<p>CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. </p><p>Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1&alpha;, MIP-1&alpha;P, MIP-1&beta;), interferon gamma (IFN-&gamma;), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1). </p><p>Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant. </p><p>This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-speci&#64257;c memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-speci&#64257;c anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.</p> / Dissertation

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs. MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation. Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life.

In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below.I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate.Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate in response to any stalled ribosome.I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.

HuR est une protéine liant l’ARN, qui contrôle l’expression des gènes au niveau post-transcriptionnel. Dans le cytoplasme, HuR module la stabilité et la capacité de traduction des ARNm sur lesquels elle se fixe. Nos résultats montrent que HuR est surexprimée dans le carcinome hépatocellulaire (CHC) humain et dans des lignées de CHC en culture. HuR est anormalement retrouvée dans le cytoplasme des cellules hépatiques tumorales, et participe à leur prolifération. En combinant l’analyse globale des gènes régulés par l’extinction d’HuR, celle des ARNm liés à HuR et celle du transcriptome des CHC humains, nous avons identifié 2 gènes dont l’expression est régulée par HuR. Ces gènes sont sous-exprimés dans les tissus de CHC et participent à la mise en place du phénotype cancéreux (résistance à l’apoptose, prolifération cellulaire, invasion,...). / HuR is a RNA binding protein that controls gene expression at post-transcriptional level. In the cytoplasm, HuR modulates the stability and capacity of mRNA translation upon which it binds. Our results show that HuR is overexpressed in hepatocellular carcinoma (HCC) and in human HCC cell lines in culture. HuR is abnormally found in the cytoplasm of liver tumor cells, and contribute to their proliferation. By combining the global analysis of genes regulated by the extinction of HuR, the mRNAs associated with HuR and the transcriptome of human HCC, we identified two genes whose expression is regulated by HuR. These genes are under-expressed in HCC tissues and participate in the development of cancerous phenotype (resistance to apoptosis, cell proliferation, invasion ,...).

In humans, the functional regulation of facilitative glucose transporters (GLUTs) by insulin plays a central role in the maintenance of glucose homeostasis. The insensitivity of tissues to this regulation results in diabetes mellitus, however, the underlying mechanisms remain largely unknown. To establish Caenorhabditis elegans (C. elegans) as a model system to study the mechanisms of insulin regulation of GLUTs because of the well-conserved insulin/IGF-like signaling (IIS) and many unique advantages of this organism, we functionally characterized 9 candidate genes of human GLUT homologues in C. elegans based on their sequence homologies to GLUTs. We found that FGT-1 is the only functional GLUT homologue with the ability to transport 2-deoxy-D-glucose (2DG) in Xenopus oocytes. FGT-1 mediated 2DG transport could be inhibited by the GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM, which is smaller than the Km values of human GLUT1 and GLUT4. In addition to glucose, FGT-1 could also transport mannose, galactose, and fructose. Using a FGT-1::GFP fusion construct under the control of the 5 kb promoter sequence of the fgt-1a gene, FGT-1 was shown to be ubiquitously expressed in C. elegans tissues and cells, including the digestive tract, neurons, and body wall muscle. Two FGT-1 alternative splicing isoforms, FGT-1A and FGT-1B, showed similar transport activity and tissue localization. To study the function of FGT-1 and its regulation by IIS, the changes in several phenotypes that are known to be regulated by IIS were observed in FGT-1-knockdown worms or null strains in the presence or absence of IIS activity. FGT-1 knockdown resulted in fat accumulation but had no effects on dauer formation or brood size in both wild-type and daf-2 (insulin receptor) gene mutant strains. However, the function of FGT-1 in animal growth and aging was dependent on the IIS background, suggesting IIS regulation of FGT-1 function. Consistently, FGT-1 mediated glucose uptake was almost completely defective in the daf-2 and age-1 (PI3 kinase) mutants, and phloretin could only marginally inhibit 2DG uptake in these strains. This defect was only partially related to the approximately 60% decrease in FGT-1 protein levels in these mutants, suggesting the involvements of both post-transcriptional and post-translational regulatory mechanisms. We also found that OGA-1, an O-GlcNAcase, is essential for the function of FGT-1, implying possible regulation of FGT-1 function by glycosylation. In summary, our study has established C. elegans as a powerful model to study the mechanism by which insulin regulates glucose transporters and has provided insights into the mechanism of defective glucose uptake by tissues in patients with diabetes.

La famille des protéines IMPs (IGF2 mRNA binding proteins) compte trois membres IMP1, 2 et 3. Les IMPs participent au développement embryonnaire. IMP1 et IMP3 sont considérées comme des protéines oncofoetales. En effet, malgré leur faible expression dans les tissus adultes, elles se retrouvent fortement surexprimées dans des cellules tumorales. Malgré la forte homologie entre les membres de la famille, les IMPs présentent des différences fonctionnelles qui restent très mal comprises jusqu’à présent. De nombreuses études montrent que la protéine IMP3 est très abondante dans de nombreux cancers tels que les carcinomes utérin, rénal, pulmonaire, les hépatocarcinomes et les rhabdomyosarcomes. Ces dernières années, IMP3 est devenu un marqueur de mauvais pronostique pour les patients atteins de cancer. Au cours de ma thèse j’ai principalement travaillé sur une lignée cellulaire de rhabdomyosarcomes (RMS). Les RMS sont des tumeurs principalement pédiatriques mais qui peuvent survenir à tout âge. En outre, la moitié des patients atteints des RMS meurent dans l'année suivant leur rechute et 90% des patients meurent dans les cinq ans suivant leur rechute. De nouvelles approches thérapeutiques sont absolument nécessaires. Mon sujet de thèse consiste à comprendre par quels mécanismes moléculaires les IMPs participent au processus oncogénique des RMS embryonnaires (eRMS). Pour cela, je me suis intéressée à la régulation des cyclines par les IMPs. Dans le cadre de mon projet, j’ai étudié l’effet des IMPs sur trois cyclines différentes : D1, D3 et G1. J’ai montré qu’IMP3, à la différence des deux autres, est capable de contrôler l’expression des cyclines D1, D3 et G1 dans les eRMS, ainsi que dans huit autres lignées de cancer humain différentes. Cette régulation a également des effets sur le cycle cellulaire des eRMS, expliquant l’importance d’IMP3 dans les cancers. Par diverses approches biochimiques, j’ai démontré que, sur les trois IMPs, seule IMP3 est très enrichie dans le noyau des eRMS, dans lequel elle forme des complexes avec les ARNm des CCND1, D3 et G1. Les différents résultats obtenus suggèrent un modèle selon lequel ces interactions au sein du noyau semblent indispensables à la régulation de la traduction des trois cyclines en protégeant leurs ARNm du complexe de silencing RISC (RNA induced silencing complex) et constituent donc la clé du mécanisme par lequel IMP3 contrôle la prolifération des cellules cancéreuses. / RNA-binding proteins of the IMP family (IGF2 mRNA-binding proteins 1-3) are key post-transcriptional regulatory factors of gene expression. They are known to control cell motility, adhesion, and proliferation. In our previous work, we show that all three IMP proteins can directly bind the mRNAs of cyclins D1, D3, and G1 (CCND1, D3, and G1) in vitro. Nevertheless, only IMP-3 regulates their expression in a significant manner in vivo, thus controlling proliferation of a number of human cancer cell lines. Importantly, the nuclear localization of IMP-3 is essential for the post-transcriptional regulation of the expression of CCND1, CCND3, and CCNG1 (CCNs). To elucidate the molecular mechanisms of IMP-3- specific regulation, we have identified its protein partners in human embryonic rhabdomyosarcoma (RMS) cells. We now show that in the nucleus and in the cytoplasm, IMP-3 interacts with a number or RNA-binding nucleocytoplasmic proteins, including DHX9, PTBP1, NF90, NF110, HNRNPA1, HNRNPA2/B1 and HuR. These IMP-3 partners have a dramatic impact on the protein levels of the cyclins. Interestingly, the decrease of CCNs protein synthesis in IMP-3 depleted cells can be fully reversed by down-regulating the key proteins of RNAi machinery, such as AGO2 and GW182. These findings suggest that IMP-3- dependent RNP complexes pre-assembled in the nucleus can protect their target mRNAs from cytoplasmic RNAi-dependent repression in human cancer cells.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

The expression of numerous plant mRNAs is attenuated by RNA sequence elements located in the 5' and 3' untranslated regions (UTRs). For example, in plants and many higher eukaryotes, roughly 35% of genes encode mRNAs that contain one or more upstream open reading frames (uORFs) in the 5' UTR. For this dissertation I have analyzed the pattern of conservation of such mRNA sequence elements. In the first set of studies, I have taken a comparative transcriptomics approach to address which RNA sequence elements are conserved between various families of angiosperm plants. Such conservation indicates an element's fundamental importance to plant biology, points to pathways for which it is most vital, and suggests the mechanism by which it acts. Conserved motifs were detected in 3% of genes. These include di-purine repeat motifs, uORF-associated motifs, putative binding sites for PUMILIO-like RNA binding proteins, small RNA targets, and a wide range of other sequence motifs. Due to the scanning process that precedes translation initiation, uORFs are often translated, thereby repressing initiation at the an mRNA's main ORF. As one might predict, I found a clear bias against the AUG start codon within the 5' untranslated region (5' UTR) among all plants examined. Further supporting this finding, comparative analysis indicates that, for ~42% of genes, AUGs and their resultant uORFs reduce carrier fitness. Interestingly, for at least 5% of genes, uORFs are not only tolerated, but enriched. The remaining uORFs appear to be neutral. Because of their tangible impact on plant biology, it is critical to differentiate how uORFs affect translation and how, in many cases, their inhibitory effects are neutralized. In pursuit of this aim, I developed a computational model of the initiation process that uses five parameters to account for uORF presence. In vivo translation efficiency data from uORF-containing reporter constructs were used to estimate the model's parameters in wild type Arabidopsis. In addition, the model was applied to identify salient defects associated with a mutation in the subunit h of eukaryotic initiation factor 3 (eIF3h). The model indicates that eIF3h, by supporting re-initation during uORF elongation, facilitates uORF tolerance.

<p>MicroRNAs are key post-transcriptional regulators that have been found to play critical roles in the regulation of cellular functions. There is an emerging concept that microRNAs may be just as essential for fine-tuning physiological functions and responding to changing environments and stress conditions as for viability or development. In this dissertation, two studies are presented: The first study demonstrates a role for microRNA in the regulation of oxidative stress response in erythroid cells and the functional consequences of dysregulated microRNA expression in Sickle Cell Disease (SCD) pathobiology. The second study examines a functional role for microRNA in the cellular response to changes in cellular iron concentration. Together these studies illustrate the scope of importance of microRNAs in the coordination of cellular responses to diverse stresses. </p><p>Homozygous Sickle Cell (HbSS) erythrocytes are known to have reduced tolerance for oxidative stress, yet the basis for this phenotype has remained unknown. Here we use erythrocyte microRNA expression profiles to identify a subset of HbSS patients with higher miR-144 expression and more severe anemia. We reveal that in K562 erythroid cells and primary erythroid progenitor cells, miR-144 directly regulates NRF2, a central regulator of cellular response to oxidative stress, and modulates the oxidative stress response. We further demonstrate that increased miR-144 is associated with the reduced NRF2 levels, decreased glutathione regeneration, and attenuated antioxidant capacity found in HbSS erythroid progenitors, thereby providing a mechanism for the reduced oxidative stress tolerance and increased anemia severity seen in HbSS patients. </p><p>The post-transcriptional regulation of the IRP2 regulon in the cellular response to iron deficiency is well characterized. Here we examine the potential role for microRNA-mediated regulation in the coordinated response to cellular iron deficiency.</p> / Dissertation

<p>The ability of iron (Fe) to easily transition between two valence states makes it a preferred co-factor for innumerable biochemical reactions, ranging from cellular energy production, to oxygen transport, to DNA synthesis and chromatin modification. While Fe is highly abundant on the crust of the earth, its insolubility at neutral pH limits its bioavailability. As a consequence, organisms have evolved sophisticated mechanisms of adaptation to conditions of scarce Fe availability. </p> <p>Studies in the baker's yeast Saccharomyces cerevisiae have shed light into the cellular mechanisms by which cells respond to limited Fe-availability. In response to Fe-deficiency, the transcription factors Aft1 and Aft2 activate a group of genes collectively known as the Fe-regulon. Genes in this group encode proteins involved in the high-affinity plasma membrane Fe-transport and siderophore uptake systems, as well as Fe-mobilization from intracellular stores and heme re-utilization. Concomitant with the up-regulation of the Fe-regulon, a large number of mRNAs encoding Fe-dependent proteins as well as proteins involved in many Fe-dependent processes are markedly down regulated. Thus, in response to low Fe-levels the cell activates the Fe-uptake and mobilization systems, while down-regulating mRNAs involved in highly Fe-demanding processes leading to a genome-wide remodeling of cellular metabolism that permits the funneling of the limiting Fe to essential Fe-dependent reactions. </p> <p>The Fe-regulon member Cth2 belongs to a family of mRNA-binding proteins characterized by an RNA-binding motif consisting of two tandem zinc-fingers of the CX8CX5CX3H type. Members of this family recognize and bind specific AU-rich elements (AREs) located in the 3'untranslated region (3'UTRs) of select groups of mRNAs, thereby promoting their rapid degradation. In response to Fe-limitation, Cth2 binds ARE sequences within the 3'UTRs of many mRNAs encoding proteins involved in Fe-homeostasis and Fe-dependent processes, thereby accelerating their rate of decay. </p> <p>Work described in this dissertation demonstrates that the Cth2 homolog, Cth1, is a bona fide member of the Fe-regulon, binds ARE-sequences within the 3'UTRs of select mRNAs and promotes their decay. Cth1 and Cth2 appear to be only partially redundant; Cth1 preferentially targets mRNAs encoding mitochondrial proteins, while Cth2 promotes the degradation of most of Cth1 targets in addition to other mitochondrial and non-mitochondrial Fe-requiring processes. The coordinated activity of Cth1 and Cth2 results in dramatic changes in glucose metabolism. In addition, experiments described in this dissertation indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE-mediated regulation by the Cth1 and Cth2 proteins, creating an auto- and trans-regulatory circuit responsible for differences in their expression. Finally, work described here demonstrates that Cth2 is a nucleocytoplasmic shuttling protein and that shuttling is important for the early determination of cytosolic mRNA-fate.</p> / Dissertation