Dissertations / Theses on the topic 'Housekeeping expression'

Since the initial genome sequencing projects along with the recent advances on technology, molecular biology and large scale transcriptome analysis result in data accumulation at a large scale. These data have been provided in different platforms and come from different laboratories therefore, there is a need for compilation and comprehensive analysis. In this thesis, we addressed the automatization of biological data acquisition and integration from these non-uniform data using machine learning techniques. We focused on two different mining studies in the scope of this thesis. In the first study, we worked on characterizing expression patterns of housekeeping genes. We described methodologies to compare measures of housekeeping genes with non-housekeeping genes. In the second study, we proposed a novel framework, bi-k-bi clustering, for finding association rules of gene pairs that can easily operate on large scale and multiple heterogeneous data sets. Results in both studies showed consistency and relatedness with the available literature. Furthermore, our results provided some novel insights waiting to be experimented by the biologists.

This thesis reports the study of chromatin composition and conformation on the expression of integrated reporters at thousands of genomic locations in the Drosophila genome. We have adapted and improved a technology (Thousands of Reporters Integrated in Parallel TRIP) to randomly integrate barcoded reporters allowing us to measure the context effects on transcription at ~80.000 different loci. We have focused on housekeeping promoter-reporters due to their relative autonomy from distal regulatory elements. Taking advantage of published genome-wide localization maps of chromatin protein and histone marks, together with the three-dimensional genome structure of the Drosophila Kc167 cell line, we have been able to computationally extract the features that best predict the expression of the integrated reporters. Centromeric heterochromatin is highly silencing but position effects were also observed along chromosome arms, away from heterochromatin. Chromatin states such as Black and Blue (Polycomb, H3K27me3) were found to be refractory to expression while Green (HP1, H3K9me) was found to be permissive or refractory depending on the location. Yellow (MRG15, H3K4me) chromatin was the most permissive while Red (Brm, H3K4me) could also be repressive or activating depending on the integration point. Surprisingly we discovered that the housekeeping reporters are maximally expressed when they land on loci engaging in contacts with promoters and terminators of active genes. The low dependence on enhancers confirms the hypothesis that the requisites for developmental regulation are different that for broadly expressed genes. Moreover our results bring experimental evidence to the observation that housekeeping genes tend to cluster during evolution along the chromatin fiber, providing an explanation to the spatial contacts among these clusters observed in Hi-C experiments.<br>Esta tesis recoge los resultados del estudio del efecto de la composición y conformación de la cromatina en la expresión génica mediante la integración de miles de reporteros en el genoma de Drosophila. A tal efecto hemos adaptado y mejorado una técnica (Thousands of Reporters Integrated in Parallel, TRIP) permitiendo la integración aleatoria de genes reportero marcados (barcoded) con el fin de medir su expresión dependiente de contexto en ~80.000 loci distintos. Gracias a los numerosos mapas de ocupación a escala genómica de proteínas asociadas a cromatina y marcas de histonas, así como la estructura tridimensional del genoma en la linea celular utilizada Drosophila Kc167, hemos podido extraer las variables que mejor explican la expresión de los genes reportero integrados. La Heterocromatina pericentromérica demostró su capacidad represora aunque los efectos de posición también pudieron observarse en los brazos cromosómicos, lejos de dicha cromatina. Estados de la cromatina como Black y Blue (Polycomb, H3K27me3) se mostraron refractarios a la expresión mientras que la de tipo Green (HP1, H3K9me) demostró tener efecto ambivalente en función del lugar de integración. La cromatina Yellow (MRG15, H3K4me) mostró ser la mas permisiva mientras que la de tipo Red (Brm, H3K4me) evidenció el mismo carácter ambivalente en función del punto de integración. Sorprendentemente descubrimos que los reporteros housekeeping se expresan de forma óptima cuando se integran en loci contactando promotores y terminadores de genes activos. La escasa dependencia de enhancers confirma la hipótesis según la cual los requisitos para la regulación de genes del desarrollo difieren de los utilizados por genes de expresión ubicua. Por ultimo nuestros resultados brindan evidencia experimental a la observación de la agrupación de genes housekeeping a lo largo de la fibra de ADN durante la evolución. De mismo modo aportan una explicación para el elevado numero de contactos que muestran dichas agrupaciones en experimentos Hi-C.

Les genes de maintenance sont exprimes de maniere ubiquitaire par un mecanisme encore incompris. Pour aborder ce probleme, les auteurs ont choisi comme modele d'etude un gene de maintenance typique, le gene de l'hydroxymethylglutaryl coa reductase (hmgcr), enzyme cle de la biosynthese du cholesterol. Le gene hmgcr de souris a ete isole et sa region promotrice caracterisee; l'expression et le profil de methylation de plusieurs genes chimeriques, dans lesquels le gene marqueur cat est sous le controle de sequences promotrices hmgcr de taille decroissante, ont ete etudies in vitro (par transformation de cellules en culture) et in vivo (dans les souris transgeniques)

Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246.

Magister Scientiae (Medical Bioscience) - MSc(MBS)<br>Purpose: Breast cancer is one of the most frequent and fatal diseases women all around the globe are challenged with today. In women, breast cancer has the highest mortality rate of all cancers and the incidence rate is on the increase. It is estimated that by the year 2025, 19.3 million women will become a victim of this grave health problem. This disease is a result of the formation of malignant tumours caused by genetic alterations that are involved in the proliferation of cells, cellular differentiation and the disturbance in homeostasis which subsequently leads to the abnormal multiplication and growth of cells. Breast cancer is considered a multifactorial disease with various risk factors such as age, radiation exposure, hormone therapy, oral contraceptives, dietary factors, environmental exposure and genetic predispositions. Breast cancers can be subdivided and classified based on their cellular surface receptors such as Estrogen Receptors, Progesterone Receptors and Human Epidermal Growth Factor Receptor 2. Of the various subtypes, the triple-negative breast cancer subtype which is negative for all 3 surface receptors and presents as the most aggressive form of breast cancer with a poor prognosis. Between 10-20% of all breast cancer cases are classified as triple-negative breast cancer. Due to the hormonal status of triple-negative breast cancer, treatment options are limited and thus of great concern. Chemotherapy remains the most common treatment modality, but prognosis is poor with relapse within years ultimately leading to poor survival outcome. Due to this lack of effective treatment plans, an alternative treatment with minimal side effects and better survival remains an imperative area to explore. A wide scope of literature highlights red palm oil and its health benefits, with its growth inhibitory potential drawing great attention. Red palm oil, extracted from the Elaeis guineensis palm tree is red in colour due to the abundance of carotenoids, tocotrienols and tocopherols found in the oil. Various compounds make up the oil such as lycopene, carotenes, vitamin E and coenzyme Q10. Most studies have researched the effects of vitamin E extracted from the oil as a contributor to its growth inhibitory activity. This study focuses on the effects of the commercial red palm oil as a whole with all its compounds on the proliferation of breast cancer cells as well as the effect it has on various genes associated with breast cancer. Method: This study investigated the effect of red palm oil concentrations (1, 10, 100, 500 and 1000 μg/ml) on breast cancer cells—MCF-7 and MDA-MB-231 with comparison to a non-cancerous cell line—MCF-12A for 24-, 48- and 72-hour treatment periods. The parameter investigated was cell proliferation through the CCK-8 cell proliferation assay and the morphology following red palm oil treatment was observed and captured. Additionally, this study also investigated the effect of red palm oil on the expression of Human Mammaglobin (hMAM) and Maspin genes through the PCR assay and results visualised through agarose gel electrophoresis. Data was statistically analysed using GraphPad version 6.0 software. Results: Following treatment of red palm oil, no apparent changes in the cell morphology was observed despite using variable treatment concentrations over variable times for MCF-7, MDA-MB-231 and MCF-12A cells relative to their respective controls. Immortalised MCF-12A cells showed a significant increase in proliferation with the varying treatment concentrations, but more prominently with the highest concentration at 24, 48 and 72 hours. MCF-7 cells showed significant decreases at 24 and 72 hours. Decreased proliferation was observed at all dosages used, particularly at 10, 100, and 500 μg/ml. Furthermore, MDA-MB-231 cells demonstrated a gradual increase in cell proliferation for the 3 selected time periods in the varying concentrations. Additionally, red palm oil did not alter the gene expression of Maspin at any of the varying treatments for MDA-MB-231 nor MCF-7 cells. However, changes in hMAM gene expression were observed at treatment concentration of 100 μg/ml in MDA-MB-231 cells that were incubated for 24 and 48 hours. However, the hMAM expression was not affected in treated MCF-7 cells. Conclusion: Red palm oil, as an alternative dietary oil, seems to have potential growth inhibitory properties as demonstrated by the change in the cell proliferation of the MCF-7 cells. Literature show that various individual compounds extracted from red palm oil have anti-proliferative and inhibitory effects on breast cancer cells making them good candidates for therapy. However, this study concludes that red palm oil as a whole component would not be a suitable therapeutic agent for highly aggressive triple-negative breast cancer.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior<br>Este trabalho tem como objetivo avaliar a estabilidade de genes de referÃncia e a expressÃo das proteÃnas morfogenÃticas Ãsseas (BMP-2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e seus mensageiros intracelulares (SMADs-1, 5 e 8) em folÃculos caprinos antes e apÃs cultivo por 18 dias. Para avaliar a estabilidade dos genes de referÃncia e o nÃvel de expressÃo das BMPs, receptores e SMADs, folÃculos com aproximadamente 0,2, 0,5 e 1 mm foram isolados mecanicamente de ovÃrios caprinos. AlÃm disso, folÃculos com aproximadamente 0,2 mm foram isolados e cultivados por 18 dias em meio de cultura suplementado com FSH. ApÃs a extraÃÃo do RNA total e sÃntese de cDNA, foi realizada a quantificaÃÃo do RNAm, por PCR em tempo real, utilizando-se primers especÃficos para genes de referÃncia (&#946;-actina, PGK, GAPDH, &#946;-tubulina, UBQ, RPL-19, rRNA18S), e para as BMPs (2, 4, 6, 7 e 15) receptores de BMPs (BMPR-IA, IB e II) e SMADs (1, 5 e 8). Os resultados mostraram que &#946;-tubulina e PGK sÃo os genes de referÃncia mais estÃveis em folÃculos frescos prÃ-antrais e antrais caprinos. Os RNAs mensageiros para as BMPs (2, 4, 6, 7 e 15), seus receptores (BMPR-IA, IB e II) e SMADs (1, 5 e 8) sÃo expressos em diferentes nÃveis em folÃculos prÃ-antrais e antrais caprinos, sendo que a expressÃo do RNAm para BMP-4, BMP-6 e BMP-7 em folÃculos de 1 mm sÃo significativamente maiores do que em folÃculos de 0,2 e 0,5 mm. Entretanto, os nÃveis de RNAm para BMP-2 foi reduzido em folÃculos de 1 mm, jà os nÃveis de BMP-15 nÃo diferiram entre as categorias foliculares analisadas. Os nÃveis de RNAm para BMPR-IB foram maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm, enquanto que o RNAm para BMPR-II foi significativamente maior em folÃculos de 0,5 mm do que em folÃculos de 0,2 e 1 mm. Por outro lado, nÃveis de RNAm para BMPR-1A nÃo diferiram entre folÃculos analisados. Os nÃveis de RNAm para SMAD-5 foram significativamente maiores em folÃculos de 0,2 mm do que em folÃculos de 0,5 e 1 mm. Contudo, folÃculos de 0,5 mm mostraram nÃveis maiores de RNAm para SMAD-8 do que folÃculos de 0,2 e 1 mm. Os nÃveis de RNAm para SMAD-1 nÃo diferiram entre os folÃculos. ApÃs as comparaÃÃes dentro de cada categoria folÃcular, BMP-15 foi mais expressa do que BMP-7 em folÃculos de 0,2 e 0,5 mm. Em folÃculos de 0,5 mm a expressÃo do BMPR-IB foi maior do que BMPR-II. Em todas as trÃs categorias foliculares estudadas, a expressÃo da SMAD-5 foi superior a SMAD-8. ApÃs o cultivo, os folÃculos apresentaram reduÃÃo dos nÃveis de RNAm para BMP-2, BMP-4, BMP-7, BMPR-IA e SMAD-5. Em conclusÃo, &#946;-tubulina e PGK sÃo os dois genes housekeeping mais estÃveis para folÃculos frescos caprinos com 0,2, 0,5 e 1 mm de diÃmetro. BMPs, seus receptores e SMADs apresentam padrÃes de expressÃo especÃficos em cada categoria folicular estudada. No entanto, em folÃculos cultivados hà uma variaÃÃo na expressÃo dos componentes do sistema BMP, diferindo da expressÃo in vivo de folÃculos com o mesmo tamanho.<br>The aims this study to evaluate the stability of reference genes and the expression of bone morphogenetic protein (BMP-2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and intracellular messengers (SMADs- 1, 5 and 8) in goat follicles before and after culture for 18 days. To evaluate the stability of reference genes and the expression of BMPs, receptors and SMADs, follicles of approximately 0.2, 0.5 and 1 mm were mechanically isolated from goats ovaries. In addition, approximately 0.2 mm follicles were isolated and cultured for 18 days in culture medium supplemented with FSH. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA, the quantification of mRNA was carried out by real-time PCR using specific primers for genes of reference (GAPDH, &#946;-tubulin, &#946;-actin, PGK, UBQ, RPL - 19, rRNA18S) and BMPs (2, 4, 6, 7 and 15) receptors of BMPs (BMPR-IA, IB and II) and SMADs (1, 5 and 8). Results showed that &#946;-tubulin and PGK are the most stable reference genes in goats preantral and antral follicles. The messengers RNA for BMP (2, 4, 6, 7 and 15), their receptors (BMPR-IA, IB and II) and Smads (1, 5 and 8) are expressed at different levels in preantral and antral goats, and mRNA expression for BMP-4, BMP-6 and BMP-7 in 1-mm follicles are significantly higher than in follicles of 0.2 and 0.5 mm. However, the levels of mRNA for BMP-2 were reduced in follicles 1 mm, as BMP-15 did not differ between follicular categories. The levels of mRNA for BMPR-IB were higher in follicles of 0.2 mm than in follicles of 0.5 and 1 mm, whereas the mRNA for BMPR-II was significantly higher in follicles than 0.5 mm in follicles of 0.2 to 1 mm. Moreover, mRNA levels for BMPR-1A did not differ between follicles examined. The levels of mRNA for SMAD-5 were significantly higher in 0.2 mm follicles than in follicles of 0.5 and 1 mm. However, follicles of 0.5 mm showed higher levels of mRNA for SMAD-8 than follicles 0.2 and 1 mm. The levels of mRNA for SMAD-1 did not differ between follicles. After the comparisons within each category follicle, BMP-15 expression was higher than BMP-7 in follicles between 0.2 and 0.5 mm. Follicles 0.5 mm in the expression of BMPR-IB was greater than BMPR-II. In all three follicular categories studied, the expression of SMAD-5 was superior to SMAD-8. After culture, follicles showed reduced levels of mRNA for BMP-2, BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, &#946;-tubulin and PGK genes are the two most stable housekeeping for fresh goat follicles 0.2, 0.5 to 1 mm in diameter. BMPs, their receptors and SMADs have specific expression patterns in each category follicular studied. However, in cultured follicles showed a variation in the variation in the expression of BMP system components, differing from in vivo expression of follicles with the same size.

碩士<br>國立臺灣海洋大學<br>資訊工程學系<br>102<br>High throughput RNA-seq analysis provides a powerful tool for revealing relationships between gene expression level and biological function of proteins. To discover differentially expressed genes among various RNA-seq datasets obtained from different experimental designs, an appropriate normalization method for calibrating multiple experimental datasets is the first challenging problem. In this thesis, a novel normalization method to facilitate biologists in selecting a set of suitable housekeeping genes for inter-sample normalization is proposed. The approach is achieved by adopting user defined experimentally related keywords, gene ontology (GO) annotations, orthologous housekeeping genes, and stability of housekeeping genes at different time periods. By identifying the most distanced GO terms from query keywords and selecting housekeeping gene candidates with low coefficients of variation among different spatio-temporal datasets, the proposed method can automatically enumerate a set of functionally irrelevant housekeeping genes for practical normalization. By employing benchmark RNA-seq datasets to evaluate our developed system, the results showed that different selections of housekeeping gene set would lead to strong impact on differential gene expression analysis. The compared results have shown that our proposed method outperformed other traditional approaches in terms of both sensitivity and specificity. The proposed mechanism of selecting appropriate housekeeping genes for inter-dataset normalization is robust and accurate for differential expression analyses.

博士<br>國立陽明大學<br>生物醫學資訊研究所<br>100<br>Abstract Housekeeping (HK) genes fulfill the basic needs for a cell to survive and function properly. Owing to their ubiquitous expression and fairly constant expression levels, they have been used as references in mRNA quantification experiments. However, there is an increasing number of studies showing significant, albeit small, variations in many so-called HK genes, particularly when the expression data are taken under multiple tissue types or experimental conditions. Such variations are largely uncharacterized. By analyzing expression data for human genes, in this thesis we uncovered a previously unnoted characteristic of HK gene expression, namely that the ranking order of their expression levels tends to be preserved from one tissue to another. Further analysis by Venn diagram decomposition and pathway stratification identified three main factors of the observed ranking preservation, namely that, compared to those of non-HK genes, the expression levels of HK genes show a greater degree of dispersion (less overlap), stableness (a smaller variation in expression between tissues), and correlation of expression. Our results shed light on regulatory mechanisms of HK gene expression that are probably different for different HK genes or pathways, but are consistent and coordinated in different tissues. Finally, based on the novel expression structure of human HK genes discovered, we developed a HK classifier, named HK_ERA, to mine HK candidate genes. In comparison to four conventional HK prediction methods, HK_ERA had the best performance on the prediction of four different HK gene lists. Advantages over conventional methods for identifying HK genes are discussed.

碩士<br>國立東華大學<br>海洋生物研究所<br>104<br>Little is known about the sub-cellular effects of low temperature exposure on gene expression of ornamental beauty shrimp, or for most crustaceans for that matter. Prior to conducting such gene expression analyses, it is necessary to validate suitable internal reference genes, more commonly referred to as housekeeping genes (HKGs), to control for different reverse transcription reaction efficiencies between samples. Therefore, this study aimed to validate five common housekeeping genes (HKGs)-18S ribosomal RNA (18S rRNA), ATPase (nak), histone 3, β-actin(actb), glyceraldehyde 3-phosphate dehydrogenase (gapdh)- for use in experiments seeking to document the molecular-scale effects of cryopreservation on beauty shrimp embryos. For chilling experiments, we incubated shrimp embryos at either room temperature (control) or 5°C for 0, 4, 8, 16, or 32 hr. GeNorm, NormFinder, and Bestkeeper were used to identify the best HKG. GeNorm selected histone 3 and 18S rRNA as the most stable, whereas NormFinder found the 18S rRNA to be HKG for eye-formation and pre-hatch stage samples. In contrast, Bestkeeper identified gapdh and actb as the best candidates. This is the first study to identify suitable HKGs for low temperature studies of beauty shrimp embryos.