70 no espermatozÃide de ovinos abre perspectivas para estudos futuros sobre os efeitos do mRNA HSP70 no desenvolvimento embrionÃrio, de modo a avaliar se essa expressÃo ocorre de modo espontÃneo, programado e seqÃencial, e se esses mecanismos se refletem na fertilidade e no desenvolvimento embrionÃrio. O segundo estudo tem como principal objetivo avaliar os efeitos de uma dieta contendo farelo de castanha de caju (FCC) na expressÃo de genes relacionados ao metabolismo dos lipÃdios no mÃsculo Longissimus dorsi de carneiros Morada Nova. Vinte carneiros maduros sexualmente foram divididos em dois grupos baseando-se no peso vivo. Os animais foram mantidos em baias individuais. Durante trÃs meses, o grupo castanha (GCA) foi alimentado com raÃÃo contendo FCC, enquanto o grupo controle (GCO) recebeu raÃÃo Ã base de milho e soja. As duas dietas eram isocalÃricas e isoprotÃicas, adicionadas de suplemento mineral. Os carneiros tambÃm receberam feno de Tifton e Ãgua Ã vontade. A quantidade de alimento ofertado (raÃÃo e feno) foi ajustada diariamente para sobra de 10%. Sete genes codificantes de proteÃnas envolvidas direta ou indiretamente foram selecionados como alvos, incluindo: GH, ACACA, CAST, CAPN3, LPL, SCD e FASN. Para normalizaÃÃo, foram selecionados cinco genes candidatos: ACTB, GAPDH, RPL4, RPS18 e TBP. Dentre os sete genes alvos selecionados anteriormente, os alvos GH, ACACA e CAST foram removidos. Os dois primeiros foram removidos devido amplificaÃÃo de alinhamento mÃltiplo (baixa especificidade do primers), enquanto CAST apresentou baixa eficiÃncia de amplificaÃÃo. Da lista de gene alvo final, a expressÃo de somente dois genes foi afetada pela dieta, SCD (p<0.01) e FASN (p<0.05), enquanto LPL (p=0,1022) e CAPN3 (p=0,0939) nÃo apresentaram diferenÃa significativa (p<0.05). Os genes SCD e FASN foram reprimidos no GCA comparada ao GCO. Este Ã o primeiro relato de que uma raÃÃo contendo FCC afetou a expressÃo gÃnica de proteÃnas envolvidas na deposiÃÃo de lipÃdios no mÃsculo em ovinos. Considerando que uma dieta contendo FCC altera a expressÃo de genes lipogÃnicos sem afetar o ganho de peso nem a eficiÃncia reprodutiva de ovinos, faz da castanha de caju uma importante alternativa para o sistema de produÃÃo de ovinos criados em regiÃes tropicais.
This thesis presents two chapters. In the first chapter, its objective was to investigate the potential use of correlative microarray-based transcript pairs as candidate markers for male fertility using dysplasia of the fibrous sheath (DFS) as an affected model. It is widely recognized that microarray technology may be limited by cost and that the quality of the transcript remains relatively unknown. To address these issues, we analyzed the stable transcript pairs by qPCR with a systematic primer design process. On this experimental study, we used men with proven fertility and men with a diagnosis of DFS. Our approach was based on primer sequences for six genes of interest were designed using Oligo7 and Primer3Plus. Primer specificity was initially assessed in silico by searching the ENSEMBL, University of California Santa Cruz, and National Center for Biotechnology Information databases for nontarget complementary sequences throughout the genome. The ability of transcript pairs to classify samples from males of proven fertility away from DFS was assessed. Our results showed that in conjunction with identifying four new stable transcript pairs, comparison of the DFS qPCR C(t) correlation coefficients revealed the disruption of four stable fertile sample transcript pairs. This suite of transcript pairs resolves DFS. In conclusion, the results show that with effectively designed primers, qPCR may provide an affordable molecular assay to assess male fertility status. Second chapter includes two studies regarding evaluations of ram feeded with supplemented diet with cashew nut. On the frist study, our goal was detect transcripts for Heat Shock Protein (HSP70), Clusterin (CLU), Ovis aries T-complex protein 1 alfa subunit-like protein (TCP1) e Ovis aries chaperonin containing TCP1, subunit 8 (theta) (CCT8) on ram sperm by. For primer designing we used published ESTs from NCBI and manually annotated by us using Primer3Plus and OligoAnalizer. PRM2 was used as internal qPCR control. Semen samples from mature Morada Nova ram were collected by eletroejaculator, washed in PBS and prepared for further RNA extraction. Selected samples followed quality recommendatios from ColÃgio Brasileiro de ReproduÃÃo Animal regarding motility, vigor and concentration. Our results showed presence of mRNA HSP70 on ram sperm and they can possible be envolved in early embryo development, oocyte activation and post fertilization events. Further analyses will be necessary to confirm presence of TCP1 and CCT8 on ram sperm. Our findings indicate new perpectives about the effects of these chaperones during embryo development mesuring if its expression reflects male fertility on the early embryo development. On the second study the the main goal is to evaluate the effects of a lipid-enriched diet containing cashew nut brain on the expression of genes related to lipid metabolism in the longissimus dorsi muscle of Morada Nova rams. Twenty sexually mature and reproductively sound rams were divided in two groups based on ram live weight, and each ram was kept on individual pens. During three months, group 1 (G1) rams were fed with a lipid-rich diet, containing cashew nut bran (CNB), while group 2 (G2) was fed with a meal based on corn and soy. Both diets were isocaloric and isoproteic, and had a mineral mix added-in. The rams also were offered Tifton grass hay and had free access to water. The amount of diet offered (ration plus hay) was adjusted everyday to a maximum waste of 10%. Seven genes coding for proteins directly or indirectly involved in lipid metabolism were initially selected as targets, incluiding GH, ACACA, CAST, CAPN3, LPL, SCD, and FASN. Also, five genes were selected as reference genes, ACTB, GAPDH, RPL4, RPS18 and TBP. From the seven genes originally selected as targets, GH, ACACA and CAST were removed, leaving the final list with four targets. The first two genes were removed due to alternative pairing of the primers (low specificity), while CAST showed low amplification efficiency during PCR reaction. From the final target list, the expression of only two genes was affected by diet, SCD (p<0.01) and FASN (p<0.05), while LPL (p=0,1022) and CAPN3 (p=0,0939) were not different at the p<0.05 level. Both SCD and FASN genes were down-regulated in G1 (lipid-rich diet containing CNB) compared to G2. These genes are involved in lipogenic pathways, related to tissue lipid deposition; therefore, these results were expected. This is the first time that a fat-rich diet based on CNB was shown to affect gene expression of proteins involved in fat deposition in carcass muscles of rams. Longissimus dorsi is one of the finest meat cuts. Considering that human diets rich in poli-unsaturated fatty acids (PUFA) can decrease the risk of heart and other chronic diseases, a change in the fatty acid profile of this muscle could contribute to a healthier diet, aggregating value to the end-product of the lamb meat market. The effects of CNB-based diet on the gene expression of SCD and FASN support the notion that such diet, as previously shown for other sources of lipid in ruminants, can potentially change the fatty acid composition of L. dorsi, but this hypothesis needs to be experimentally verified by profiling fatty acids in animals fed CNB versus carbohydrate-based diets. CNB use as an ingredient in animal feeding is environmentally-friendly, since it contributes to by-product recycling from the agroindustrial plants in Northeast Brazil. Also, considering that CNB-based diet changes lipogenic gene expression without affecting weight gain or reproductive status of the rams, as shown in another work from our team, makes CNB a very important alternative food in ram production systems in tropical regions.

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47

Gill, Hardeep. "The Effect of Aluminium Industry Effluents on Sediment Bacterial Communities." Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23423.

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The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

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48

Lee, Hsin-Ying, and 李欣穎. "Depressive State-related Biomarkers Detected by Whole-Blood RNA Sequencing." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/7mpa54.

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碩士
國立臺灣大學
流行病學與預防醫學研究所
107
Mood disorders, including major depressive disorder (MDD) and bipolar disorder (BPD), are severe and heritable psychiatric disorders. The high frequency of episode recurrence and disease chronicity cause great disease burden to individuals, families, and societies. For the recurrence of episode, depressive episode is suffered by both major depressive disorder (MDD) and bipolar disorder (BPD) patients. To better understand the underlying biological mechanisms of depressive episode, we aimed to investigate transcriptome changes between acute episode versus remission status for depressive episode among patients with MDD or BPD using RNA sequencing. We recruited 12 clinically diagnosed patients with BPD or MDD, who were repeatedly measured for their severity of depressive symptoms using Hamilton Depression Rating Scale (HAMD) during episode (HAMD score>=16), and follow-up for at least two-months till remission (HAMD score<=8). Blood samples were drawn at the time of depression severity assessment. We employed differential expression analysis with intra-individual comparison and gene co-expression analysis to identify differential expression genes (DEGs) and modules related to depressive episode. We further explore possible explanations of the biological functions in the DEGs and modules using functional enrichment analysis. In differential expression analysis, several DEGs were identified, such as glucosylceramidase beta (GBA, log2FC = -1.54, P = 9.06*10-5) and glutamate ionotropic receptor NMDA type subunit associated protein 1 (GRINA, log2FC = -0.54, P = 9.84*10-4), both of which have been mentioned in previous depression studies. A variety of functions were overrepresented in our DEGs, such as GOs related to morphogenesis, cellular development, cell movement, as well as immune system. In co-expression network, we detected two modules related to depressive state, and the immune system also stood out in modules associated with depressive state. Another aim of our study is to find out overlapped genes between depressive state markers and trait markers, including genetic variants and brain expression markers associated with MDD. Results revealed that trait markers were enriched in our DEGs, but not in modules related to depressive state. This indicated that the DEGs might give clues to the connection of biological functions between depressive state and trait, while the modules implied state-specific information. Further studies with a bigger sample size are needed to examine the targets detected in our study in order to identify reliable biomarkers for depressive state and confirm the biological connection between depressive state and trait.

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Magro, Martim Ramos Chaves Lopes. "Effect of temperature on embryonic development, larval viability and biomarkers in the first stages of life of Octopus Vulgaris (Cuvier, 1797)." Master's thesis, 2015. http://hdl.handle.net/10400.1/8233.

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Dissertação de Mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015
O cultivo de Octopus vulgaris, apresenta diversos problemas, principalmente durante os primeiros estádios de desenvolvimento. Nas últimas décadas, muitos grupos de investigação têm feito um esforço no sentido de conseguir perceber quais as soluções para os problemas do cultivo desta espécie. O presente estudo tem como objetivo analisar o efeito de diferentes temperaturas de incubação (19ºC e 22ºC) no desenvolvimento embrionário da postura de apenas uma fêmea. Foram analisados biomarcadores de crescimento, stresse fisiológico, defesas antioxidantes e atividade neuronal (RNA/DNA, HSP70, GST e AChE, respetivamente). Os ovos foram incubados a partir do estádio XV até à eclosão e posteriormente foram mantidos à temperatura ambiente (22±1ºC) durante 14 dias. Sobrevivência, crescimento específico, biomassa e peso seco foram medidos nesse período. Foram recolhidas amostras com o intuito de analisar os diferentes biomarcadores por individuo. Para analisar HSP70, utilizaram-se “pools” de paralarvas (7-8). Os resultados apresentam diferenças significativas relativamente à sobrevivência e crescimento específico entre temperaturas. Os resultados do rácio de RNA/DNA e GST (0 e 14 dias) e HSP70 (0,7 e 14 dias) apresentam diferenças significativas entre diferentes idades e temperaturas. Na análise efetuada à AChE não foram encontradas diferenças significativas de atividade entre os diferentes grupos (idades e temperaturas). É notável a ampla variabilidade de resposta aos biomarcadores das paralarvas provenientes da mesma postura. Os resultados demonstram que o rácio RNA/DNA, GST e HSP70 como biomarcadores sensíveis para o crescimento, stress térmico e defesas antioxidantes em paralarvas. No entanto, o crescimento e a temperatura parece não alterar o sistema neurotransmissor dos indivíduos.
European Research COST AQUAGAMETE FA1205, CEPHSINACTION FA1301

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50

Benčová, Simona. "Transkriptomická analýza zápalových kožných biomarkerov u myší s neuropatiou malých nervových vlákien (Transkriptomická analýza zánětlivých kožních biomarkerů u myší s neuropatií malých nervových vláken)." Master's thesis, 2018. http://www.nusl.cz/ntk/nusl-388798.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Simona Benčová Supervisors: Dr. Claire Demiot, Dr. Aurore Danigo Assoc. Prof. Přemysl Mladěnka, Ph.D Title of diploma thesis: Transcriptomic analysis of cutaneous inflammatory biomarkers in a mouse model of small fiber neuropathy. Peripheral neuropathy is an expanding public health problem conditioned by various diseases and associated with several adverse effects such as the occurrence of chronic pain or increased risk of pressure ulcers (PUs). The aim of this study is to explore, whether the inflammatory state of the skin is modified during peripheral neuropathy and in the course of the formation of a pressure ulcer. The transcriptomic analysis was performed with two different models of mice: PU model and uninjured model, to determine genes that differ in expression and in particular, those involved in inflammation. Small fiber neuropathy was induced in young mice by intraperitoneal injection of resiniferatoxin (50 µg/kg, i.p.) - transient receptor potential vanilloid 1 (TRPV1) agonist. PUs were induced by applying two magnetic plates on the dorsal skin. Gene expression was obtained based on RNA microarray and the results were subsequently verified by qPCR. The transcriptomic analysis of PU...

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Dissertations / Theses on the topic 'RNA biomarkers'

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