It was previously demonstrated that miR-199a was down-regulated in testicular germ cell tumor (TGCT) partly caused by hypermethylation of its promoter. More detailed analyses showed that miR-199a-5p, one of its two derivatives, suppressed TGCT invasiveness and proliferation via directing targeting PODXL and MAFB. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this project we identified DNMT3A, the de novo methyltransferase, as a direct target of miR-199a-3p using a 3’-UTR reporter assay. In NT2 (NTera 2) and HT (Hs 1.Tes) cells, miR-199a-3p regulated the expression of endogenous DNMT3A (both DNMT3A1 and DNMT3A2 isoforms), especially DNMT3A2 isoform. In clinical samples, the expression of DNMT3A2 and miR-199a-3p were reciprocally regulated. However, DNMT3A did not regulate miR-199a expression. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. MiR-199a-3p could restore the expression of APC and MGMT via de-methylation in their promoters. Our studies demonstrated the dysregulation of miR-199a-3p in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potential therapeutic use of synthetic miR-199a-3p oligonucleotides as effective demethylation agent in the treatment of TGCT. However, since DNMT3A expression did not regulate miR-199a expression, the mechanism of promoter DNA hypermethylation of miR-199a in TGCT needs further investigation. / MiR-199a is encoded by two loci in the human genome, namely, miR-199a-1 on chromosome 19 and miR-199a-2 on chromosome 1. Another microRNA, miR-214, also locates on chromosome 1. Previous study revealed that it is co-transcribed with miR-199a-2, which is directed by miR-199a-2 promoter. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this project, it was determined that miR-199a and miR-214 were concordantly expressed in TGCT. Silencing of DNMT1 increased the expression of miR-199a and miR-214, accompanied by de-methylation in the promoters of miR-199a-1/2. Overexpression of TP53 down-regulated the expression of DNMT1 and increased the expression of mature miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which caused the down-regulation of miR-199a, miR-214 and TP53, as well as the up-regulation of DNMT1 and PSMD10 in TGCT. These observations partly explain the mechanism of promoter DNA hypermethylation in miR-199a in TGCT. They also suggest a potential therapeutic approach by targeting the miR-199a/miR-214/PSMD10/TP53/DNMT1 regulatory network in the treatment of TGCT. / 先前的研究證實miR-199a在睾丸生殖細胞腫瘤 (簡稱睾丸癌) 中是低表達的，部分歸因於其啟動子區域過度甲基化。對其功能研究發現miR-199a能抑制睾丸癌細胞的生長，侵襲和轉移，且miR-199a的抑癌屬性應歸功於它的兩個衍生物之一miR-199a-5p。然而，miR-199a的另一個衍生物miR-199a-3p在睾丸癌中的生物學功能仍然在很大程度上是未知的。此研究中，DNMT3A被鑒定為miR-199a-3p的直接靶定目標。在NT2和HT細胞中，miR-199a-3p能調控內源性DNMT3A(DNMT3A1和DNMT3A2)的表達水準，尤其是DNMT3A2。在臨床樣本中，DNMT3A2的表達水準與miR-199a-3p的表達水準呈負相關。但DNMT3A並不能調控miR-199a的表達水準。進一步研究顯示過表達miR-199a-3p能減少APC和MGMT啟動子區域甲基化而恢復其表達水準。研究證實異常表達的miR-199-3p可能在睾丸癌的癌變過程中發揮作用，並提出一個潛在的治療方案，即使用miR-199a -3p作為有效的去甲基化藥劑治療睾丸癌。然而睾丸癌中導致miR-199a啟動子區域過度甲基化的機制有待進一步研究。 / 在人類基因組中，miR-199a-1(位於19號染色體)和miR-199a-2(位於1號染色體)都編碼miR-199a。同時miR -214也位於1號染色體，研究表明miR-214與miR-199a-2由miR-199a-2啟動子介導共同轉錄，但miR-199a和miR- 214共同表達的生物學意義仍未知。此研究中，miR-199a和miR-214在睾丸癌中的表達呈現一致性。沉默DNMT1後miR-199a和miR-214的表達水準顯著提高，並伴隨著miR-199a-1/2啟動子區域的DNA去甲基化。在NT2細胞中。過表達TP53能下調DNMT1的表達水準，同時上調miR-199-3p/5p和miR- 214的表達水準。此外，過表達miR -214能導致PSMD10表達水準的下調以及TP53表達水準的上調。綜上所述，我們提出一個miR-199a/miR-214/PSMD10/TP53/DNMT1自我調控網路，此調控通路能引起睾丸癌中miR-199a，miR-214和TP53表達水準的下調，以及DNMT1和PSMD10表達水準的上調，且部分解釋睾丸癌中miR-199a啟動子區域過度甲基化的機制，同時該調控網路可作為治療睾丸癌的一個潛在靶點。 / Chen, Bifeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 103-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 20, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

La Lipoprotéine lipase (LPL) est une enzyme essentielle de la lipolyse intravasculaire dont la régulation est complexe. La découverte des miRs, régulateurs de l'expression posttranscriptionnelle des gènes via leurs interactions avec les régions 3' non traduite (3'UTR), apporte de nouvelles perspectives pour la compréhension de la régulation de la LPL et de ses gènes régulateurs. Nous présentons à travers deux études, l'implication des microARNs (miRs) dans la régulation de la LPL et d'un de ses gènes activateurs APOA5. Dans le premier travail, nous avons mis en évidence la création d'un site de liaison fonctionnel du miR-485-5p par expliquons ainsi le mécanisme potentiellement impliqué dans l'association de ce polymorphisme aux hypertriglycéridémies sévères et modérées en population générale. Dans un second travail, nous avons identifié un haplotype de la LPL, incluant la mutation p.Ser474Ter (rs328) et sept single nucleotide polymorphisme (SNPs) de la région 3'UTR, significativement associés à une diminution des triglycérides (TG) plasmatiques en population générale. Nous avons ensuite démontré la fonctionnalité des sept SNPs de la région 3'UTR par la suppression de sites de liaison de plusieurs miRs. Ainsi ces résultats suggèrent que l'association du variant p.Ser474Ter (rs328) à la triglycéridémie pourrait au moins partiellement être liée à son déséquilibre de liaison avec les sept SNPs fonctionnels de la région 3'UTR. Nos travaux sont parmi les premiers à mettre en évidence l'implication des miRs dans la régulation de la LPL et de ses gènes régulateurs chez l'homme. Ils permettent ainsi d'accroitre la connaissance des mécanismes impliqués dans la régulation de la lipolyse intravasculaire. Enfin, ils éclairent les mécanismes fonctionnels mis en jeu par deux polymorphismes significativement associés à la triglycéridémie / The lipoprotein lipase (LPL) is a key enzyme which regulates plasma triglycerides (TG) intravascular lipolysis involving a complex regulation. The microRNAs (miR) are implicated in gene post-transcriptional regulation through their interaction with the 3’untranslated region (3’UTR). Their discovery provides new insights in the understanding of the LPL regulation and its regulator genes. We present two works regarding the implication of miRs in the regulation of the LPL and one of its activator APOA5. First, we identified a functional miR-485-5p binding site creation induced by the minor C allele of the c.*158C>T (rs22667882) located in APOA5 3’UTR.We therefore provide an explanation of the mechanism potentially involved in this polymorphism association with both mild and severe hypertriglyceridemia in general population. In a second work, we identified a LPL haplotype harboring p.Ser474Ter (rs328) polymorphism and seven single nucleotide polymorphisms (SNPs) located in the 3’UTR. This haplotype is significantly associated with lower plasma triglycerides (TG) concentration in general population. We demonstrated that the SNPs located in the 3’UTR induce several functional miRs binding-site suppressions that could lead to an increase of LPL expression. Finally, p.Ser474Ter association with triglyceridemia could be at least partially explained by its strong linkage disequilibrium with these functional 3’UTR SNPs. These works are amongst the first studies to bright to light the miRs implication in the regulation of LPL or its regulator genes in human. They provide a better knowledge of the mechanisms involved in intravascular lipolysis. Finally, they also explain the functional mechanisms of two polymorphisms, significantly associated with the plasma TG concentration

La dystrophie musculaire facioscapulohumérale (FSHD) est la troisième myopathie la plus fréquente en Europe. La maladie atteint progressivement les muscles du visage, des bras et des jambes. Les symptômes apparaissent dans la majorité des cas avant 20 ans et la maladie, souvent douloureuse, provoque fréquemment un handicap majeur qui nécessite de se déplacer en fauteuil roulant. J'ai démontré que la protéine KLF15 est surexprimée dans les tissus des patients FSHD et dans les myoblastes cultivés in vitro et prélevés sur des patients FSHD. Des résultats préliminaires indiquent que la surexpression du facteur KLF15 pourrait être expliquée par le stress oxydant induit par DUX4 et la surexpression de certains facteurs de myogenèse induits par DUX4c. J'ai démontré que KLF15 interagit directement avec les répétitions D4Z4 et contrôle leur fonction d'activation de transcription. Ainsi KLF15 sert de médiateur entre les répétitions D4Z4 et deux gènes dans la région 4q35: FRG2 et DUX4c ce que explique la surexpression de ces deux gènes dans la FSHD. La surexpression du gène DUX4c, homologue du DUX4, perturbe le programme de différentiation musculaire en activant certains facteurs de myogenèse (myomiRs ou microRNAs myogéniques). En somme, la découverte du rôle du facteur KLF15 dans la FSHD met en évidence une boucle de rétrocontrôle positif qui relie la surexpression du facteur KLF15 avec la surexpression des gènes codés dans la région 4q35. Le rôle central du facteur KLF15 dans ce nouveau modèle de la maladie permet d'envisager une nouvelle piste thérapeutique pour la dystrophie FSHD basé sur l'inhibition du facteur KLF15. / Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We here report that the enhancer within the D4Z4 repeat binds the Krüppel-like factor KLF15. KLF15 was found to be upregulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15 binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.

Au cours de ma thèse, j’ai étudié la contribution de miR-205 dans le développement du cancer du sein. MiR-205 a été choisi suite à l'analyse comparative de l'expression du miRome entre la lignée « normale » MCF10A et une lignée cancéreuse dérivée MCF10A-CA1a. J’ai démontré que l’expression de miR-205 augmente durant la tumorigenèse tandis que miR-205 est non détectable dans la lignée cellulaire ayant un potentiel métastatique. De plus, j’ai montré que les cellules souches du cancer du sein expriment miR-205, contrairement à la population non souche. En utilisant des cultures de cellules épithéliales 3D, j’ai corrélé la fonction tumorigène de miR-205 à la répression de l'apoptose et non à une prolifération accrue. De plus, le niveau d'expression de la E-Cadhérine dépend de la quantité de miR-205 dans les différentes lignées cellulaires de MCF10A. Les études de perte de fonction suggèrent que la E-Cadhérine est impliquée dans le phénotype acini miR-205-Dépendant, en corrélation avec la transformation de cellules épithéliales du sein. L’ensemble de ces résultats met en lumière la complexité et la duplicité des miRNA durant le processus de cancérisation. Ce type d’étude ouvre des perspectives d’utilisation des miRNA dans le cadre des diagnostics et/ou thérapeutiques. / During the time I was working on my thesis, I aimed to understand the role of miR-205 in breast cancer development. MiR-205 was chosen from the comparative analysis of total micro-RNAs expression in non-Transformed and tumorigenic cell lines of the MCF10A breast epithelial cell model. I demonstrated the complexity of miR-205 functions during breast epithelial cell transformation by showing miR-205 overexpression in transformed non-Invasive cell lines and miR-205 down-Regulation in cell line with metastatic potential. Moreover, we demonstrated increased level of miR-205 expression in breast cancer stem cells in comparison with non-Stem cells. Using 3D cultures of breast epithelial cells, I succeeded to correlate the tumorigenic function of miR-205 with its role in modulation of acinar size, and to attribute it to the apoptosis repression but not increased proliferation. Further, I was able to show that miR-205 exercises its oncogenic functions via targeting ZEB1, an inhibitor of E-Cadherin. Indeed, E-Cadherin expression level depends on the amount of miR-205 in different MCF10A cell lines. Downregulating E-Cadherin restored normal acinar morphology in miR-205 expressing cells, consistent with E-Cadherin being involved in the miR-205-Dependent acini phenotype that correlates with tumorigenic breast epithelial cell transformation.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.

Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.

Eukaryotic gene expression is a complex process that can be controlled at the level of transcription, post-transcription, translation, or post-translation. In recent years there has been growing interest in understanding the role of the 3'-untranslated region (UTR) in post-transcriptional regulation. The 3'-UTR contains many regulatory sequences, including microRNA (miR) target sites and AU-rich elements (AREs). Although a relatively recent discovery, miRs have been shown to downregulate target gene expression and have important roles in regulating many cellular processes, including cellular growth. Cellular growth consists of two distinct states, proliferation and quiescence. The proliferative state consists of G1, S, G2, and M phases while quiescence is the G0 phase. In response to mitogenic stimuli, quiescent cells enter the proliferative cycle and may transit back to the quiescent state. Reentry into quiescence is essential to prevent aberrant proliferation as well as to protect the cellular life span. Cells that remain in quiescence for an extended period of time lose their ability to proliferate. It has been shown that the redox status of the cells may regulate quiescence and proliferative capacity since overexpression of SOD2 protects the proliferative capacity of quiescent cells. We hypothesized that the redox environment regulates proliferative capacity through miR expression and regulation of miR targets. Early results showed treatment with hydroxytyrosol (HT), an olive-derived catechol, was able to protect the proliferative capacity of quiescent normal human fibroblasts. HT was shown to use hydrogen peroxide and produce superoxide in a catechol-semiquinone-quinone redox cycle. Interestingly, HT also induced SOD2 expression. Further results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular growth states. Results from a Q-RT-PCR and dual luciferase-3'-UTR reporter assays identified ARID4a (AT-Rich Interacting Domain 4a, also known as RBP1) and CCL5 (C-C motif Ligand 1) as targets for miR-302. Ionizing radiation, that is well known to induce oxidative stress and delay cell cycle progression, decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide, SOD2 overexpressing cells, and HT treated cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as a redox-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.

microRNAs are single-stranded small RNAs that function as post-transcriptional regulators of gene expression. We are studying the mir-44 family, specifically mir-44 and mir-45, which have identical sequence. Loss of mir-44 and mir-45 results in defects that suggest that the mir-44 family acts to negatively regulate the MAPK pathway. The MAPK pathway regulates sex myoblast migration, a process which is required for normal egg laying. We hypothesized that the mir-44 family of microRNAs is necessary for normal sex myoblast migration and subsequent formation of the functional egg laying structure in the hermaphrodite. We created a mutant that had mutations in both mir-44 and mir-45 and a transgene that expresses GFP in the sex myoblast cells. Then we observed the migration and division of the sex myoblasts in wild-type and mutant worms using fluorescence microscopy. In all cases, the mutant worms displayed a greater percent difference from average sex myoblast migration and division. However, a two-tailed two-proportions z-test found no significant difference between wild type and mutant sex myoblast migration (p=0.9148), nor in mutant sex myoblast division along the axial (p=0.4205) and sagittal (p=0.3583) planes of the body. This allows us to conclude that mir-44 and mir-45 are unlikely to be responsible for the migration nor division of the sex myoblasts, and the defects are likely due to interference with a different biological mechanism.

CD4+ T cells are critical in the fight against parasitic, bacterial, and viral infections, but are also involved in many autoimmune and pathological disorders. Ligation of the T Cell Receptor (TCR) is the primary signal required for T cell activation proliferation, differentiation and cytokine release. Upon TCR activation, several kinases and adaptor proteins are assembled at the TCR/linker for activation of T cells (LAT) signaling complexes, a process indispensable for optimal signal transduction. One important group of proteins recruited to the TCR/LAT complexes is the GRB2 family of adaptors. Due to their role in mediating signaling complexes, the GRB2 family of adaptors are critical for development, proliferation, and survival of diverse cell types. These proteins have been linked to the initiation and progression of numerous pathological conditions including diabetes, asthma/allergy, and solid and hematopoietic malignancies. Therefore, it is essential to characterize and understand the complete functions of these proteins for the generation of safe and efficient targeting treatments for diseases mediated by these proteins. In T cells, GRB2 and its homologs, GADS and GRAP, are crucial for the propagation of signaling pathways through the TCR and adaptor protein LAT. These proteins recruit distinct sets of proline-rich ligands to LAT thereby inducing multiple signaling pathways such as MAP kinase activation, calcium influx and cellular adhesion. However, the role of GRB2 family members in controlling TCR and LAT mediated signaling in mature human T cells is not completely understood. Moreover, the relative role of GRB2 family members in the extent and timing of the recruitment of SH3 domain ligands to the LAT complex is unknown. Our hypothesis is that these proteins recruit distinct sets of ligands to the LAT complex that can drive differential downstream signaling events. As presented in CHAPTER III, we developed microRNA and shRNA targeting viral vectors to effectively inhibit the expression of GRB2 and GADS in human CD4+ T cells to examine the role of these adaptors in mature human T cells. We also established optimized protocols for high efficacy retro or lentiviral transduction of human T cell lines, activated and "hard-to-transduce" non-activated primary human CD4+ T cells. In CHAPTER IV, we demonstrate the requirement for GRB2 in TCR-induced IL-2 and IFN-γ release. The defects in cytokine release in the absence of GRB2 were attributed to diminished formation of LAT signaling microclusters, which resulted in reduced MAP kinase activation, calcium flux and PLC-γ1 recruitment to LAT signaling clusters. Overall, the data presented in this chapter demonstrate that the ability of GRB2 to facilitate protein clustering is as important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. In CHAPTER V, we describe the role for GADS in mediating TCR-induced IL-2 and IFN-γ production. GADS was critical for the recruitment of SLP-76 and PLC-γ1 to the LAT complex and subsequent calcium influx. We also show, in contrast to the current paradigm, that recruitment of GADS/SLP-76 complexes to LAT is not required for TCR-mediated adhesion and cytoskeletal arrangement. Overall, our studies reveal novel mechanisms for the role of GRB2 family members in TCR-mediated signaling. They also provide insight into the mechanisms that regulate growth factor, cytokine and insulin receptors. Importantly, studies presented in this thesis will help us understand the mechanisms of T cell activation and highlight potential new therapies for T cell-mediated diseases, including leukemia, lymphomas, autoimmune disorders and cardiovascular disease.