Dissertations / Theses on the topic 'MicroRNA profiling'

I linfomi a cellule T periferiche rappresentano circa il 12% di tutte le neoplasie linfoidi.In questo studio, abbiamo effettuato un’analisi di miRNA profiling (TaqMan Array MicroRNA Cards A) su 60 campioni FFPE suddivisi in: PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) e cellule T normali (N=13). Abbiamo identificato 4 miRNA differenzialmente espressi tra PTCLs e cellule T normali. Inoltre, abbiamo identificato tre set di mirna che discriminano le tre entita di PTCLs nodali
AIMs: Here, we performed an extensive miRNA profiling of PTCLs in order to identify differentially expressed miRNA, either involved in their pathogenesis or potentially useful for their differential diagnosis. Methods: We studied by miRNA profiling (TaqMan Array MicroRNA Cards A) 60 samples including PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) and normal T cells (N=13); in addition, 40 independent PTCL cases were studied by qRT-PCR as validation. We assess a GEP and miRNA Profiling. Findings: we identified 256 miRNA differentiating the two groups. In conclusion, miRNA profiling allowed to identify miRNA possibly involved in PTCL pathogenesis and to develop a novel diagnostic tool for the differential diagnosis of the commonest subtypes.

I linfomi a cellule T periferiche rappresentano circa il 12% di tutte le neoplasie linfoidi.In questo studio, abbiamo effettuato un’analisi di miRNA profiling (TaqMan Array MicroRNA Cards A) su 60 campioni FFPE suddivisi in: PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) e cellule T normali (N=13). Abbiamo identificato 4 miRNA differenzialmente espressi tra PTCLs e cellule T normali. Inoltre, abbiamo identificato tre set di mirna che discriminano le tre entita di PTCLs nodali
AIMs: Here, we performed an extensive miRNA profiling of PTCLs in order to identify differentially expressed miRNA, either involved in their pathogenesis or potentially useful for their differential diagnosis. Methods: We studied by miRNA profiling (TaqMan Array MicroRNA Cards A) 60 samples including PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) and normal T cells (N=13); in addition, 40 independent PTCL cases were studied by qRT-PCR as validation. We assess a GEP and miRNA Profiling. Findings: we identified 256 miRNA differentiating the two groups. In conclusion, miRNA profiling allowed to identify miRNA possibly involved in PTCL pathogenesis and to develop a novel diagnostic tool for the differential diagnosis of the commonest subtypes.

Background: Arrhythmogenic cardiomyopathy (AC) is a clinically and genetically heterogeneous myocardial disease, characterised by a progressive myocardial dystrophy with fibro-fatty replacement, and represents one of the major causes of sudden cardiac death in the young and athletes. Although half of AC patients harbour private desmosomal gene mutations, their low and age-dependent penetrance suggests the involvement of other regulatory molecules. MicroRNAs (miRNAs) are a group of endogenous short noncoding RNAs that regulate gene expression by sequence-specific recognition of their target transcripts. They have been associated with numerous pathophysiological conditions, including cardiovascular diseases; however, their role as key regulatory molecules in AC as well as their impact on the onset and progression of the disease is largely unknown. Purpose: miRNA profiling in genotype-positive AC-patients with different gene mutations in order to identify their potential as AC biomarkers. Methods: The study involved 59 subjects with a definite AC diagnosis, previously genotyped, and 14 healthy controls. 84-miRNA array was applied on 8 frozen right-ventricle (RV) myocardial tissue samples, from heart transplanted AC patients; 9 whole blood samples, from patients with definite AC diagnosis, and 6 healthy controls (HC). In the validation study, seven miRNAs were analysed on 42-AC and 8-HC blood samples. miRNA analysis was performed by qPCR, relative quantification ΔΔCt method and in silico target prediction. All data were expressed in fold-change values. Receiver operating characteristic (ROC) analysis was performed on validated miRNAs. Results: miRNA profiling on AC-tissue samples displayed a genotype-related profile, 19 miRNAs were differentially expressed in PKP2 carriers, 15 in DSP carriers and 14 in DSG2 carriers, when compared to healthy controls. A common signature between PKP2 and DSP carriers was identified with 14 miRNAs in common (PKP2/DSP profile). None of these miRNAs were shown within DSG2 profile. In silico target prediction identified Hippo Signaling Pathway as a common target for both profiles. Analysis of AC-tissue samples as a unique group confirmed 26 differentially expressed miRNAs (AC-tissue profile) with predicted targets in the AC pathway. AC-blood miRNA profiling demonstrated a 14-miRNA signature, with 10 miRNAs differentially expressed in common with AC-tissue profile. Hsa-miR-144-3p, -122-5p, -208a-3p and -494-3p as well as hsa-miR-21-5p, -155-5p and -320a were analysed on a larger cohort of 42-AC and 8-HC. Only hsa-mir-122-5p was significantly overexpressed (p-value<0,05). ROC analysis showed hsa-miR-122-5p to be a potential AC biomarker (area under the curve: 0.83). Conclusions: A genotype-related miRNA profile was observed in AC-tissue samples, as to reflect clinical variability. In addition, 10 miRNAs in common were identified between AC-tissue and AC-blood profiles, proving a specific miRNA signature for AC. These miRNA profiles targeted pathways involved in AC pathogenesis demonstrating their key roles in the onset and progression of the disease. Circulating level of hsa-miR-122-5p was significantly elevated in AC subjects, demonstrating its potential as a prognostic marker for heart failure in AC.
Introduzione. La Cardiomiopatia Aritmogena (AC) è una malattia clinicamente e geneticamente eterogenea del miocardio, caratterizzata da una progressiva distrofia miocardica con sostituzione fibro-adiposa, e rappresenta una delle principali cause di morte improvvisa nei giovani e negli atleti. Nonostante circa la metà dei pazienti affetti da AC presentino mutazioni nei geni desmosomiali, la bassa penetranza e dipendenza dall’età della patologia suggeriscono il coinvolgimento di altre molecole regolatrici. I microRNA (miRNA) sono un gruppo di molecole endogene, di RNA non codificante, che regolano l'espressione genica mediante lo specifico riconoscimento di sequenze target dei trascritti. Sono stati associati a numerose condizioni patofisiologiche, tra cui malattie cardiovascolari; tuttavia, il loro ruolo come molecole regolatrici nella AC e il loro impatto sull'insorgenza e sulla progressione della malattia è in gran parte sconosciuto. Scopo dello studio. Analizzare il profilo di espressione dei miRNA in pazienti affetti da AC genotipicamente positivi allo scopo di studiare il loro potenziale come biomarcatori prognostici. Materiali e metodi. Lo studio ha coinvolto 59 soggetti con una diagnosi clinica di AC, precedentemente genotipizzati, e 14 controlli sani (HC). Un array composto da 84-miRNA è stato testato su: 8 campioni di tessuto miocardico congelato del ventricolo destro, proveniente da pazienti trapiantati affetti da AC; 9 campioni di sangue intero congelato, da pazienti con diagnosi clinica di AC e 6 controlli sani. Nella fase di validazione sono stati analizzati sette miRNA su campioni di sangue provenienti da 42-AC e 8-HC. L'analisi è stata eseguita mediante qPCR seguita da quantificazione relativa con il metodo ΔΔCt e predizione in silico dei geni target. I risultati sono stati espressi in valori di “fold-change” e le curve ROC (Receiver Operating Characteristic) analizzate sui miRNA validati. Risultati. L’analisi dei miRNA su 8 campioni di tessuto di pazienti affetti da AC mostrava un profilo correlato al genotipo rispetto ai controlli sani, in particolare: 19 miRNA erano differenzialmente espressi nei portatori di una mutazione in PKP2, 15 nei portatori di una mutazioni in DSP e 14 nei portatori di una mutazione in DSG2. E’ stato identificato un profilo d’espressione in comune tra i portatori della mutazione in PKP2 e i portatori della mutazione in DSP, con 14 miRNA alterati (profilo PKP2/DSP). Nessuno di questi miRNA è stato trovato nel profilo DSG2. Lo studio in silico dei possibili geni target ha identificato la via di segnale “Hippo Signaling Pathway” come target comune per entrambi i profili (PKP2/DSP- DSG2). Considerando i campioni di tessuto AC come un unico gruppo indipendentemente dal gene mutato sono emersi 26 miRNA differenzialmente espressi (profilo AC-tessuto) che hanno come target geni coinvolti nel pathway AC. Lo studio dei miRNA nei 9 campioni di sangue dei pazienti affetti da AC ha dimostrato un profilo costituito 14-miRNA alterati, dei quali 10 alterati anche nel profilo AC-tessuto. Hsa-miR-144-3p, -122-5p, -208a-3p e -494-3p così come hsa-miR-21-5p, -155-5p e -320a sono stati infine validati su una coorte più ampia di 42-AC e 8-HC. Solo hsa-mir-122-5p è stato riscontrato come significativamente sovraespresso (valore p <0,05). L'analisi di curve ROC ha mostrato che hsa-miR-122-5p è un potenziale biomarcatore di AC (AUC: 0.83). Conclusione. Nei campioni AC di tessuto è stato osservato un profilo di miRNA correlato al genotipo, tale da rispecchiare la variabilità clinica della patologia. Inoltre, sono stati identificati 10 miRNA in comune tra i profili dei campioni AC di tessuto e sangue, evidenziando un profilo di espressione di miRNA specifico per AC. Entrambi i profili infatti (tessuto e sangue) hanno come target vie di segnale coinvolte nella patogenesi della AC, dimostrando un ruolo chiave nella insorgenza e la progressione della malattia. In particolare il livello di hsa-miR-122-5p in circolo era significativamente elevato nei soggetti affetti da AC, dimostrando il suo potenziale come marcatore prognostico della malattia.

Lo sviluppo dei neuroni dopaminergici mesencefalici (mDA) è un fenomeno complesso e non ancora pienamente compreso. Molti studi hanno focalizzato la loro attenzione sul ruolo svolto da diversi fattori di trascrizione specifici e ben noti. L'obiettivo della mia tesi di dottorato è focalizzato su una classe relativamente nuova di regolatori post-trascrizionali denominati microRNA (miRNAs), in grado di regolare l'espressione genica legando le sequenze parzialmente complementari nelle regioni 3' non tradotte (UTR) degli mRNAs target. Per studiare il ruolo svolto dai miRNAs durante la differenziazione dei neuroni mDA, abbiamo scelto di analizzare il profilo di espressione dei miRNA usando piattaforme di Array. A tale scopo, abbiamo utilizzato un protocollo ottimizzato da cellule staminali di epiblasto di topo (epiSC) differenziate in neuroni mDA (Jeager et al.,2011). Dall'analisi bioinformatica dei dati dell'array, ottenuti dalle epiSC differenziate in neuroni mDA, abbiamo identificato alcuni candidati molto probabilmente implicati nella differenziazione e nella funzione dei neuroni DA. I miRNA candidati sono stati sottoposti a screening per la loro capacità di indurre il fenotipo DA. A questo scopo, ho generato vettori lentivirali inducibili per ciascun miRNAs e ho infettato colture primarie mesencefaliche di topo allo stadio E12.5. Tra tutti i miRNA candidati, miR-218 e miR-34b/c aumentano il numero di cellule TH + positive, suggerendo il loro possibile contributo nei neuroni mDA. Inoltre, miR-218 e miR-34b/c, risultano arricchiti sia nel mesencefalo dei topi (E13.5) che nelle cellule GFP + sortate al FACS, isolate da embrioni E13.5 Pitx3-GFP di topo, rispetto al controllo. I dati ottenuti dal saggio di Luciferasi e dal saggio di reporter a doppia fluorescenza suggeriscono che miR-34b/c legano e sopprimono la 3'UTR di Wnt1 e viene espresso durante la differenziazione dei neuroni mDA. Tramite analisi di ibridazione in situ e dati d’ immunoistochimica ho potuto verificare che miR-218 è espresso in particolare nel mesencefalo di topo allo stadio E14, dove co-localizza rostralmente con Isl-1 (marcatore di motoneuroni) e caudalmente con TH, Pitx3, Lmx1a (marcatori dopaminergico). Questi dati suggeriscono che miR-218 è espresso anche nei motoneuroni craniali, come descritto in altri recenti studi (Thiebes, K.P. et al., 2014; Amin, N.D et al., 2015). Per comprendere ulteriormente il ruolo di miR-218 nello sviluppo e nella funzione dei neuroni dopaminergici ho generato topi knock-out condizionali (cKO) per miR-218-2. Accoppiando miR-218-2 flox / flox con topi En1Cre /+ che esprimono Cre sotto il controllo del promotore di Engrailed 1 (En1, marker pro-dopaminergico), sarò in grado di comprendere il contributo di miR-218 nel sistema dopaminergico. I topi miR-218-2 flox / flox En1Cre /+ da osservazioni preliminari, hanno mostrato un fenotipo con danno motorio, ma per confermare questi dati sto attualmente effettuando test comportamentali e analisi in vivo. Attraverso il profilo di espressione di miRNAs, siamo in grado di comprendere il meccanismo e la funzione del sistema dopaminergico, poiché i miRNAs sono regolatori chiave nelle reti di espressione genica, possono influenzare molti processi biologici e in futuro potrebbero essere utilizzati come biomarkers per diagnosticare patologie legate al sistema nervoso.
Midbrain dopaminergic neurons (mDA) development is a complex and still not fully understood phenomenon. Many studies till now concentrated their attention on the roles played by several, specific and well-known transcription factors. The aim of my PhD thesis is focus on a relatively new class of post-transcriptional regulators named microRNAs (miRNAs) able to regulate gene expression by targeting partially complementary sequences in the 3’untranslated regions (UTRs) of the target mRNAs. To investigate the role played by miRNAs during mDA differentiation, we choose to analyze miRNAs expression profile by using miRNA Array platforms. To this purpose we used an optimized protocol from mouse Epiblast stem cells (epiSC) differentiated into DA neurons (Jeager et al. 2011). By bioinformatics analysis of the array data, obtained from epiSC differentiated into mDA neurons, we identified few candidates most likely implicated in the DA neurons differentiation and function. The candidate miRNAs were screened for their ability to induce DA phenotype. To this purpose, I generated inducible lentiviral vectors for each miRNA and I have infected mesencephalic primary cultures from mice at stage E12.5. Among all candidate miRNAs, miR-218 and miR-34b/c increase the number of TH+ positive cells, showing their possible contribution in the mDA neurons. Moreover, miR-218 and miR-34b/c, were enriched both in midbrain of mice (E13.5) and in FACS sorted GFP+ cells isolated from E13.5 Pitx3-GFP mice embryos when compared with control. Data obtained from Luciferase Assay and Dual Fluorescence Reporter Assay suggest that miR-34b/c target and suppress Wnt1 3’UTR and it is expressed during DA neurons differentiation. By performing In situ hybridization analysis and immunohistochemistry, I was able to detect miR-218 in particular in the mouse midbrain at stage E14, where co-localize rostrally with Isl-1 (motor neuron marker) and caudally with TH, Pitx3, Lmx1a (dopaminergic marker). This data suggests that miR-218 is expressed also in cranial motor neurons, as described in others recent studies (Thiebes, K.P. et al. 2014; Amin, N.D et al. 2015). To further understand the role of miR-218 in development and function of dopaminergic neurons I have generated the conditional knock-out (cKO) mice for miR-218-2. By mating miR-218-2 flox/flox with En1Cre/+ mice expressing the Cre under Engrailed 1 promoter (En1 is a pro-dopaminergic marker) I will be able to investigate the contribution of miR-218 in dopaminergic system. Preliminary observations on miR-218-2 flox/flox En1Cre/+ mice shown motor impairment phenotype, but to confirm this data I’m currently performing behavior tests and in vivo analysis. Through miRNA expression profiling we be able understand mechanism and function of dopaminergic system, because miRNAs are as key regulators in gene expression networks, can influence many biological processes and have also shown promise as biomarkers for neuro-disorders.

Papillary thyroid cancer (PTC) is commonly diagnosed in Australia and has an increasing incidence; it is projected to be the 10th most commonly diagnosed cancer by the end of 20191. Fortunately it has a good outcome, with the mortality rate stable over the last 25 years at around 2%2. The challenge to the clinician is to determine the best diagnostic tools to aid in appropriate management of the disease. MicroRNA (miRNA) is an emerging tool in the diagnosis and management of cancers. We examined the expression of miRNAs in an Australian population with PTC, both those known to have altered expression and novel miRNAs. Tissues of 30 subjects were collected, 18 from whom we also had access to serum. The miRNAs were studied in thyroid tumour and normal tissue, lymph nodes and serum. MiRNAs mir-10b, miR-31, miR-100, miR-146b, miR-221, miR-222 and let-7d were studied, with miR-16, RNU48 and Cel-39 used as controls. Our study demonstrated significantly elevated expression of miRs -146b, -221 and -222 in tumour tissue vs normal tissue and a significantly decreased expression of miR-10b. We also showed that handling of serum samples has a significant impact on the results when analysing miRNAs, due to the effect of haemolysis. 1. Ferlay J SI, Ervik M, et al. GLOBOCAN 2012 v1.0, Cancer incidence and mortality worldwide: IARC cancerbase no. 11 In. Lyon, France: International Agency for Research on Cancer; 2013. 2. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9-29.

MicroRNAs (miRNAs) are emerging as important, albeit poorly characterized, regulators of gene expression. Here, I review the current knowledge of miRNAs in humans, including their biogenesis, modes of action and various methods for studying them. To fully elucidate the various functions of miRNAs in humans, we require a more complete understanding of their numbers and expression changes amongst different cell types. This document includes a description of a new method for surveying the expression of miRNAs that employs the new Illumina sequencing technology. A set of methods is presented that enables identification of sequences belonging to known miRNAs as well as variability in their mature sequences. As well, a novel system for miRNA gene discovery using these data is described. Application of this approach to RNA from human embryonic stem cells (hESCs) obtained before and after differentiation into embryoid bodies (EBs) revealed the sequences and expression levels of 362 known plus 170 novel miRNA genes. Of these, 190 known and 31 novel microRNA sequences exhibited significant expression differences between these two developmental states. Owing to the increased number of sequence reads, these libraries currently represent the deepest miRNA sampling in any human cell type spanning nearly six orders of magnitude of expression. Predicted targets of the differentially expressed miRNAs were ranked to identify those that are likely under cooperative miRNA regulation in either hESCs or EBs. The predicted targets of those miRNAs enriched in either sample shared common features. Included amongst the high-ranked predicted gene targets are those implicated in differentiation, cell cycle control, programmed cell death and transcriptional regulation. Direct validation of these predicted targets or global discovery of miRNA targets should reveal the functions of these sequences in the differentiation of hESCs.
Science, Faculty of
Graduate

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
All cells in a multicellular organism carry the same genes, yet these same genes direct the differentiation of many different cell types. This is facilitated by differential gene expression, the control of which can be exerted at the transcriptional, as well as post-transcriptional, level. MicroRNAs (miRNAs) are -22- nucleotide small RNAs that mediate post-transcriptional regulation of gene expression by base pairing to their target mRNAs to direct repression. In animals, this repression is usually mediated through translational repression and/or mRNA destabilization. In studies that investigate miRNA-mediated repression with reporter constructs or individual endogenous genes, translational repression and mRNA destabilization have been observed to contribute variably to the overall level of repression. This led to the question of whether the same was true for endogenous targets at a genome-wide level. While changes in mRNA levels can be easily captured by microarray measurements, it is harder to measure translational repression on a genome-wide scale. To address this gap, we used ribosome profiling to measure effects on protein production and compared these to simultaneously measured effects on mRNA levels. The latter were also quantified by a deep-sequencing approach (mRNA-Seq). This enabled us to obtain a snapshot of changes in translational efficiency at the genome-wide level. For both ectopic and endogenous miRNA regulatory interactions, we observed that lowered mRNA levels account for most (>84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output. The slight reduction in translational efficiency is likely mediated by an inhibition of translation initiation. For studying miRNA repression in an endogenous system, we had initially used in vitro differentiated neutrophils from mir-223 knockout mice and compared these to cells from wild-type mice. Because neutrophils have a shorter lifespan than most differentiated cell types, we selected another endogenous system, antigen-stimulated B cells from mir-155 knockout mice, and similarly compared these to cells from wild-type mice. In addition to mRNA-Seq and ribosome profiling, we made parallel proteomics measurements. Our results show that miR- 155 in antigen-stimulated B cells primarily mediates mRNAlevel changes, though the contribution from changes in translational efficiency was larger than previously observed. In addition, we observed widespread translation of upstream open reading frames initiated from canonical and non-canonical start codons. These upstream open reading frames are also translationally repressed by miR-155.
by Huili Guo.
Ph.D.

Huntington's Disease (HD) is an inherited neurodegenerative disorders caused by CAG repeat expansions in exon 1 of the huntingtin gene (htt). Patients with HD experience profound region specific neural degeneration for reasons that remain incompletely understood. Early studies in HD brain suggest that transcriptional misregulation occurs early in disease, before significant tissue loss and degeneration has occurred. However, a comprehensive understanding of the events that contribute to this remain poorly understood. In this study, we investigate a functional role for small RNA or miRNAs in the central nervous system (CNS) of patients with HD. Our work identifies subsets of miRNAs misregulated in HD. A functional role for these miRNAs was investigated by identifying their predicted targets. We identify a subset of differentially detected miRNAs which are inversely correlated with predicted downstream predicted 3'UTR target genes. We also identify targets of these differentially detected miRNAs including transcriptional regulators REST, CoREST and cFOS. The transcription factor REST silences neuronal gene expression in non-neuronal cells. Polyglutamine expansions in Huntingtin, which cause HD, abrogate REST-mediated Huntingtin binding, and as a result REST translocates to the nucleus, occupies RE1 consensus sites and represses the expression of both coding and non-coding RNAs. In this work, we identify miRNAs (miRNAs) with upstream REST consensus sites that are decreased in HD patient primary motor cortices (BA4). One of these miRNAs, miRNA-9/miRNA-9* is capable of regulating the expression of two components of the REST complex: miRNA-9 targets REST and miRNA-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates. In addition to these studies, we identify CNS enriched miRNAs which may differentially regulate human versus non-human primate gene expression. We computationally identified a single nucleotide change from G to A in the 3'UTR of human cFOS 3'UTR which is predicted to be regulated by the brain enriched miRNA-7. A regulatory role for the single nucleotide change in humans (G->A) was assessed by mutating the single nucleotide in the human cFOS 3'UTR (from A->G), as well as by introducing the corresponding human mutation (G->A) into the rhesus and chimpanzee cFOS 3'UTRs. The presence of the A nucleotide in the predicted MRE for miRNA-7 was sufficient to partially abrogate miRNA-7 activity in reporter plasmids. Finally, overexpression of artificial precursor miRNAs in human HEK293 and mouse N2A cell lines confirmed differential targeting of cFOS in human versus mouse cell lines. These data provide evidence for the potential contribution of a single nucleotide change in humans as regards changes in cFOS regulated gene expression. Since cFOS is a transcription factor, downstream affects from altered expression could be significant. Together, this work provides new support for the role of brain enriched miRNAs in the CNS and identifies functional support that their misregulation or altered expression can impact expression of protein coding transcripts in disease brain, and may be relevant to primate brain evolution.

MicroRNAs are short RNA molecules composed of 20-22 nucleotides. They highly accurately indicate cell identity and hence they are useful in labeling cells and tacking lineage commitment. However, this requires accurate microRNA profiling of cells in individual developmental stages. Since microRNAs are important negative regulators of eukaryotic gene expression, microRNA profiling allows better understanding of molecular regulatory networks of important cellular events, such as adult neurogenesis. Adult neurogenesis is the process in which neurons, as well as glia, are generated from neural stem cells. It was found to be responsible for brain regeneration, olfactory discrimination, memory formation and learning. Depression was suggested to be related to dysregulation of neurogenesis. Thus, knowledge in cellular and molecular mechanisms of adult neurogenesis will lay solid foundation to develop therapies to regenerate neural cells after injuries or onsets of neurodegenerative diseases and to understand the cognitive ability, memory formation and learning of the brain. In spite of its importance, investigation into the miRNA profiles and functions in neurogenesis is still infant. This project aimed to establish a preliminary microRNA profile on neurogenesis. Although this was not completed, the project could be extended to a large-scale microRNA profiling in neurogenesis. This would enable future workers to track the lineage commitment, the migration, and the distribution of NSCs and their derived cells accurately by in situ hybridization. Also, the future workers may construct a 2D representation of the changes in miRNA profiles and this may lead to discovery of previously unknown molecular and cellular differences among cells of same cell identity.
published_or_final_version
Anatomy
Master
Master of Medical Sciences

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, accounting for a total of 2-3% of adult neoplasias, and it arises from the renal epithelium. Clear cell renal cell carcinoma (ccRCC) is the most common, invasive and metastatic among RCC subtypes, representing 75-80% of kidney primary malignancies. The von Hippel-Lindau (VHL) gene, which is the main tumor suppressor gene involved in early steps of RCC tumorigenesis, undergoes complete inactivation by mutation, deletion, and promoter methylation in the majority of sporadic ccRCCs and in all inherited forms. Defining the prognosis for RCC cases is important for both decision-making and counseling patients, but sometimes the diagnosis is difficult because tumor subtypes have overlapping histo-pathological features, thus resulting undistinguishable by microscopy investigation. In recent years, in several human cancers, microarray gene expression profiling proved to be a powerful tool to better classify tumor subtypes and to identify novel molecular biomarkers potentially useful for clinical applications. In fact, tumor transcriptomic profiling may identify patterns of genes that are functionally related to patients’ prognosis, response to therapy and overall survival. Recent evidences have shown that microRNA (miRNA) molecules are involved in tumorigenesis, indicating that miRNAs might function as both tumor suppressors and oncogenes, and their role in RCC pathogenesis is now emerging. miRNAs are small single-stranded non-protein-coding RNA molecules, that function as negative post-transcriptional gene regulators in animals, plants and viruses, and are involved in many biological processes, also including haematopoietic cell differentiation, apoptosis, cell proliferation and organ development. miRNA and gene expression patterns are closely related, since they cooperatively work to create gene regulatory networks. Therefore, integrative genomics approach might be a useful tool to elucidate the complex relationships underlying these networks. The aim of my PhD fellowship work was to reconstruct miRNA-gene post-transcriptional regulatory networks involved in RCC biology, using miRNA and gene expression profiles of three RCC cell lines compared to a normal one, obtained by Affymetrix high-density microarray technology. We calculated differentially expressed genes and miRNAs, and, by functional enrichment analysis, we identified genes and miRNAs that were already known to be associated with RCC and involved in relevant pathways for this pathology, such as hypoxia, p53 signaling, focal adhesion, angiogenesis and mTOR signaling. Through integrated analysis of miRNA-gene expression profiles, we reconstructed potentially active regulatory networks involving miRNAs and their predicted target genes. We validated some miRNA-gene pairs by quantitative PCR, thus confirming their anti-correlated expression levels. Our results demonstrated that RCC cell lines can be an useful in vitro model for RCC pathology, since they showed gene and miRNA expression profiles similar to renal tumoral tissues, as obtained by comparing our results with published data. The analysis of the correlations between gene and miRNA expression profiles using a genome-wide integrative approach could help the identification of both post-transcriptional regulatory networks and novel candidate markers functionally relevant for RCC pathology. However, further investigations are necessary to elucidate the actual role of miRNA-gene networks in the context of RCC progression and outcome.

Abstract Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the Western world. Although it shares mainly the same risk factors as coronary heart disease (CHD), i.e. similar initial events in both diseases but with time, they lead to different clinical outcomes. Thus, when it affects the coronary arteries, the disease leads to an obstructive or rupture-prone plaque whereas in the aortic valve, it causes massive calcification and ossification. This obstructs the blood flow from the left cardiac ventricle, causing myocardial hypertrophy, and if left untreated, heart failure and death. Many of the pathobiological differences between CAVD and CHD remain unknown. Currently, there are no effective lifestyle- or pharmacologic treatments for CAVD and the only therapy is a valve replacement operation. In this thesis, several studies utilizing large-scale methods were undertaken to profile the molecular events leading to CAVD. Surgically removed valves from patients in different stages of the disease were obtained and gene transcripts, microRNA-molecules and several proteins were identified as being differentially expressed. Several of these were investigated further, including two pro-inflammatory CC-type chemokine ligands 3 and 4 (CCL3 and CCL4), microRNA-125b, several granzyme-proteins and heat-shock protein 90. The results of this thesis provide a large dataset of hundreds of molecular changes associated with CAVD. It is proposed that they can be used as a basis for the generation of new hypotheses and assist in the design of experiments to clarify the mechanisms driving CAVD
Tiivistelmä Aorttaläpän kalkkeutuva ahtauma on länsimaiden yleisin sydänläppäsairaus. Riskitekijät ovat pääosin samat kuin sepelvaltimotaudissa, ja molemmat saavat alkunsa samalla tavalla. Ajan myötä ne kuitenkin johtavat varsin erilaisiin kliinisiin ilmenemismuotoihin: sepelvaltimoihin kasvaa ahtauttavia ja repeytymisherkkiä plakkeja, kun taas aorttaläppään muodostuu runsaasti kalkkia ja luuta. Se haittaa verenvirtausta sydämen vasemmasta kammiosta aorttaan, mikä aiheuttaa sydänlihaksen paksuuntumista. Hoitamattomana tauti johtaa lopulta sydämen vajaatoimintaan ja kuolemaan. Monet syyt eroihin sepelvaltimotaudin ja aorttaläpän ahtauman välillä ovat edelleen tuntemattomia. Tällä hetkellä aorttaläpän ahtaumaan ei ole olemassa tehokasta elintapa- tai lääkehoitoa, ja ainoa hoitomuoto onkin vioittuneen aorttaläpän korvaaminen proteesilla. Tässä väitöskirjatyössä tehtiin useita laaja-alaisia molekyylitason profilointitutkimuksia, joilla selvitettiin aorttaläpän ahtaumaan mahdollisesti johtavia mekanismeja. Aineistona oli leikkauksessa potilailta poistettuja, erilaisissa taudin vaiheissa olevia aorttaläppiä. Niistä kerättiin tietoja kaikkien geenien ilmentymisestä, mikroRNA-molekyyleistä sekä koko proteomitason muutoksista. Useat tunnistetuista molekyyleistä valittiin jatkotutkimuksiin niiden tarkempien ominaisuuksien selvittämiseksi. Näitä olivat tulehdusta välittävät kemokiinit CCL3 ja CCL4, mikroRNA-125b, useat grantsyymiproteiinit sekä lämpöshokkiproteiini 90. Väitöskirjatyön tuloksista voidaan muodostaa ainutlaatuinen aineisto sadoista erilaisista aorttaläpän ahtaumaan johtavista molekyylitason muutoksista. Sitä voidaan hyödyntää uusien tutkimushypoteesien muodostamisessa sekä aorttaläpän ahtauman tarkempien mekanismien selvittämiseen tähtäävien kokeellisten tutkimusten suunnittelussa

Epithelial Ovarian Cancer (EOC) is a very malignant neoplasm, accounting for 5% of cancer mortality in women. Although progress has been made in EOC treatments by improved debulking surgery and platinum-taxane regimens, the 5-year survival rate of advanced-stage EOC remains below 30%. This poor prognosis relies on the one hand on the late diagnosis and on the other on the chemo-resistance occurring after only a few months from the completion of treatment. The reasons for recurrence and cancer drug resistance remain uncertain. Recent evidence suggests that EOC, akin most tumors, contains a tiny population of cells, named cancer stem cells (CSC), probably responsible for chemotherapy resistance and tumor recurrence. Ovarian CSC are characterized by the co-expression of two surface markers: CD44 (hyaluronic acid receptor) and CD117 (stem cell factor receptor or c-kit). Recently, our research group demonstrated that ovarian CD44/CD117 co-expressing cells, which represent 1-2% of cancer cells from ascitic effusions of EOC-bearing patients, are endowed with canonical stemness properties and are able to resist in vitro and in vivo glucose starvation. We reported that this glucose deprivation resistance is mostly due to the ability of ovarian CSC to privilege oxidative phosphorylation, rather than the aerobic glycolysis (Warburg effect) exploited by the non-stem tumor bulk. However, independently of CSC fraction, our experiments also highlighted that not all the analyzed EOC samples presented a similar glucose addiction. Thus, investigating this issue and its related metabolic aspects is the first aim of the current project. Concerning this aim, we showed that tumor cells from EOC patients can be categorized, according to their in vitro viability under glucose starvation, into glucose deprivation-sensitive (glucose-addicted, GA) and glucose deprivation-resistant (glucose non-addicted, GNA). Although deregulated glucose metabolism is usually observed in cancer, whether this metabolic trait influences response to or is modulated by cytotoxic drugs is unknown; therefore, we addressed the possible correlation of these glucose addiction profiles with the patient response to platinum (PLT) regimens. In this regard, when EOC cells were cultured in the absence of glucose, all samples from PLT-sensitive patients felt into the GA group; compared to GNA samples, they disclosed higher expression of glucose metabolic enzymes, higher proliferation rates and in vitro sensitivity to PLT, as well as reduced multi-drug resistance pump expression. On the other hand, the samples derived from PLT-resistant patients felt into the GNA category. The close association between PLT sensitivity and glucose metabolic profile was confirmed in a xenograft model, where a stringent parallelism between PLT sensitivity/resistance and glucose metabolism was identified. Finally, in a cohort of naïve EOC patients categorized as GA or GNA at diagnosis, Kaplan Meier curves showed that the GA phenotype was associated with significantly better progression-free survival, compared to GNA patients. Overall, these results suggest that in vitro glucose addiction of EOC cells could be regarded as a reliable marker to predict the patient response to platinum regimens. Investigating the molecular traits leading to the distinctive glucose metabolism of EOC samples is the second aim of this project. In this regard, microRNA (miRNA), that are small non-coding RNA molecules, represent a promising field, in view of their ability to modulate many genes and pathways. Moreover, their involvement in cancer development and progression has already been reported in ovarian cancer, and recently miRNA also emerged to regulate cell metabolism in several normal and cancer tissues. Thus, we performed a miRNA profile on EOC patient-derived samples, comparing both GA versus GNA samples and CSC versus non-CSC, in order to establish whether the miRNA signature behind the metabolic differences of EOC samples is associated with the total tumor bulk or rather with a specific cell fraction. The data did not reveal any miRNA differentially expressed in GA compared to GNA cells, but several miRNA resulted significantly deregulated in CSC versus non-CSC. We focused our attention on mir-602, which was found up-regulated in CSC; indeed, despite little information about this miRNA, its target Casein Kinase 1 Delta (CSNK1D), which displays a key role in cell proliferation and asymmetric division, seemed very interesting in view of its involvement in breast cancer progression. We demonstrated that CSNK1D is down-regulated in CSC, according to mir-602 over-expression. Moreover, the in vitro inhibition of mir-602 reduced the expression of the major stemness-associated genes in CSC, suggesting that mir-602 could regulate some cell stemness pathways. Since none of the analyzed stemness genes is directly targeted by mir-602, it is reasonable to advance that mir-602 could indirectly activate the expression of such genes through its inhibitory function on CSNK1D; thus, we propose that cell stemness signaling is inhibited by casein kinase, whose translation is in turn repressed by mir-602. Although a few further experiments are needed to validate our idea, this project highlights a possible miRNA-mediated regulatory mechanism of cell stemness features in EOC.
Il tumore ovarico di tipo epiteliale (EOC – Epithelial Ovarian Cancer) rappresenta la prima causa di morte per neoplasia ginecologica. Sebbene la chirurgia e la chemioterapia a base di carbo/cis-platino abbiano migliorato la prognosi delle pazienti affette da tale carcinoma, il tasso di sopravvivenza a 5 anni dalla diagnosi di EOC in stadio avanzato rimane inferiore al 30%. Tale elevata mortalità è riconducibile alla diagnosi in fase tardiva ed all’insorgenza di resistenza alla chemioterapia di prima linea, che porta a frequenti ricadute entro pochi mesi dalla conclusione dei trattamenti. Le cause della comparsa di fenomeni di resistenza alla terapia, e della conseguente ricorrenza del tumore, è ancora incerta. Recenti studi hanno indicato che la crescita del carcinoma epiteliale dell’ovaio è sostenuta da una minima popolazione di cellule, dette cellule tumorali staminali (CSC - Cancer Stem Cells), probabilmente responsabili della ricomparsa del tumore al termine delle sedute farmacologiche. Un consenso generale supporta l’idea che l’eliminazione di questa popolazione rappresenti uno dei più importanti obiettivi della terapia anti-tumorale. Tuttavia, si ha ancora una scarsa conoscenza dei meccanismi che conferiscono un vantaggio di sopravvivenza alle CSC sulle cellule tumorali non staminali, rendendo così arduo lo sviluppo di terapie mirate anti-CSC. Le CSC del carcinoma epiteliale ovarico sono caratterizzate dall’espressione di due marcatori di superficie cellulare: CD44 (recettore dell’acido ialuronico) e CD117 (c-kit o recettore del fattore cellulare staminale SCF). Recentemente, il nostro gruppo di ricerca ha dimostrato che le cellule ovariche co-esprimenti CD44 e CD117, che rappresentano l’1-2% delle cellule tumorali provenienti dall’ascite delle pazienti affette da EOC, possiedono le canoniche proprietà staminali e sono in grado di sopravvivere in vitro ed in vivo alla deprivazione di glucosio. Abbiamo inoltre osservato che tale resistenza all’assenza di glucosio è principalmente dovuta alla capacità delle CSC, contrariamente alle cellule tumorali non staminali, di privilegiare la fosforilazione ossidativa anziché la glicolisi aerobica (Warburg Effect). Tuttavia, indipendentemente dalla frazione delle cellule tumorali staminali, l’analisi comparativa tra i diversi campioni di EOC ha evidenziato che non tutti presentano la stessa dipendenza dai glucidi; per alcuni, infatti, pochi giorni in vitro senza glucosio sono sufficienti a ridurre significativamente la vitalità cellulare, mentre per altri lo stesso effetto è ottenibile solo dopo molte settimane di coltura nelle stesse condizioni. Dunque, approfondire tale questione e gli aspetti metabolici ad essa correlati è il primo scopo di questo progetto. A tal proposito, sulla base della vitalità cellulare in condizioni di coltura senza glucosio, abbiamo potuto suddividere le cellule derivanti da asciti di pazienti con EOC in due categorie: sensibili alla deprivazione di glucosio (GA – Glucose-Addicted) e resistenti a tale deprivazione (GNA – Glucose Non-Addicted). Sebbene variazioni nella regolazione del metabolismo del glucosio siano state frequentemente osservate nei casi di neoplasia, non è ancora noto se questo diverso tratto metabolico influenzi la risposta dei pazienti alle terapie, o se sia da queste modulato. Pertanto, abbiamo deciso di ricercare una eventuale correlazione tra i diversi profili di dipendenza dal glucosio da noi riscontrati e la risposta dei pazienti al trattamento a base di carbo/cis-platino. Infatti, da un punto di vista clinico, i pazienti vengono categorizzati come platino-resistenti o platino-sensibili, a seconda che la neoplasia recidivi entro od oltre i 6 mesi, rispettivamente, dalla fine della chemioterapia di prima linea. I nostri esperimenti hanno rivelato che, quando le cellule di EOC vengono coltivate in assenza di glucosio, tutti i campioni provenienti da pazienti platino-sensibili ricadono all’interno del gruppo GA; confrontati con i campioni GNA, i GA mostrano una maggiore produzione degli enzimi del metabolismo glucidico, un maggior tasso di proliferazione, e una minore espressione delle pompe cellulari per l’espulsione dei farmaci. Parallelamente, i campioni derivanti dai pazienti platino-resistenti rientrano nella categoria GNA. La stretta associazione tra la sensibilità ai chemioterapici e il profilo cellulare di utilizzo del glucosio è stata confermata in un modello murino di xenotrapianti, nel quale è stato identificato uno stringente parallelismo tra risposta al platino e metabolismo glucidico. Infine, in una coorte di pazienti non chemio-trattate affette da EOC, le quali erano state categorizzate come GA o GNA alla diagnosi, le curve di Kaplan Meier hanno messo in luce che il fenotipo GA, rispetto a quello GNA, è associato con un maggior periodo di sopravvivenza senza recidive, in modo statisticamente significativo. Nel complesso, questi dati suggeriscono che il grado di dipendenza dai glucidi delle cellule di EOC, osservabile in vitro, può rappresentare un valido marcatore per predire la risposta dei pazienti alla chemioterapia a base di platino. Analizzare il tratto molecolare che determina il peculiare metabolismo glucidico dei campioni di EOC costituisce il secondo obiettivo del nostro progetto di ricerca. A tal riguardo, i microRNA (miRNA), ossia piccole molecole di RNA non codificante, rappresentano un promettente settore di studio, in qualità della proprietà di queste strutture molecolari di regolare molti geni e vie di segnale. Inoltre, il loro coinvolgimento nello sviluppo e nella progressione tumorale è già stato dimostrato per il carcinoma ovarico, e recentemente i miRNA sono risultati essere importanti modulatori del metabolismo cellulare in molti tessuti normali e neoplastici. Pertanto, il nostro gruppo di ricerca ha prodotto un profilo di espressione di miRNA su cellule derivanti da pazienti affette da EOC, confrontando sia campioni GA contro GNA, sia CSC contro non-CSC; il nostro fine è stabilire se il pattern di miRNA alla base delle differenze metaboliche tra i campioni di EOC sia associato alla totale massa tumorale o piuttosto ad una specifica frazione cellulare neoplastica. Questi dati non hanno rivelato alcun miRNA differentemente espresso tra cellule GA e GNA; tuttavia, molti miRNA sono risultati deregolati nelle CSC rispetto alle non-CSC. Noi ci siamo focalizzati sul mir-602, up-regolato delle CSC; infatti, nonostante la scarsa conoscenza su questo miRNA, il suo target chinasi Caseina 1 Delta (CSNK1D), che detiene un ruolo chiave nella proliferazione cellulare e nella divisione asimmetrica, ci è parso molto interessante in virtù del suo già dimostrato coinvolgimento nella progressione del carcinoma mammario. In questo contesto, i nostri esperimenti hanno dimostrato che CSNK1D è down-espressa nelle CSC, in accordo alla up-modulazione del mir-602. Inoltre, l’inibizione in vitro del mir-602 riduce nelle CSC l’espressione della maggior parte dei geni associati alle proprietà staminali, suggerendo che il mir-602 potrebbe controllare alcune delle vie di segnale correlate alla staminalità. Dato che nessuno dei geni di staminalità analizzati si lega direttamente al mir-602, appare ragionevole supporre che suddetto miRNA possa indirettamente attivare l’espressione di tali geni tramite la sua funzione inibitoria su CSNK1D. Dunque, secondo la nostra ipotesi, le caratteristiche staminali sarebbero inibite dalla chinasi Caseina, la quale sarebbe a sua volta repressa dal mir-602. Nonostante molti altri esperimenti siano necessari per confermare questa nostra teoria, il progetto qui presentato mette in rilievo la possibile esistenza di un meccanismo di regolazione, mediato dal mir-602, responsabile delle proprietà di staminalità delle cellule del carcinoma epiteliale ovarico.

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.

Nell’ultimo decennio la scoperta dei microRNA ha messo in luce un nuovo e fine meccanismo di regolazione post-trascrizionale, che interviene in molti processi quali lo sviluppo, il differenziamento la proliferazione e la morte cellulare. Inoltre, numerose evidenze hanno dimostrato il coinvolgimento dei microRNA in diverse patologie. Nella maggior parte degli studi effettuati, e' stata utilizzata la tecnologia microarray per identificare i microRNA coinvolti nei meccanismi patogenetici, ma anche per ottenere dei profili di espressione caratteristici di patologia con valore diagnostico o prognostico. Questi studi suggeriscono che l'analisi dei profili di espressione dei microRNA può essere considerata uno strumento utile per comprendere quale ruolo essi svolgono nella regolazione dei processi fisiopatologici. In questo lavoro abbiamo studiato il profilo di espressione di microRNA, mediante tecnologia microarray in due diversi modelli sperimentali: 1) un modello in vitro, utile per la comprensione dei meccanismi molecolari alla base della risposta immunitaria; 2) un modello in vivo, idoneo per lo studio della patogenesi di una patologia epatica molto diffusa nota come NAFLD ("Non-alcoholic fatty liver disease"). E' di recente scoperta la relazione tra infiammazione, immunità innata e microRNA, che sono descritti essere coinvolti nella regolazione della risposta cellulare all’infezione microbica. Noi abbiamo, così, analizzato lo specifico profilo di espressione di microRNA in cellule dendritiche umane, utilizzando un modello in vitro di stimolazione e attivazione mediante agonisti di differenti recettori Toll-like (TLRs): R848/Resiquimod, ligando del TLR7/8; LPS, ligando del TLR4; e poly(I:C), ligando del TLR3. Questa analisi ha permesso di identificare gruppi di microRNA espressi specificamente in risposta a determinati stimoli e puo' risultare utile per chiarire il possibile ruolo dei microRNA nei meccanismi attraverso i quali le cellule dendritiche discriminano i diversi patogeni. La steatosi epatica di origine non-alcolica, o NAFLD, è una patologia emergente caratterizzata da un ampio spettro di condizioni epatiche: dalla semplice steatosi, alla steatoepatite con fibrosi più o meno avanzata (NASH, non-alcoholic steatohepatitis). La patologia nelle sue forme più gravi può evolvere fino alla cirrosi e all’epatocarcinoma. La NAFLD ha una complessa eziopatogenesi ancora poco chiara. Per individuare i possibili meccanismi molecolari coinvolti nello sviluppo della NAFLD abbiamo utilizzato un modello animale, capace di riprodurre i vari aspetti della patologia umana. In prticolare, in questo studio abbiamo effettuato l’analisi dei profili di espressione dei microRNA nel tessuto epatico di ratti sottoposti a diversi regimi dietetici. I nostri risultati hanno dimostrato che il trattamento con i diversi regimi ipercalorici causa un aumento significativo del peso corporeo e del fegato, e di alcuni parametri metabolici rispetto agli animali controllo, come anche differenti danni epatici. L’analisi dei microRNA ha dimostrato la significativa deregolazione di 3 microRNA down-regolati (miR-122, miR-451 e miR-27a) e 3 up-regolati (miR-200a, miR-200b e miR-429) negli animali sottoposti alle diete ipercaloriche rispetto alla dieta standard. Fra i potenziali bersagli di tali microRNA emergono alcune molecole coinvolte nel controllo dell’apoptosi e dell'infiammazione, ma soprattutto proteine del segnale intracellulare, e del metabolismo lipidico e glucidico.
Over the last decade, the discovery of microRNAs revealed a new mechanism of post-transcriptional regulation. MicroRNAs are involved in many biological processes such as development, differentiation, proliferation and cell death. Moreover, several evidences showed the pathogenic role of microRNAs in various diseases. A lot of studies used microarray technology to identify miRNAs involved in the pathogenesis, but also to obtain the expression pattern characteristic of pathology with diagnostic or prognostic assessment. These studies suggest that profiling of microRNAs may be used to understand the role they play in regulating pathophysiological processes. In this work we employed microarray technology to investigate the expression profile of microRNAs in two different experimental models: 1) an in vitro model, useful for understanding the molecular mechanisms underlying the immune response, 2) a in vivo model, suitable for studying the pathogenesis of non-alcoholic fatty liver disease, also known as NAFLD. Recently, has been explored the relationship between inflammation, innate immunity and microRNAs, which are described to be involved in regulating cellular response to microbial infection. Thus, we identified the specific expression profile of microRNAs in human dendritic cells, using an in vitro model of stimulation and activation by agonists of different Toll-like Receptors (TLRs): R848/Resiquimod, ligand of TLR7/8; LPS, ligand of TLR4; and poly(I: C), ligand of TLR3. Analysis of expression profiles identified groups of miRNAs expressed specifically in response to treatments with LPS, R848, or their combination with respect to control dendritic cells. This analysis will help to clarify their possible role in mechanisms of dendritic cells to discriminate pathogens. The non-alcoholic fatty liver disease or NAFLD is an emerging disease characterized by a wide spectrum of liver conditions from simple steatosis, steatohepatitis with or without fibrosis (NASH, non-alcoholic steatohepatitis). The etiopathogenesis of NAFLD is complex and still unclear. To identify possible molecular mechanisms involved in the development of NAFLD, we used an animal model, able to reproduce various aspects of human pathology. In this study we performed the analysis of microRNAs expression profiles in liver tissue of rats subjected to different diets. Our results showed that treatment with different ipercaloric regimens caused a significant increase in body weight and liver, and some metabolic parameters, compared to control animals, as well as different liver damage. The analysis of microRNAs showed the significant downregulation of three microRNAs (miR-122, miR-451 and miR-27a) and the up-regulation of other three microRNAs (miR-200A, miR-429 and miR-200B) in animals treated with ipercaloric diets respect to those with a standard diet. Among the potential targets of these microRNAs we found some molecules involved in the regulation of apoptosis and inflammation, but also intracellular signaling proteins, and lipid and glucose metabolism.

Rainbow trout are an important salmonid species whose poor utilization of dietary carbohydrates spurred research investigating molecular and physiological components of its glucoregulation. Among the environmental factors described to exert robust changes in glucose metabolism in rainbow trout, nutrition and social stress are among the most studied: Diets exceeding 20% of carbohydrates and chronic social stress induce hyperglycemia in adult and juvenile rainbow trout, respectively. Common to both responses is a contribution of hepatic de novo gluconeogenesis, which has been described to evade repression in response to high dietary carbohydrate content and to be stimulated in subordinate rainbow trout. Compared to previous studies investigating the regulation of hepatic gluconeogenesis at the molecular level, the recent publication of the annotated rainbow trout genome has opened novel possibilities to investigate paralogue-specific and posttranscriptional regulation of gluconeogenesis. In this thesis, I identify and describe the regulation of the novel phosphoenolpyruvate carboxykinase paralogue pck2b in rainbow trout and identify specific miRNA candidates predicted to contribute to gene paralogue-specific regulation of gluconeogenesis in nutritional and social contexts using small RNA next generation sequencing, real-time RT-PCR and in silico target prediction approaches. In nutritional and social status experiments, in silico predicted targets of differentially expressed hepatic miRNAs are enriched for gluconeogenesis regulation, suggesting a posttranscriptional component in regulating gluconeogenic transcript abundance. Differentially expressed hepatic miRNAs in both experiments comprise evolutionarily conserved and teleost-specific miRNAs, and are indicative of both environmental factor-specific and common regulation of gluconeogenesis transcripts in rainbow trout liver. Together this work provides novel comparative insight into hepatic miRNA-dependent glucoregulation and identifies several specific candidate miRNAs for future functional validation in hepatic glucoregulation in rainbow trout.

Les mutations hétérozygotes du gène codant pour le facteur de transcription HNF1B sont à l'origine d'un syndrome multisystémique complexe connu sous le nom de « Renal Cysts and Diabetes » (RCAD). Un modèle de souris généré dans notre laboratoire s'est avéré reproduire plusieurs caractéristiques de la maladie humaine. Nous avons réalisé un séquençage ARNm-microARN à différents stades de développement (E14,5 ; E15,5 ; E17,5) de ce modèle. Nous avons montré que les gènes les plus dérégulés étaient impliqués dans les processus métaboliques de transport, de lipides et d’acides organiques et étaient exprimés dans les tubules proximaux et, dans une moindre mesure, dans l’anse de Henlé et les canaux collecteurs. Nous avons sélectionné quatre microARN (miR-802, 194-2, 192 et -30a), régulés à la baisse et potentiellement contrôlés par HNF1B. Des expériences de transactivation de gène rapporteur dans des cellules HEK-293 ont montré que HNF1B était capable de transactiver la transcription de ces microARN via des sites de liaison présents dans les séquences régulatrices de ces gènes. En utilisant des microARN MIMICS nous avons par la suite montré que mir-802, mir-194-2 et mir-192 étaient capables d'inhiber l’expression d’un gène rapporteur contenant la région 3'UTR de HNF1B. L'analyse d'échantillons d'urine de 22 patients RCAD et de 22 contrôles sains a permis d'identifier 146 peptides excrétés de manière différentielle et associés au syndrome. En utilisant ces résultats dans un modèle mathématique, classificateur prédit efficacement le syndrome RCAD avec une sensibilité de 91.7% et une spécificité de 91.1% sur une large population de patients
Heterozygous mutations in the gene encoding the transcription factor HNF1B are the cause of a complex multisystem syndrome known as Renal Cysts And Diabetes (RCAD). A mouse model generated in our laboratory was shown to reproduce several features of the human disease. We performed high-throughput mRNA-microRNA sequencing at different developmental stages (E14.5, E15.5, E17.5). We showed that the most down-regulated genes were involved in transport, lipid and organic acid metabolic processes and expressed in proximal tubules and to a lesser extent in the loop of Henle and collecting ducts. We then selected four microRNAs (mir-802, 194-2, 192 and -30a), which were down-regulated and potentially controlled by HNF1B. Luciferase assays in HEK-293 cells showed that HNF1B was able to specifically transactivate in a dose response mode these microRNAs through binding HNF1B-binding sites in their regulatory promoter/enhancer upstream sequences. We subsequently showed by luciferase assays using miRNA MIMICS that mir-802, mir-194-2 and mir-192 were able to inhibit luciferase vectors containing the 3’UTR of Hnf1b. Analysis of urine samples from 22 RCAD patients and 22 healthy controls led to the identification of 146 peptides differentially excreted and associated with RCAD including a similarity regarding collagen and uromodulin fragments with the RCAD mouse model. Combining the peptides into a mathematical model we used independent cohorts of patients to validate the prediction of the RCAD syndrome. Our classifier efficiently predicted RCAD syndrome with 91.7% sensitivity and 91.1% specificity on a wide population

Biological systems, their physical structure and their functions, are built, maintained, and controlled by the activity of enzymes. Understanding how enzymes contribute to the regulation of various pathways and processes allows us to gain a deeper understanding of the entirety of the biological system. As changes in enzyme activity are often essential for the pathogenesis of multiple and varied diseases, identifying these changes represents a crucial step to both understanding the disease and preventing its progression within the individual. Enzymes’ functional output can be controlled by numerous different mechanisms, including control of transcription and translation, subcellular localisation, co-factor interactions, or chemical modification to specific amino acids. Activity-based protein profiling allows the potential for activity of target enzymes to be measured, thereby gaining a more accurate representation of the functional state of the biological system. In this work, profiling differential enzyme activity allows the discovery of previously unknown links between metabolic regulatory enzymes and infection by the hepatitis C virus (HCV). The novel probe wortmannin-yne is described and is shown to be able to report on the activity multiple kinases, including MAPK1, whose activity is dysregulated during HCV replication. Novel probes designed to target a smaller selection of kinases, phosphatidylinositol kinases, are reported and are shown to be capable of measuring HCV-induced changes to not only kinase activity but also regulatory protein-protein interactions with the phosphoinositide kinases. Lastly, the role of microRNA-27b in the HCV-induced dysregulation of lipid metabolic enzymes is examined. Three novel targets of microRNA-27b are identified, and their dysregulation is shown to have an effect on the life cycle of HCV. Altogether, this work has developed new tools for the study of metabolic enzymes and identified new avenues of investigation into the dysregulation of lipid metabolism.

Philosophiae Doctor - PhD
2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, accounting for an estimated half million deaths annually, and represents one of the major health problems. Although much is known about the cellular changes that lead to HCC, the molecular pathogenesis of HCC is not yet well understood. Gene expression studies conducted with microarray techniques and real-time PCR, suggest that tumors are characterized by an aberrant activation of signal transduction pathways involved in proliferation, survival, cell differentiation and angiogenesis. However, for HCC, these studies don’t allow the identification of a "signature" or a single specific pathway that is predominantly involved in the development and prognosis of the malignancy. Recently it has become clear that the classification and stratification of cancer can be performed not only through the analysis of gene expression, but also by analyzing the expression of microRNAs, small non-coding RNA molecules that negatively control gene expression and protein synthesis. In the present study we performed an integrated analysis of genome-wide mRNA and microRNA (miR) expression profiles to characterize the molecular events involved in the step-by-step progression (preneoplastic nodules-adenoma-early HCC-advanced HCC) of hepatocellular carcinoma (HCC) in the rat Resistant-Hepatocyte (R-H) model. Interestingly, while analysis of the transcriptome clustered together preneoplastic lesions and advanced HCC, suggesting that the majority of the genes dysregulated in HCC are already aberrantly expressed in early lesions, miRNome analysis did not co-cluster the two populations but, very interestingly, stratified the lesions according to their stage of progression to HCC. The results also unveiled specific genes/miRs, altered in the very early steps of the carcinogenic process, in the transition from adenoma to early HCC or in the progression to advanced HCC. By assessing the correlation between the expression of each miRNA and its targets, we determined that distinct pathways are aberrantly activated in different stages of the carcinogenic process. This integrated approach was also able to identify molecular events discriminating the preneoplastic lesions that will progress to HCC from those that spontaneously regress. Finally, 110 orthologous genes were almost super imposable between rat and human HCC signatures, supporting the value of the R-H model in recapitulating human liver cancer. Conclusions: This systematic analysis deciphered the molecular phenotypes of the several steps involved in the onset and progression of HCC and investigated their variations at mRNA and miR levels. In view of the striking similarity between mRNA and miRs commonly dysregulated in rat and human HCC, our results provide a valuable source for future studies and highlight promising genes, miRNAs, pathways and processes which may be useful for diagnostic or therapeutic applications.

Introduction. Alors qu’elle constitue le seul traitement curatif du cholangiocarcinome intrahépatique (CCIH), la résection reste associée à un taux de récidive supérieur à 60% et un taux de survie réelle à 5 ans inférieur à 20%. Une estimation fiable du pronostic ainsi qu’une meilleure compréhension de la biologie tumorale est essentielle pour améliorer le pronostic.Méthodes. A l’appui des données clinico-biologiques de deux larges cohortes de patients avec CCIH réséqué (MSKCC, n=189 et AFC, n=522), trois objectifs ont été explorés. Tout d’abord, définir quel modèle pronostique publié est le plus performant. Ensuite, définir la fiabilité de l’évaluation pronostique préopératoire à partir de, respectivement, l’imagerie, des microARN (miR) circulants diagnostiques et du profil génomique tumoral. Enfin, évaluer l’impact pronostique de la survenue d’événements périopératoires tels que transfusion et morbidité.Résultats. Premièrement, les nomogrammes apportaient une meilleure estimation pronostique en comparaison à la classification AJCC 7ème édition. Deuxièmement, la taille et la multifocalité tumorale sur l’imagerie préopératoire permettaient de différencier deux groupes de patients de pronostic clairement distincts (p<0,001). L’existence d’une mutation d’un gène de remodelage de la chromatine (BAP1, ARID1A, PBRM1) tendait à être associé à une survie sans récidive plus favorable qu’en l’absence de mutation (p=0,09). Alors qu’ayant un potentiel comme marqueur diagnostique circulant, miR21 et miR221 n’étaient pas associé à la survie. Troisièmement, la transfusion peropératoire n’impactait pas la survie à long terme alors que la survenue d’une complication sévère (grade Dindo-Clavien > 2) était indépendamment associée à une survie globale plus courte (p=0,002).Conclusion. Alors que les nomogrammes postopératoires apportent une meilleure estimation pronostique, le développement de modèles pronostiques préopératoires est faisable notamment à partir de l’imagerie et de marqueurs biologiques tumoraux complémentaires
Introduction. Complete resection stands as the only curative option for intrahepatic cholangiocarcinoma (IHCC). Still, prognosis remains poor after resection due to a recurrence rate over 60% leading to actual 5-year survival rates below 20%. Reliable prognostic estimation and better understanding of tumor biology would be of interest for improving IHCC prognosis.Methods. Using clinical and biological data from two large cohort of resected IHCC (MSKCC, n=189 and AFC, n=522), three objectives have been explored. First, assessing the performances of different published prognostic models. Second, defining the reliability of preoperative prognostic estimation using imaging, tumoral genomic profiling and circulating tumoral microRNA (miR). Third, evaluating the prognostic impact of perioperative events such as blood transfusion and morbidity.Results. First, nomograms displayed better prognostic accuracy over the AJCC 7th edition staging system. Second, tumor size and multifocality on preoperative imaging allowed patient stratification in groups statistically different regarding prognosis (p<0.001). Further, the presence of chromatine remodeling gene mutations (BAP1, ARID1A, PBRM1) tended towards longer recurrence-free survical (p=0,09). Some diagnostic circulating miR such as miR21 and miR221 were not associated with survival. Third, in contrast with intraoperative transfusion, the occurrence of severe morbidity (Dindo-Clavien grade > 2) was independently associated with shorter overall survival (p=0.002).Conclusion. Nomograms outperform conventional staging sytem. Preoperative prognostic estimation is feasible and reliable using imaging. Identifying new prognostic biomarkers would help refining preoperative prognostic estimation

Colorectal cancer is the third most common cancer and the leading cause of cancer deaths in Western industrialised countries. Despite recent advances in the screening, diagnosis, and treatment of colorectal cancer, an estimated 608,000 people die every year due to colon cancer. Our current knowledge of colorectal carcinogenesis indicates a multifactorial and multi-step process that involves various genetic alterations and several biological pathways. The identification of molecular markers with early diagnostic and precise clinical outcome in colon cancer is a challenging task because of tumour heterogeneity. This Ph.D.-thesis presents the molecular and cellular mechanisms leading to colorectal cancer. A systematical review of the literature is conducted on Microarray Gene expression profiling, gene ontology enrichment analysis, microRNA and system Biology and various bioinformatics tools. We aimed this study to stratify a colon tumour into molecular distinct subtypes, identification of novel diagnostic targets and prediction of reliable prognostic signatures for clinical practice using microarray expression datasets. We performed an integrated analysis of gene expression data based on genetic, epigenetic and extensive clinical information using unsupervised learning, correlation and functional network analysis. As results, we identified 267-gene and 124-gene signatures that can distinguish normal, primary and metastatic tissues, and also involved in important regulatory functions such as immune-response, lipid metabolism and peroxisome proliferator-activated receptors (PPARs) signalling pathways. For the first time, we also identify miRNAs that can differentiate between primary colon from metastatic and a prognostic signature of grade and stage levels, which can be a major contributor to complex transcriptional phenotypes in a colon tumour.

ABSTRACT In the first part of our work we focused our attention on the biological question about the differences between two pathologies: T-cell lymphoblastic leukemia and T-cell lymphoblastic lymphoma. These two diseases share many features such as immunophenotypic features, lymphoblast morphology and clinical characteristics and are differentially diagnosed only on the base of bone marrow involvement. We tried to understand whether T-cell leukemia and lymphoma are a unique pathology with a different manifestation or whether they are two different diseases. The results obtained by gene expression profiling revealed an intrinsic difference in the expression of 78 genes between T-ALL and T-LBL. In particular since these genes belong to the angiogenesis and the chemotactic response we supposed that the two malignancies have different ability to respond to several cyto- and chemokines and that T-LBL need to modulate transcription to promote angiogenesis as well as to deal with hypoxic conditions. Also by analysis of copy number we were able to identify some abnormalities that seemed to be specific for each group regardless the limited data set of patients. Although this work provides additional elements in the characterization of these two pathologies, many studies have yet to be done, especially on the comprehension of the different capability of cells to migrate and invade the bone marrow compartment. The complete understanding of the molecular characteristics of T-LBL and T-ALL represents the driving element toward the design of fully successful therapeutic approaches. The second part of the study focused on the genetic characterization of two different groups of T-ALL patients on the basis of MRD response. With the aim to find biological correlates with the outcome for HR and nonHR patients, we performed many analyses, starting from copy number analysis to microRNAs expression profiling. Furthermore, we tried to integrate all the data in order to delineate common characteristics for each group of patients. First of all, the study of copy number revealed the presence of multiple abnormalities in all patients: we found known and unknown lesions, and in some cases we were able to associate them with HR or nonHR patients. The improvement of copy number results was obtained by the study of translocations. Also in this case we found one or more translocation in the majority of patients and we identified that the most recurrent were SIL-TAL1 and TLX3 translocations. Moreover, we tested the Notch1 mutations and, as expected, about 60% of patients were mutated for Notch1, with a tendency for Notch1 mutations to be more frequent in the nonHR group. The second step was the analysis of gene and microRNAs expression. The initial unsupervised analysis between HR and nonHR group failed to distinguish the two groups; but the successively supervised analysis revealed the distribution of MR patients in an equal manner between HR and SR patients. Thus an unsupervised analysis without MR patients showed a specific pattern of expression for each group (SR and HR). The GSEA analysis performed highlighted the enrichment of two specific pathways: the mir-215/192 pathway and the methylation pathway. The results obtained by the expression profile of about 700 microRNAs in the HR and nonHR group and those achieved by the combined analysis of GEP and microRNAs suggested miR-215 and miR-107 as the most differentially expressed and provided some possible target genes of these microRNAs. Moreover, to delineate specific pattern of expression not driven by MRD but by other alterations, we used the data derived from copy number, translocation and mutational analyses to supervise genetic subgroups. Significant results were obtained for Nocth1 mutated vs non-mutated, TLX3 translocated vs non-translocated, PTEN and LEF1 deleted vs non-deleted. We also tried to integrate all the data provided by both genomic and trasncriptomic analyses to understand whether the distribution of MR and the different signature of HR and SR were correlated with specific gene lesions. SIL-TAL1 fusion gene and the deletion of LEF1 and PTEN seemed to be specific for the HR group while the TLX3-translocation seemed to be peculiar for the nonHR group of patients. In conclusion HR and nonHR patients seemed to show some peculiar lesions and patterns of expression that could be justify the different response to therapy. In summary, several high throughput methodologies have been applied to the selected subgroup of patients to study the biological correlates of the different response to therapy. By this work we tried to provide a better characterization of T-ALL and to give a way of interpretation for the different outcome of T-ALL patients.

Le bulbe olfactif (BO) des mammifères adultes est le siège d'une intense neurogenèse tout au long de la vie. L'intégration des nouveaux neurones dans le BO est alimentée par la génération continuelle de progéniteurs immatures dans la zone periventriculaire (ZPV) du ventricule latéral du cerveau antérieur. Au cours de leur différentiation, ceux-ci migrent « en chaine » de la ZPV vers le BO. Une fois dans le BO ils migrent alors radialement vers leur localisation finale et achèvent leur différentiation. Le phénotype des neurones néoformés est divers et est déterminé par la position des cellules souche dans la ZPV. Outre un intérêt spécifique, cette neurogenèse offre des perspectives uniques pour étudier la neurogenèse en général. En effet, dans ce système, les étapes successives du processus de différentiation sont distinctement séparées dans l'espace.Durant ma thèse j'ai étudié le rôle des microARN dans la neurogenèse du BO. Les microARN sont des ARN d'environs 22 nucléotides qui régulent négativement l'expression des gènes au niveau post-transcriptionnel. En utilisant des souris mutantes conditionnelles pour une enzyme clé dans la synthèse des microARN, j'ai démontré que les microARN étaient essentiels à la génération de nouveaux neurones. Par la suite, pour identifier des microARN candidats, le profil d'expression de l'ensemble des microARN durant la neurogenèse a été réalisé. Cette étude s'est faite par séquençage haut-débit des petits ARN sur un panel d'échantillons représentatifs des différentes étapes de la neurogenèse du BO et des différents compartiments de cellules souche de la ZPV
New neurons are continuously and extensively generated in the adult mammalian olfactory bulb (OB). The constant integration of new neurons into the OB circuitry is fueled by the continuous generation of immature progenitors in the periventricular zone (PVZ) of the lateral ventricle of the forebrain. Immature precursor cells leave the PVZ and migrate in interwoven chains to the OB. After arrival in the OB they migrate radially to their final positions and undergo terminal differentiation. The phenotype of these new neurons is diverse and determined by the position of the stem cells in the PVZ. Beyond its specific interest, this system of postnatal neurogenesis provides unique, advantageous properties to study neurogenesis in general, as the distinct steps of the neurogenic sequence (stem cell, amplification, migration, final differentiation) are clearly spatially separated. During my PhD I aimed to elucidate the roles of microRNA mediated regulation of gene expression in the OB neurogenesis. MicroRNAs are a class of small regulatory RNAs around 22 nucleotides in length. They act as negative regulators of gene expression on a post-transcriptional level thereby restricting protein output. Using a conditional knock-out mouse line for a key enzyme of microRNAs synthesis, I first demonstrated that microRNAs are absolutely required to complete the neuronal differentiation process. Subsequently, in order to identify candidates playing a role in neurogenesis, a miRNome profiling was performed by deep sequencing of small RNAs in tissues representative for different stem cell compartments and steps of neurogenesis

Il carcinoma epatocellulare (HCC) è il più frequente tumore maligno del fegato e rappresenta il sesto tipo di tumore più comune nel mondo. Spesso i pazienti con HCC vengono diagnosticati a stadi piuttosto avanzati, quando le uniche opzioni terapeutiche in grado di migliorarne la sopravvivenza sono la chemoembolizzazione dell'arteria epatica ed il trattamento con l'inibitore multi-cinasico, Sorafenib. In questo contesto, la scoperta del ruolo centrale dei microRNA (miRNA) nella tumorigenesi umana risulta di fondamentale importanza per lo sviluppo di nuovi marcatori diagnostici e bersagli terapeutici. I microRNA (miRNA) sono delle piccole molecole di RNA non codificante, della lunghezza di 19-22 nucleotidi, filogeneticamente molto conservati, ed esercitano un ruolo cruciale nella regolazione di importanti processi fisiologici, quali sviluppo, proliferazione, differenziamento, apoptosi e risposta a numerosi segnali extracellulari e di stress. I miRNA sono inoltre responsabile della fine regolazione dell'espressione di centinaia di geni bersaglio attraverso il blocco della traduzione o la degradazione dell'mRNA target. Studi di profiling hanno evidenziato l'espressione aberrante di specifici miRNA in numerosi tipi di tumore umano. Lo scopo del presente lavoro è stato quello di individuare un pannello di miRNA deregolati nell'epatocarcinoma umano e di caratterizzare il ruolo biologico di tre miRNA deregolati nell'HCC, al fine di individuare alcuni dei meccanismi molecolari alla base della trasformazione maligna miRNA-associata. La nostra ricerca è stata inoltre focalizzata nell'individuazione di nuovi bersagli e strumenti terapeutici, quali i microRNA, per il trattamento combinato di HCC in stadio intermedio-avanzato.

Il carcinoma epatocellulare (HCC) è il più frequente tumore maligno del fegato e rappresenta il sesto tipo di tumore più comune nel mondo. Spesso i pazienti con HCC vengono diagnosticati a stadi piuttosto avanzati, quando le uniche opzioni terapeutiche in grado di migliorarne la sopravvivenza sono la chemoembolizzazione dell'arteria epatica ed il trattamento con l'inibitore multi-cinasico, Sorafenib. In questo contesto, la scoperta del ruolo centrale dei microRNA (miRNA) nella tumorigenesi umana risulta di fondamentale importanza per lo sviluppo di nuovi marcatori diagnostici e bersagli terapeutici. I microRNA (miRNA) sono delle piccole molecole di RNA non codificante, della lunghezza di 19-22 nucleotidi, filogeneticamente molto conservati, ed esercitano un ruolo cruciale nella regolazione di importanti processi fisiologici, quali sviluppo, proliferazione, differenziamento, apoptosi e risposta a numerosi segnali extracellulari e di stress. I miRNA sono inoltre responsabile della fine regolazione dell'espressione di centinaia di geni bersaglio attraverso il blocco della traduzione o la degradazione dell'mRNA target. Studi di profiling hanno evidenziato l'espressione aberrante di specifici miRNA in numerosi tipi di tumore umano. Lo scopo del presente lavoro è stato quello di individuare un pannello di miRNA deregolati nell'epatocarcinoma umano e di caratterizzare il ruolo biologico di tre miRNA deregolati nell'HCC, al fine di individuare alcuni dei meccanismi molecolari alla base della trasformazione maligna miRNA-associata. La nostra ricerca è stata inoltre focalizzata nell'individuazione di nuovi bersagli e strumenti terapeutici, quali i microRNA, per il trattamento combinato di HCC in stadio intermedio-avanzato.

Autism Spectrum Disorder is the name for a heterogeneous group of neurodevelopmental conditions, clinically defined by: defects in social interaction and communication; fixed interests and repetitive behaviors. Molecular basis of ASD is heterogeneous and only partially known. ASD-associated variants have been characterized in hundreds of genes and separate transcriptome studies have identified points of convergence among these loci, proving that common biological processes play a role in this disorder. However, no common ASD-associated variants with large effect size, that would be appropriate for its molecular diagnosis, have been identified to date, and therefore, diagnosis just relies on clinical assessment and confirmation. Many factors, including disorders comorbid with ASD, like Tourette Syndrome, complicate ASD behavior-based diagnosis and make it vulnerable to bias. Extracellular microRNAs have attracted researchers for their potential as non-invasive tools for diagnosis, prognosis, and treatment evaluation of human diseases and disorders. Circulating miRNAs can be detected in all mammalian body fluids, from serum to saliva. Stability and general consistency of levels among individuals, along with the existence of specific expression signatures in association with both physiological and pathological conditions, make circulating miRNAs appropriate biomarkers. To investigate ASD etiology and to identify potential biomarkers to support its diagnosis, we used TLDA technology to profile serum miRNAs from ASD, TS, and TS+ASD patients and NCs (unaffected controls). Through validation assays, we demonstrated that miR-140-3p is upregulated in ASD vs: NC, TS, and TS+ASD. We found that delta Ct values for miR-140-3p and YGTSS scores are positively correlated. Our network functional analysis showed that nodes controlled by miR-140-3p, especially CD38 and NRIP1, are involved in processes convergingly dysregulated in ASD, such as synaptic plasticity, immune response, and chromatin binding. Through biomarker analysis, we proved that serum miR-140-3p can discriminate among ASD and NC, ASD and TS, and ASD and TS+ASD, showing that it could be useful to strengthen the behavior-based diagnosis of either ASD or TS+ASD, which can be challenging in some clinical cases. Among all body fluids, saliva represents the most accessible and complete source of different types of molecules that could reflect genetic, epigenetic, environmental, metabolic, emotional, and behavioral alterations in ASD. Therefore, we also used NanoString nCounter technology to profile supernatant saliva circulating miRNAs from ASD patients and NCs. Through validation assays, we demonstrated that both miR-29a-3p and miR-141-3p are upregulated in ASD saliva compared to NC one. We observed that delta Ct values for both miRNAs are correlated with neuropsychiatric scores evaluating ASD defects in social interaction and verbal communication. Target genes of these miRNAs represent main components and regulators of pathways and processes known to be dysregulated in ASD. Through biomarker performance evaluation, we proved that saliva miR-29a-3p and miR-141-3p when used in combination could be useful and non-invasive tools for discriminating ASD patients. In particular, these miRNAs could be used as supportive means for the recognition of ASD verbal and social defects. Overall, our findings suggest that profiling of circulating miRNAs in body fluids can represent an easy and innovative approach to address important biomedical issues, such as the need for biomarkers and the necessity to investigate neurodevelopmental disorders through more accessible patient biopsies. In fact, through the characterization of miRNAs in ASD serum and saliva, we identified three miRNAs that could facilitate ASD clinical assessment and that are worth being further investigated for their potential role in neurodevelopment.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that regulate gene expression at both the mRNA and protein levels. MiRNAs have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. We investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N =35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain-specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. Given that miRNAs can bind to their targets with imperfect complementarity, computational prediction of true miRNA:mRNA interactions has been difficult and therefore, functional validation of miRNA:mRNA interactions has been relatively sparse. Thus, it was the goal of this study to demonstrate biological functionality of miRNAs on their targets by evaluating transcriptional and translational levels of gene expression(real-time PCR, western blot) as well as determining miRNA target-site specificity (luciferase reporter gene assays). We investigated two miRNAs, miR-132 and miR-137, both of which have been shown to regulate neuronal function and development, and are believed to be associated with schizophrenia from two distinct avenues of research, miR-132 from expression studies and miR-137 from genetic studies. We demonstrated miR-132 down-regulates NTF3, DISC1, and GRIK5 at the transcript level and down-regulates GRIK5 at the protein level as well. Furthermore, we demonstrated miR-137 down-regulates TCF4, CACNA1C, CDK6, ANK3, and ZNF804A at the transcript level, and down-regulates TCF4, CACNA1C, and CDK6 at the protein level. Going further, we also demonstrated miR-137 binds specifically to target sites in the 3'-UTR of CACNA1C, TCF4, and CDK6, suggesting repression of these genes is directly mediated by miR-137. In total, this study provides strong evidence that miRNA dysregulation may contribute to schizophrenia pathogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs playing an important role as post-transcriptional regulators of gene expression. A growing body of evidence has shown that alterations in miRNA expression pattern are associated with several pathological processes. In particular, post-mortem brain studies support miRNA involvement in the pathophysiology of psychiatric disorders but their role as peripheral biomarkers or targets of pharmacological treatments has been scarcely investigated. Our purpose was to identify potential biological correlates of suicide and to evaluate their role in the pathogenesis and pharmacotherapy of suicide. We conducted a high-throughput profiling of miRNAs in lymphoblastoid cell lines (LCLs), not-treated or treated with lithium in vitro, derived from bipolar suicide completers (S, n=7) and non-suicidal bipolar patients (NS, n=12). Global miRNA expression was measured using nCounter® miRNA expression Assay (NanoString Technologies). In total, 28 miRNAs were differentially expressed or regulated by lithium between the two groups at a false discovery rate of 0.1. Two miRNAs were validated with quantitative Real Time-PCR (qRT-PCR): miR-4286, up-regulated in non-treated LCLs from S versus NS, and miR-186-5p, down-regulated in lithium-treated LCLs from S versus NS. Treatment of human neural progenitor cells with lithium down-regulated both miRNAs. Then, we evaluated the expression of these miRNAs in post-mortem brains from 12 suicidal bipolar subjects (Sbrain) and 13 healthy controls (Cbrain), showing an under-expression of miR-4286 in Sbrain and no differences for miR-186-5p. Finally, we performed a Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to clarify the possible role of the identified miRNAs in biological mechanisms involved in suicide. Results from GO analysis indicated that miR-4286 and miR-186-5p regulate the expression of key genes involved in neuronal processes as neurogenesis and synaptic plasticity. Whereas, KEGG pathway analysis showed that both miRNAs are involved in several biological pathways that could be related to suicide, such as TGF-beta signaling pathway and long-term potentiation pathway. In conclusion, our study suggests that miR-4286 and miR-186-5p could constitute potential biomarkers of suicide and be involved in the mechanism of action of lithium.

>Magister Scientiae - MSc
Type 2 diabetes mellitus (T2DM), a metabolic disease characterized by chronic hyperglycemia, is the most prevalent form of diabetes globally, affecting approximately 95 % of the total number of people with diabetes i.e. approximately 366 million. Furthermore, it is also the most prevalent form in South Africa (SA), affecting approximately 3.5 million individuals. This disease and its adverse complications can be delayed or prevented if detected early. Standardized diagnostic tests for T2DM have a few limitations which include the inability to predict the future risk of normal glucose tolerance individuals developing T2DM, they are dependent on blood glucose concentration, its invasiveness, and they cannot specify between T1DM and T2DM. Therefore, there is a need for biomarkers which could be used as a tool for the early and specific detection of T2DM. MicroRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies show that dysregulation of microRNAs may lead to various diseases including T2DM, and thus, may be useful biomarkers for disease detection. Therefore, identifying biomarkers like microRNAs as a tool for the early and specific detection of T2DM, have great potential for diagnostic purposes. The main focus of this investigation, therefore, is the early detection of T2DM by the identification and validation of novel biomarkers. Furthermore, based on previous studies, the aim of the investigation was to identify differentially expressed miRNAs as well as identify their potential target genes associated with the onset and progression of T2DM. An in silico approach was used to identify miRNAs found to be differentially expressed in the serum/plasma of T2DM individuals. Three publically available target prediction software were used for target gene prediction of the identified miRNA. The target genes were subjected to functional analysis using a web-based software, namely DAVID. Functions which were clustered with an enrichment score > 1.3 were considered significant. The ranked target genes mostly had gene ontologies linked with “transcription regulation”, “neuron signalling, and “metal ion binding”. The ranked target genes were then split into two lists – an up-regulated (ur) miRNA targeted gene list and a down-regulated (dr) miRNA targeted gene list. The in silico method used in this investigation produced a final total of 4 miRNAs: miR-dr-1, miR-ur-1, miR-ur-2, and miR-ur-3. Based on the bioinformatics results, miR-dr-1 and its target genes LDLR, PPARA and CAMTA1, seemed the most promising miRNA for biomarker validation, due to the function of the target genes being associated with T2DM onset and progression. The expression levels of the miRNAs were then profiled in kidney tissue of male Wistar rats that were on a high fat diet (HFD), streptozotocin (STZ)-induced T1DM, and non-diabetic control rats via qRT-PCR analysis. The hypothesis was that similar miRNA expression would be found in the HFD kidney samples compared to serum expression levels of the miRNA obtained from the two databases, since kidneys are involved in cleansing the blood from impurities. This hypothesis proved to be true for all miRNAs except for miR-ur-2. Additionally, miR-ur-1 seemed the most significant miRNA due to it having different expression ratios for T1DM and T2DM (i.e. -7.65 and 4.2 fold, respectively). Future work, therefore, include validation of the predicted target genes to the miRNAs of interest i.e. miR-dr-1: PPARA and LDLR and miR-ur-1: CACNB2, using molecular approaches such as the luciferase assays and western blots.

In the Helicobacter hepaticus (Hh) colitis model, Hh infection of either wild-type mice treated with a blocking antibody to the IL-10 receptor (anti-IL-10R) or IL-10 KO mice results in intestinal inflammation associated with inflammatory Th1 and Th17 responses. Recent findings suggest that altered expression of the post-transcriptional gene regulators microRNAs contribute to pathogenic immune responses during intestinal inflammation. Here, examination of microRNA expression during Hh colitis showed that microRNAs are differentially expressed in the inflamed large intestine of Hh+ IL-10 KO mice compared to uninfected controls, both at the tissue-level and the CD4+ T-cell level. Kinetic examination of the cecal and colonic levels of miR-155, miR-326 and miR-132 (microRNAs previously shown to augment Th1 and/or Th17 responses) demonstrated that miR-155 was up-regulated and miR-326 and miR-132 were down-regulated at different time points post Hh infection. Furthermore, the change in expression of these microRNAs coincided with inflammation development. Microarray profiling of large intestine LP CD4+ T cells revealed that two microRNAs were significantly up-regulated (miR-21a and miR-31) and seven microRNAs were significantly down-regulated (miR-125a, miR-125b, miR-139, miR-181a, miR-192, miR-30a and miR-467c) in colitic IL-10 KO mice compared to uninfected controls. The anti-inflammatory cytokine IL-10 is necessary for protection against intestinal inflammation. Here, the phenotype of IL-10-producing LP CD4+ T cells was examined in a non-inflammatory immune response (Hh+ WT mice) and in an inflammatory immune response (Hh+/anti-IL-10R-treated WT mice). Compared to uninfected controls, the Hh+ mice showed a slight expansion in IL-10+ IL-17A+FoxP3+/- cells whereas the Hh+/anti-IL-10R-treated mice showed a significant expansion in all the IL-10+ LP CD4+ T cells co-expressed both inflammatory cytokines IL-17A and/or IFN-γ and/or the Treg transcription factor FoxP3. The experiments carried out in this thesis demonstrate that the profile of two important regulatory factors, microRNAs and IL-10, is markedly different in LP CD4+ T cells from the colitic setting compared to uninfected controls.

BACKGROUND Maternal obesity (MO) is expanding worldwide, contributing to Gestational Diabetes Mellitus (GDM) prevalence. The altered placental function and intrauterine environment of MO and GDM are associated with adverse maternal and fetal outcomes, with short- and long-term complications. Growing evidence suggests that both MO and GDM are characterized by placental autophagy disturbances, mitochondrial (mt) dysfunctions and epigenetic alterations, which could contribute to the pathogenesis of pregnancy-related metabolic diseases. For these reasons the aim of this work was to characterize placental autophagy and mitochondrial bioenergetics, and maternal plasmatic microRNAs profile in the context of MO and GDM. METHODS 60 women with spontaneous singleton pregnancies delivering by elective Caesarean section were enrolled: 25 normal-weight (NW), 22 obese without comorbidities (OB/GDM(-)), 13 obese with GDM (OB/GDM(+)). The expression of antioxidant metabolism markers and autophagy-related genes expression was evaluated in placental villous tissue by Real-time PCR with SYBR green chemistry. In placenta, we also quantified ATP by CellTiter-Glo Luminescent Cell Viability Assay adapted to placental villous tissue, and mt Complexes I-V protein content by Western Blot experiments. Moreover, in a subgroup of 7 NW, 6 OB/GDM(-) and 6 OB/GDM(+), microRNA (miRNA) profiling in maternal plasma at delivery was performed with miRCURY LNA Serum/Plasma miRNA Focus PCR Panel (Qiagen). Statistical analysis and bioinformatics predictive tools: SPSS v.27 (IBM); GeneGlobe (Qiagen); miTALOS v.2; miRPath v.3. RESULTS AND CONCLUSIONS Placental antioxidant genes expression (CAT, GPX1, GSS, GSR) tended to decrease, the pro-autophagic ULK1 increased and the chaperone-mediated autophagy (CMA) regulator PHLPP1 decreased in OB/GDM(-) vs NW. On the other hand, PHLPP1 expression increased in OB/GDM(+) vs OB/GDM(-). These preliminary findings showed placental alterations of autophagy in MO and GDM, paving the way for further analyses aimed at elucidating the role of placental autophagy in metabolic disorders during pregnancy. Moreover, placentas of OB/GDM(-) and OB/GDM(+) women had reduced ATP level vs NW. All OB showed decreased Complex I vs NW. Furthermore, lower expression of Complexes III and V was found in OB/GDM(+) group (vs NW; vs NW and OB/GDM(-), respectively), along with a reduced fetal/placental weight ratio (i.e. placental efficiency) vs NW. These results might suggest impaired placental mt function in obese subjects, especially with GDM. Sexual dimorphism in terms of the above-mentioned placental molecular markers was also investigated, with intriguing results suggesting that females and males might respond differently to insults and/or have differential, sex-dependent compensatory mechanisms in order to deal with the perturbed environment. MiRNA profiling in maternal plasma highlighted patterns of significantly differentially expressed miRNAs between the three groups. In particular, OB/GDM(-) vs NW differed in the expression of 4 miRNAs, OB/GDM(+) vs NW of 1, and OB/GDM(+) vs OB/GDM(-) of 14. Pathway enrichment analysis by miRPath and miTALOS revealed many pathways associated with the set of miRNAs of each comparison, among which the most interesting and relevant in our context were related to nutrients and hormones metabolism, inflammation and oxidative stress. Indeed, miRNAs could be promising biomarkers of metabolic alterations in MO and GDM. Nevertheless, future investigations are needed to further deepen the pregnancy epigenetic landscape.

Orientador: Erika Veruska Paiva Ortolan
Resumo: Introdução: Por volta de oitenta por cento dos acidentes envolvendo ingestão de cáusticos ocorrem em crianças. A estenose cáustica do esôfago é a sua principal complicação com grande morbidade. Lesões neoplásicas esofágicas podem desenvolver-se como uma complicação tardia desta estenose com um tempo médio de aparecimento entre o acidente e o desenvolvimento neoplásico de 15 a 30 anos. Considerando este risco, biopsias seriadas do esôfago são recomendadas com o objetivo de detecção precoce de displasias. Assim, um conhecimento abrangente da relação biológica entre cáusticos e neoplasia esofágica é de grande importância na identificação de novos biomarcadores que possibilitariam tratamento precoce. MicroRNAs (miRNAs) são RNAs pequenos, não codificadores de proteínas que regulam importantes processos celulares e têm se mostrado como robustos biomarcadores. O perfil global de expressão de miRNAs nesta população, seguida da identificação dos miRNA-alvo, pode levar à identificação da presença e magnitude do dano ao material genético em amostra de tecido esofágico obtido de pacientes portadores de estenose cáustica. Objetivos: Determinar o perfil global da expressão de miRNAs em células da mucosa esofágica de crianças portadoras de lesões por ingestão de cáusticos, com o objetivo de identificar miRNAs como biomarcadores associados a tumorigênese esofágica nesta população específica. Materiais e Métodos: Vinte e sete amostras esofágicas fixadas em formalina e embebidas em parafina (F... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Background: 80% of the caustic ingestions occur in children. Esophageal stricture is a major chronic complication with great morbidity. Esophageal neoplasms may develop as a late complication of caustic injury with a mean time between caustic ingestion and cancer development of 15-30 years. Serial biopsies are recommended aiming early detection of premalignant changes. Thus a comprehensive knowledge of biological relation between caustic and esophageal cancer is of major importance to identify the biomarkers that could enable an early treatment. MicroRNAs (miRNAs) are small non-coding RNA molecules and regulate key cellular processes during tumorigenesis and have been demonstrated as an useful diagnostic, prognostic and predictive biomarkers. Global miRNA expression profiling analysis in this population, followed by the identification of miRNA target genes, may lead to the identification of the presence and magnitude of damage to genetic material in a sample of esophageal tissue obtained from patients with caustic stenosis. Objectives: We aimed to identify global microRNA (miRNA) expression changes in cells of the esophageal mucosa from children with caustic lesions compared to paired macroscopic and microscopically normal esophageal tissue. Patient and Methods: 27 formalin fixed, paraffin embedded (FFPE) esophageal samples from 15 patients were divided into two groups according to the time elapsed after the injury (Group A: less than 5 years, Group B: more than 5 years). Tho... (Complete abstract click electronic access below)
Doutor

Background: Heart Transplantation (HTX) is the only curative treatment available for patients with end-stage heart failure (HF).During the first year post-transplantation more than 25% of patients will go through rejection episodes and will face the risk of developing rejection with consequent graft dysfunction with an increased morbidity and mortality. Preventing and treating acute rejection is the most central task for clinicians working with transplanted patients. The ISHLT 2005 and 2013 working formulations defined the histopathologic profile of three types of rejection: Cellular (ACR) Humoral (AMR) and Mixed (MIX). Nowadays serial endomyocardial biopsies (EMB) at decreasing intervals during the first year after transplantation and laboratory tests, such as Donor Specific Antibody (DSA) measurements, remain the gold-standard in diagnosing and monitoring acute rejection but they are morbid and prone to artefacts of sampling, interpretation and testing methodologies. Therefore this histopathological assessment needs integrative new biomarkers to characterize risk stratification for outcomes in heart transplantation. To date, the exact mechanisms involved in rejection after solid transplantation are not completely understood, so investigating process that contribute to acute allograft rejection and find effective biomarkers to diagnose, monitoring and predicting rejection will be of great value for the development of improved anti-rejection strategies. The advent of sequencing technology such as Next Generation Sequencing (NGS) is changing medical genomics by accelerating new disease biomarkers discovery. MicroRNAs (miRNAs) are small non-coding RNA molecules (19-24 nucleotides), highly conserved, which regulate genes expression at the post transcriptional level. Aim: The aim of this study is to identify MicroRNA (miRNAs) expression profile in the first year after heart transplantation (HTX) with Next Generation Sequencing (NGS) technology in formalin fixed paraffin-embedded (FFPE) endomyocardial Biopsies (EMBs), to characterize the three different types of allograft rejection classified as Cellular, Humoral and Mixed. Methods: Two groups of pts. were included: a study group of 19 pts. and a validation group of 14 pts. For each patient we selected the the first formalin fixed paraffin-embedded (FFPE) monitoring endomyocardial biopsies (EMBs) positive for each types of rejection. We excluded presensitized patients (pts) with previous implantation of Left Ventricular Assistance Device (LVAD) and with previous infections. EMBs were examined for the presence of rejection according to updated international classification criteria (ISHLT 2005 and 2013).The EMBs were classified in four groups: Acute Cellular Rejection (ACR) with 12 pts ACR: >=2R, pAMR:0, DSA: Neg ; Mixed with 6 pts ACR: >=2R, pAMR>1 (i+), DSA: Pos; Antibody Mediated Rejection (AMR) with 5 pts ACR: 0, pAMR>1 (i+), DSA: Pos; Control with 10 pts : ACR:0, pAMR:0, DSA: Neg. Small RNA fraction from the study group was sequenced with NGS Ion Proton in order to define the expression of mature miRNAs. We performed subsequent analysis with edgeR package comparing in pairs the groups to identify differentially expressed miRNAs in the different rejections. We selected 13 microRNAs according to bionformatic analysis as possible biomarckers and they have been confirmed by qRT-PCR in all the pts. With multivariate logistic regression analysis we created unique miRNA signatures as predictive model of each rejection. Moreover in situ PCR was carried out on the same EMBs to detect miRNAs expression and localization in cell types within the EMBs. Results: The identification of the best method of extraction for short non coding RNAs in FFPE EMBs was the first result I achieved. I tested different methods in house and commercial available kits and I modified the protocols to obtain good quality and adeguate quantity of RNA from FFPE tissue of small EMBs for the downstream application. With NGS we obtained and analysed more than 2257 mature microRNAs in all the biopsies of the study group. The three types of rejection and control groups were compared in pair with the un-supervised analysis showing a typical profile for each group of differentially expressed miRNAs; in particular: Mixed vs AMR: only 2 miRNAs overexpressed in the Mixed group suggesting a similarity between the two types. ACR vs AMR: 18 miRNAs overexpressed and 2 miRNAs under-expressed in the ACR. Mixed vs ACR : 7 miRNAs underexpressed and 39 miRNAs over-expressed in the ACR group. The analysis revealed that there are de-regulated microRNAs between the three rejections confirming our hypothesis that microRNAs can characterize the three pathological conditions. MiRNAs have been selected for further evaluation and validation, based on the number of reads resulting by NGS, on their highly significant FDR (< 0.05) or fold change, p-value and their involvement in relevant processes related to rejection as shown by a bioinformatic analysis based on validated target genes and reported in public databases such as TarBase (version 6.0) (111) , miRTarBase (112) , miRWalk (113), miRecords (114), DIANA-microT-CDS (115) , miRmap (116), miRDB (117) , TargetScan (118), and miRanda (119). At the end we selected 13 microRNAs. To validate the NGS data through qRT-PCR we enrolled other EMBs from 14 pts selected according to our criteria and we tested on all the 33 EMbs, both the study and validation cohort, the selected microRNAs. The validation analysis has shown a similar expression pattern for all microRNAs in particular: 6 hsa-miRNAs: 29c-3p/-29b-3p/199a-3p/190a-5p/27b-3p/302b-3p can differentiate all rejections compared to controls; 3 hsa-miRNAs: 31-5p/144-3p/218-5p are peculiar of AMR and MIX compared to control and ACR 2 hsa-miRNAs: 451a/208a-5p identify MIX compared to controls. Using miRNAs expression as co-variate and disease status as dependent variable we created logistic regression models: MIX:(miR-208a ,126-5p, 135a-5p); ACR:(miR-27b-3p, 29b-3p,199a-3p, 208a, 302b-3p); AMR: ( miR-208a, 29b-3p, 135a-5p, 144-3p) identifying with high specificity and sensitivity each types of rejection. Finally with in situ PCR we detected some of these microRNAs in different cell types: miR-29b-3p was mostly expressed in smooth muscle cells in ACR; miR-144-3p was expressed in macrophages and in endothelial cells; moreover the expression of this microRNA in macrophages was predominant and diffuse in the ACR compared to AMR. miR-126-5p was expressed in ACR and AMR samples not only in in endothelial cells but also in Cardiomyocytes and smooth muscle cells. For MicroRNA 451a we found a co-localization of signal in endothelial cells and in lymphocytes. Conclusions: This study demonstrate that MicroRNAs can be obtained easily from FFPE tissues, miRNAs differentially expressed are involved in pathophysiological mechanisms of rejection such as immune system cells cycle regulation and proliferation, , inflammatory pathways NFkB mediated and endothelial remodelling. According to our results the miRNAs up or down expressed modulate these pathways in a way peculiar for the different type of rejection. The regressive models might represent a powerful diagnostic tool and in situ detection of the miRNAs casts new light on the pathophysiological mechanisms of rejection. Moreover the expression of MiRNAs 144-3p, 126-5p, 29b-3p and 451a identified by in situ PCR in endothelial cells, smooth muscle and inflammatory cells are diagnostic and are potential pharmacological targets for rejections.
Contesto: Il trapianto di cuore è l'unico trattamento curativo disponibile per i pazienti con insufficienza cardiaca allo stadio terminale. Durante il primo anno dopo il trapianto più del 25% dei pazienti può subire episodi di rigetto e affrontare il rischio di sviluppare rigetto con conseguente disfunzione dell’ organo trapiantato con un aumento della morbilità e mortalità. Prevenire e trattare il rigetto acuto è l’ obiettivo principale per i medici che lavorano con pazienti trapiantati. Le linee guida ISHLT 2005 e 2013 hanno definito il profilo istopatologico di tre tipi di rigetto: Cellulare (ACR) Humoral (AMR) e Mixed (MIX). Al giorno d'oggi le biopsie endomiocardiche seriali (EMB) a intervalli decrescenti durante il primo anno dopo il trapianto e gli esami di laboratorio, come le misurazioni di anticorpi donatore specifici (DSA), rimangono parametri di riferimento nella diagnosi e nel monitoraggio del rigetto acuto, ma sono soggetti a artefatti dovuti alle metodologie di campionamento, interpretazione e test. Pertanto questa valutazione istopatologica necessita di nuovi biomarcatori integrativi per caratterizzare la stratificazione del rischio nel rigetto da trapianto di cuore. Ad oggi, i meccanismi esatti coinvolti nel rigetto dopo il trapianto non sono completamente compresi, quindi la ricerca sui processi che governano i meccanismi di rigetto e la scoperta di biomarcatori efficaci per diagnosticare, monitorare e prevedere il rigetto sarà di grande valore per lo sviluppo e miglioramento delle terapie contro il rigetto. L'avvento della tecnologia di sequenziamento come Next Generation Sequencing (NGS) sta cambiando la genomica medica accelerando la scoperta di nuovi biomarcatori di malattie. I microRNA (miRNA) sono piccole molecole di RNA non codificanti (19-24 nucleotidi), altamente conservate, che regolano l'espressione dei geni a livello post-trascrizionale. Obiettivo: Lo scopo di questo studio è identificare il profilo di espressione di MicroRNA (miRNA) nel primo anno dopo il trapianto di cuore (HTX) con la tecnologia Next Generation Sequencing (NGS) in biopsie endomiocardiche (EMB) fissate in formalina e incluse in paraffina (FFPE), per caratterizzare i tre diversi tipi di rigetto da trapianto di cuore classificati come Cellulare, Umorale e Misto. Metodi: due gruppi di pazienti (pz.) sono stati inclusi: un gruppo di studio di 19 pz. e un gruppo di validazione di 14. Per ogni paziente abbiamo selezionato la prima biopsia endomiocardica (EMB) fissata in formalina ed inclusa in paraffina (EMB) per ogni tipo di rigetto. Abbiamo escluso i pz. presensibilizzati con precedente impianto del dispositivo di assistenza ventricolare sinistro (LVAD) e con precedenti episodi di infezione. Le biopsie sono state esaminate per la presenza di rigetto secondo i criteri di classificazione internazionali aggiornati (ISHLT 2005 e 2013). Abbiamo quindi individuato quattro gruppi: Acute Cellular Rejection (ACR) con ACR a 12 punti:> = 2R, pAMR: 0, DSA: Neg; Misto con 6 pts ACR:> = 2R, pAMR> 1 (i +), DSA: Pos; Reiezione mediata da anticorpi (AMR) con 5 punti ACR: 0, pAMR> 1 (i +), DSA: Pos; Controllo con 10 punti: ACR: 0, pAMR: 0, DSA: Neg. La piccola frazione di RNA della coorte di studio è stata sequenziata con NGS Ion Proton per definire l'espressione dei miRNA maturi. Abbiamo eseguito un'analisi successiva con edgeR confrontando a coppie i gruppi per identificare i miRNA espressi differenzialmente nei diversi rigetti. Abbiamo selezionato 13 microRNA secondo l'analisi bionformatica come possibili biomarcatori i quali sono stati confermati da qRT-PCR in tutti i pz. Con l'analisi di regressione logistica multivariata abbiamo identificato gruppi univoci di miRNA come modelli predittivi specifici per ciascun rigetto. Inoltre, la PCR in situ è stata eseguita sulle stesse EMBs per rilevare l'espressione e la localizzazione dei miRNA nei tipi di cellule all'interno delle EMBs. Risultati: l'identificazione del miglior metodo di estrazione di microRNA da EMBs FFPE è stato il primo risultato che ho raggiunto. Ho testato diversi metodi sia manuali che kit commerciali e ho modificato i protocolli per ottenere una buona qualità e una quantità adeguata di microRNA per l'applicazioni successive. Con NGS abbiamo ottenuto e analizzato oltre 2257 microRNA maturi in tutte le biopsie del gruppo di studio. I tre tipi di gruppi di controllo e di rigetto sono stati confrontati in coppia con l'analisi non supervisionata che mostra per ciascun gruppo un profilo tipico di miRNA differenzialmente espressi; in particolare: Misto vs AMR: solo 2 miRNA sovraespressi nel gruppo Misto suggeriscono una somiglianza tra i due tipi di rigetto. ACR vs AMR: 18 miRNA sovraespressi e 2 miRNA sottoespressi nell'ACR. Mixed vs ACR: 7 miRNAs non sovraespressi e 39 miRNA sovraespressi nel gruppo ACR. L'analisi ha rivelato che ci sono microRNA de-regolati tra i tre tipi di rigetto confermando la nostra ipotesi che i microRNA possano caratterizzare le tre condizioni patologiche. I MiRNA sono stati selezionati per un'ulteriore valutazione e convalida, in base al numero di reads risultanti da NGS, sulla loro FDR significativa (<0,05), fold change, p-value e il loro coinvolgimento in processi rilevanti correlati al rigetto come mostrato dalle analisi bioinformatiche basate su geni target validati e riportati in database pubblici come TarBase (versione 6.0) (111), miRTarBase (112), miRWalk (113), miRecords (114), DIANA-microT-CDS (115), miRmap (116) , miRDB (117), TargetScan (118) e miRanda (119). Alla fine abbiamo selezionato 13 microRNA. Per validare i dati NGS tramite qRT-PCR abbiamo arruolato altri EMBs da 14 pz. selezionati in base ai nostri criteri e abbiamo testato su tutte le 33 EMbs, sia quelle della coorte di studio che quelle della coorte di validazione, i microRNA selezionati. L'analisi di validazione ha mostrato un pattern di espressione simile per tutti i microRNA in particolare: 6 hsa-miRNA: 29c-3p / -29b-3p / 199a-3p / 190a-5p / 27b-3p / 302b-3p possono differenziare tutti i rigetti rispetto a controlli; 3 hsa-miRNA: 31-5p / 144-3p / 218-5p sono peculiari di AMR e MIX rispetto al controllo e ACR 2 hsa-miRNA: 451a / 208a-5p identificano MIX rispetto ai controlli. Usando l'espressione di miRNA e la condizione patologica come variabili dipendenti abbiamo creato modelli di regressione logistica: MIX: (miR-208a, 126-5p, 135a-5p); ACR: (miR-27b-3p, 29b-3p, 199a-3p, 208a, 302b-3p); AMR: (miR-208a, 29b-3p, 135a-5p, 144-3p) che identificano con alta specificità e sensibilità ciascun tipo di rigetto. Infine con PCR in situ abbiamo rilevato alcuni di questi microRNA in diversi tipi di cellule: miR-29b-3p era principalmente espresso nelle cellule muscolari lisce in ACR; miR-144-3p era espresso nei macrofagi e nelle cellule endoteliali; inoltre l'espressione di questo microRNA nei macrofagi era predominante e diffusa nell'ACR rispetto all'AMR. Il miR-126-5p è risultato espresso in campioni ACR e AMR non solo nelle cellule endoteliali ma anche nei cardiomiociti e nelle cellule muscolari lisce. Per il MicroRNA 451a abbiamo trovato una co-localizzazione del segnale nelle cellule endoteliali e nei linfociti. Conclusioni: Questo studio dimostra che i microRNA possono essere ottenuti facilmente dai tessuti fissati in formalina e inclusi in paraffina, i miRNA differenzialmente espressi sono coinvolti in meccanismi patofisiologici del rigetto quali regolazione e proliferazione del ciclo cellulare del sistema immunitario, vie infiammatorie mediate da NFkB e rimodellamento endoteliale. Secondo i nostri risultati, i miRNA sovra o sotto espressi hanno mostrato una modulazione di questi processi in un modo peculiare per ciascun tipo di rigetto. I modelli di regressione logistica identificati potrebbero rappresentare un potente strumento diagnostico e il rilevamento in situ dei miRNA getta nuova luce sui meccanismi patofisiologici del rigetto. Inoltre l'espressione di MiRNA 144-3p, 126-5p, 29b-3p e 451a identificati mediante PCR in situ in cellule endoteliali, cellule muscolari lisce e infiammatorie è diagnostica e costituisce un potenziale bersaglio farmacologico contro il rigetto da trapianto di cuore.

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains ix, 87 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 75-87).

Mammalian expression systems, particularly Chinese hamster ovary (CHO) cells, are used industrially for the production of biopharmaceuticals. Despite improvements over the last few decades with regard to the maximum cell concentrations obtainable and the amount of recombinant protein produced from such expression systems, there is still the potential to engineer and manipulate mammalian cells to generate more robust host cells that grow faster, for longer and produce higher product yields with less heterogeneity. A number of recent studies have reported that mRNA translation is a key limitation in terms of defining the product yield from in vitro cultured mammalian cells. One mechanism by which mRNA translation is controlled is via the expression of microRNAs (miRNA). miRNA are endogenous single stranded non-coding pieces of RNA of approximately 22 nt in length that can either stall mRNA translation or activate cleavage of target mRNAs. This study set out to investigate the role of specific miRNAs on determining CHO cell growth and recombinant protein productivity. Initially, Lonza Biologics undertook an extensive miRNA profiling study using the mercury LNA microarray, and found that -56 miRNA were significantly differentially expressed during the course of a state-of-the-art fed-batch bioreactor culture. In this study we determined the profile of four target miRNA (miR-15b, miR- 16, miR-21 and miR-34c) throughout batch culture in model monoclonal antibody producing CHO cell lines and the effect of transiently over-expressing the corresponding pre-miRNA individually or together. The different miRNA were expressed differentially throughout batch culture, their profile correlating with either growth or productivity characteristics. There was no relationship between pri-microRNA amounts and the mature microRNA amounts observed in the model cell lines investigated. Overexpressing/ knocking-up of the human pre-microRNAs in CHO cells resulted in an increase in the observed mature microRNA amounts. In some cases this over-expression resulted in increased recombinant protein expression and enhanced cell specific productivity when transiently expressed in model cB72.3 monoclonal antibody producing cells. Thus, the transient over-expression of pre-microRNAs resulted in increased product yields and/or changes in growth characteristics. In particular the over-expression of microRNA-34c resulted in an increase in transgene expression. Interestingly, when multiple microRNA were over-expressed transiently together an additive or synergistic effect in growth or product titre was observed in a number of cases. This is one of the first such studies to investigate the effect of manipulating multiple miRNA on recombinant protein synthesis from mammalian cells. When individual pre-microRNAs were stably expressed the amount of mature micro RNA observed in the resulting stable pools was increased. However, the amount of mature microRNA observed could be further increased by transient transfection of the stable pools with a plasmid for the expression of the target pre-microRNA suggesting that the maturation machinery for the generation of mature microRNA was not saturated. Ultimately this work has provided an improved understanding of specific microRNAs in CHO cells and their subsequent effect on industrially relevant phenotypes, providing informed strategies to manipulate microRNAs in order

MicroRNAs (miRNAs) are non-coding RNAs whose role in gene regulation has been discovered and gradually clarified in recent years. The action of miRNAs, like that of other types of non-coding RNA, is based on their physical interaction with gene transcripts (mRNA), the inhibition or decrease of their translation and, as a consequence, of their protein production. As a result, miRNAs biological effect depends on the nature of their targets. Currently, there is not a complete map of the targets for each of the 721 human known miRNAs, but several studies have already shown a direct involvement of many of them in the pathogenetic mechanisms of a large number of diseases including cancer. Hepatitis B (HBV) and C (HCV) viruses infect hundreds of thousands people worldwide every year and cause necroinflammatory diseases which, though in different ways and rates, can cause the development of a chronic disease able to progress and leading to the development of fibrosis, cirrosis and hepatic cancer (HCC); the latter is one of the most aggressive human cancers and its early diagnosis and therapy are still unsolved issues. Thus, many reasons are pressing the scientific community towards the research of novel strategies useful to effectively improve the health management of the disease. As regards miRNAs, several studies have been carried out showing the existence of a clear pattern of specific miRNA expression in hepatic cancer tissue, while their role in the onset and progression of hepatic disease remains largely unclarified and, at the moment, not much studied. Starting from these assumptions, the aims of this study are: - The definition of a protocol for the analysis of miRNAs in tissue samples. - The monitoring of miRNA expression and the identification of those which correlate with hepatic disease progression from viral hepatitis to HCC. - The identification of the roles of these molecules through the research of their specific targets, thanks to the development and the application of bioinformatic tools. The results of this study can be summarized in two main sections: first, the definition of miRNAs expression profiles in the different phases of the hepatic disease progression, starting from slight fibrosis to the onset of hepatocellular carcinoma, obtained with realtime PCR and microarray experiments. Second, the development of a bioinformatic tool able to predict miRNA targets, which has subsequently been applied in order to investigate their function. In conclusion, this study has allowed the identification of some miRNAs whose expression was correlated to distinct phases of hepatic disease and to specific clinical parameters (grading-staging, etiology). Moreover, we investigated the pathways that could be involved by miRNAs differential expression, with the aim and the hope that they will be useful as diagnostic and prognostic instruments and will bring to the development of new therapeutic strategies based on miRNA transfer or on the inhibition of their expression.
I microRNA (miRNA) sono RNA non codificanti il cui ruolo di regolazione dell'espressione genica è stato scoperto e via via delucidato in anni recenti. L'azione dei miRNA, non dissimilmente da quella di altre tipologie di RNA non codificanti, si esprime nell'interazione fisica specifica con trascritti genici (mRNA) e nell'impedimento o comunque nella riduzione quantitativa della produzione, a partire da questi ultimi, di proteine. L'effetto biologico del miRNA dipende pertanto dalla natura del suo bersaglio, chiamato target. Non è al momento disponibile una mappa completa dei target di ciascuno dei 721 miRNA umani noti ma numerosi studi hanno già  dimostrato per molti di essi un coinvolgimento diretto nei meccanismi patogenetici di numerose patologie, fra cui il cancro. I virus dell'epatite B (HBV) e C (HCV) si trasmettono ogni anno a centinaia di migliaia di persone nel mondo e sono causa di malattie necroinfiammatorie epatiche le quali, pur con percentuali e modalità  differenti, possono portare allo sviluppo di una malattia cronica in grado di progredire nel lungo periodo provocando lo sviluppo di fibrosi, cirrosi ed epatocarcinoma (HCC); quest'ultimo è uno dei tumori umani più aggressivi e tanto la sua diagnosi precoce, quanto ancor più la sua terapia, permangono alquanto problematiche. Molte ragioni, quindi, spingono la comunità  medico-scientifica alla ricerca di nuove strategie che possano portare a effettivi miglioramenti nella gestione sanitaria di questa patologia. Nel caso dei miRNA, sono già  stati effettuati numerosi studi mirati a dimostrare l'esistenza di un preciso schema della loro espressione nel tessuto epatico canceroso, mentre il ruolo che essi possono ricoprire nelle fasi precedenti all'insorgenza del cancro rimane ancora largamente incompreso e, al momento, poco studiato. Partendo da tali presupposti, gli obiettivi di questo studio sono: - La definizione di un protocollo per l'analisi dei miRNA in campioni tissutali. - Il monitoraggio dell'espressione dei miRNA e l'identificazione di quelli che risultino correlati con la progressione dalla patologia epatica ad eziologia virale all'HCC. - L'individuazione del ruolo di tali miRNA attraverso la ricerca dei target specifici, tramite lo sviluppo e l'impiego di metodi bioinformatici. I risultati dello studio si possono riassumere in due sezioni principali: in primo luogo la definizione dei profili di espressione dei miRNA nelle diverse fasi della progressione della malattia epatica a partire dalla fibrosi lieve fino all'insorgenza dell'epatocarcinoma, conseguita grazie all'impiego di metodiche di Real-time PCR e microarray. In secondo luogo lo sviluppo di un metodo bioinformatico di predizione dei geni target, il quale è stato successivamente impiegato nella ricerca della funzione di tali miRNA attraverso la definizione dei loro bersagli. In conclusione, questo studio ha permesso di individuare alcuni dei miRNA caratterizzati dal fatto di avere un'espressione correlata a fasi distinte di malattia epatica e a specifici parametri clinici (grading-staging; eziologia). Sono state inoltre investigate le pathway nelle quali essi potrebbero essere coinvolti, con l'obiettivo e l'auspicio che possano rivelarsi utili come strumenti diagnostici e prognostici e per lo sviluppo di nuove terapie basate sul trasferimento di miRNA o sull'inibizione della loro espressione.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Neurotrophins and their receptors are key molecules in the development of the
nervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor
NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive
form (150 kDa) and a truncated non-catalytic form (50 kDa). The two
variants show different 3'UTR regions, indicating that they might be differentially
regulated at the post-transcriptional level. In this work we explore how
microRNAs take part in the regulation of full-length and truncated NTRK3,
demonstrating that the two isoforms are targeted by different sets of microRNAs.
We analyze the physiological consequences of the overexpression of some of the
regulating microRNAs in human neuroblastoma cells. Finally, we provide
preliminary evidence for a possible involvement of miR-124 - a microRNA with no
putative target site in either NTRK3 isoform - in the control of the alternative
spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.
Las neurotrofinas y sus receptores constituyen una familia de factores cruciales
para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función
principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual
se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo
tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas
no comparten la misma región 3'UTR, lo que sugiere la existencia de una
regulación postranscripcional diferente. En el presente trabajo se ha explorado
como los microRNAs intervienen en la regulación de NTRK3, demostrando que las
dos isoformas son reguladas por diferentes miRNAs. Se han analizado las
consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando
células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del
microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la
regulación de represor de splicing PTBP1.