<p>The cellular microRNA (miRNA) pathway has emerged as an important regulator of host-virus interactions. While miRNAs of viral and cellular origin have been demonstrated to modulate viral gene expression and host immune responses, reports detailing these activities in the context of mammalian retroviruses have been controversial. Using modern, high-throughput small RNA sequencing we provide evidence that the spumaretrovirus bovine foamy virus expresses high levels of viral miRNAs via noncanonical biogenesis mechanisms. In contrast, the lentivirus human immunodeficiency virus type 1 (HIV-1) does not express any viral miRNAs in a number of cellular contexts. Comprehensive analysis of miRNA binding sites in HIV-1 infected cells yielded several viral sequences that can be targeted by cellular miRNAs. However, this analysis indicated that HIV-1 transcripts are largely refractory to binding and inhibition by cellular miRNAs. In addition, we demonstrate that HIV-1 exerts minimal perturbations on cellular miRNA profiles and that viral replication is not affected by the ablation of mature cellular miRNAs. Together, these data demonstrate that the ability of retroviruses to encode miRNAs is not broadly conserved and that lentiviruses, particularly HIV-1, have evolved to avoid targeting by cellular miRNAs.</p> / Dissertation

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

miRNAs are important post-transcriptional regulators of gene expression which play vital roles in the arrays of physiological processes, including skin and hair follicle (HF) development. In this study, the role for miR-214 in the skin and HF development and their postnatal physiological regeneration was investigated. miR-214 exhibits discrete expression patterns in the epidermis and HF in developing and postnatal skin, and is highly expressed in the epithelial stem cells and their lineage-committed progenies. The effects of miR-214 on HF morphogenesis and cycle progression were evaluated by using doxycyclineinducible miR-214 transgenic mice (K14-rtTA/TRE-miR-214). Keratinocyte specific miR-214 overexpression during skin embryogenesis resulted in the partial inhibition of HF induction and formation of the HF reduced in size producing thinner hair. Overexpression of miR-214 in telogen skin caused retardation of the anagen progression and HF growth. Inhibitory effects of miR- 214 on HF development and cycling were associated with supressed activity of stem cells, reduced proliferation in the hair matrix, and altered differentiation. miR-214 induced complex changes in gene expression programs in keratinocytes, including inhibition of cyclins and cyclin-dependent kinases and several essential components of Wnt, Edar, Shh and Bmp signalling pathways, whereas β-catenin acts as a novel conserved miR-214 target. Indeed, the inhibitory effects of miR-214 on HF development were rescued by intracutaneous delivery of pharmacological Wnt activator. Thus, this study demonstrated that by targeting β-catenin and, therefore, interfering with Wnt signalling activity miR-214 may act as one of the upstream effectors of the signalling cascades which govern HF morphogenesis and cycling.

Dendritic cells are equipped with a range of different pattern-recognition receptors (PRR) aimed at recognizing foreign pathogens. Recent evidence has suggested that PRR signalling regulates the expression of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, which have been shown to fine-tune innate immune responses. This thesis describes the discovery of miR-650, a novel PRR-responsive miRNA that is down regulated in monocyte-derived dendritic cells (DCs) on PRR stimulation. Chapter 4 describes the characterisation of miR-650 expression in DCs matured by exposure to a variety of different pathogen-derived ligands, or during Influenza A virus infection. When correlating the level of miR-650 to the induction of DC activation markers on the cell surface, an inverse correlation was observed, suggesting a relationship between miR-650 down regulation and the effective dose of the ligand. Work presented in this thesis further explores the potential function of miR-650 by using a multi-pronged approach encompassing computational biology, genome-wide expression profiling and individual reporter assays, to gain insight into the gene networks regulated by miR-650. While Chapter 5 focuses on the identification and confirmation of individual miR-650:target interactions, Chapter 6 investigates both direct as well as secondary effects exerted by miR-650 on a global level. The work in these two chapters identifies a number of novel miR-650 targets and suggests a dual role for miR-650 in the innate immune response. Firstly, it is shown that miR-650 directly regulates a group of interferon-stimulated genes with known antiviral activity. Supporting its role in antiviral host defence, miR-650 is also shown to directly target components of the autophagic machinery, and even more importantly, down regulation of miR-650 induces autophagosome formation. Secondly, identified targets also include negative regulators of innate signalling suggesting that, in addition to its antiviral function, PRR-mediated down regulation of miR-650 expression may also provide a negative feedback loop controlling inflammatory responses. Notably, miR-650 displays reciprocal target regulation with miR-155, a well-studied miRNA with established functions in the innate immune system, thus suggesting cooperativity between the two miRNAs. The original aim of this thesis was to examine the effect of HIV-1 infection on the global miRNAome of DCs using a genome-wide profiling method. However, as outlined in Chapter 3, the data generated suggest that HIV-1 infection has little or no impact on miRNA expression. Further work is needed to establish if this represents deliberate immune evasion by HIV-1, or just indicates the limits of the methodology employed.

Flowering time is a carefully regulated trait that integrates cues from temperature and photoperiod to coordinate flowering at favorable times of the year. This dissertation aims to understand the evolution of genetic architecture that facilitated the transition of Pooideae, a subfamily of grass, from the tropics to the temperate northern hemisphere approximately 50 million years ago. Two traits hypothesized to have facilitated this evolutionary shift are the use of long-term low-temperature (vernalization) to ready plants for flowering, and long-day photoperiods to induce flowering. In chapter one I review literature on the regulation of grass flowering by vernalization and photoperiod, and in chapters two and three I determine the role of VERNALIZATION 1 (VRN1) and VRN2, known to confer vernalization responsiveness in core Pooideae crop species, in flowering time across Pooideae. In chapter four, I then test predictions of the hypothesis that the Brachypodium distachyon miR5200 ortholog in the ancestor of Pooideae was important for suppressing short day flowering through its negative regulation of flowering time integrator FLOWERING LOCUS T (FT)/VERNALIZATION3 (VRN3). In combination with other studies, my data demonstrate that VRN1-mediated vernalization responsiveness evolved early in the Pooideae, while VRN2-mediated vernalization responsiveness appears to have evolved much later in the diversification of Pooideae. Although miR5200 likely evolved early in the Pooideae, its transcriptional regulation by short day photoperiod appears derived within Brachypodium distachyon. This work answers important questions about the evolutionary origin of temperature- and photoperiod-mediated flowering in an economically important clade that contains crop species such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Directions for future work on this topic are discussed in chapter 5.

Prostate cancer is the leading non-cutaneous malignancy affecting men in the United States. One in every six men will be affected by prostate cancer. Due to the high incidence of prostate cancer, there is a need to develop biomarkers capable of identifying tumors from benign prostatic lesions. miRNAs are small molecules that regulate protein translation and impact cellular integrity when dysregulated. It is widely thought that miRNAs have the potential to serve as biomarkers. This study utilizes a unique combinatorial analysis of miRNA dysregulation to identify key miRNAs involved in prostate tumor initiation, progression and metastasis. Numerous dysregulated miRNAs potentially influence cancer development. A unique bioinformatically driven, network based approach was used to rank potential miRNAs that drive tumor progression. This study showed that miRNAs preferentially regulate highly connected proteins and transcription factors that affect numerous downstream targets. Thus dysregulation of a single highly connected miRNA could severely impact homeostatic maintenance of the tissue. In combination with miRNA profiling of a cancer cell progression model, the utilization of laser captured microdissection was used to separate cancer specific microRNA portraits from background differences arising from stroma cells, lymphocytes, and remaining normal epithelial cells. Integration of miRNA profiles with information gathered using networks biology and targeted proteomics resulted in the identification of a key miRNA that affects prostate cancer development and may be useful as a novel biomarker for identification/ staging of prostate cancer. Human miR-125b was identified as a potential miRNA suppressor of tumor formation. Previous work has identified miR-125-b as the post-transcriptional regulator of the ErbB2/ ErbB3 growth factor receptor family. Loss of miR-125b drives up expression of ErbB2/ ErbB3 activating downstream PI3K/AKT and RAS oncogene pathways. The level of miR-125b decreases 3-5-fold between benign and tumor epithelium. Further, miR-125b decreases during the development of prostatic intraepithelial neoplasia, which is regarded as an early indicator of prostate cancer. Thus miR-125b may be an ideal marker of early changes indicative of cancer. Restoration of miR-125b into highly tumorigenic, metastatic cells reduces mobility and invasion of underlying tissues. Taken together these data show miR-125b is a tumor suppressor in the healthy prostate.

Type 2 diabetes mellitus is a growing problem across the world and has significant pathological changes associated with it, including diabetic cardiomyopathy, wherein cardiac function is reduced. MicroRNA-21 has been shown to play a role in both the heart and diabetes so it was thought that knocking out miR-21 could have a protective effect on oxidative phosphorylation function in diabetic mice. Subsarcolemmal and interfibrillar mitochondria were isolated from adult male WT, miR-21 KO, db/db, and double knockout mice (db/db and miR-21 KO cross) and evaluated for function. Knocking out miR-21 in diabetic mice showed a restorative effect in Complex I and Complex II function even though it increased ROS production in Complex I and did not show a significant change in MPTP opening. Knocking out miR-21 could potentially restore oxidative phosphorylation function in diabetic patients but at the expense of producing more ROS.

Avec l'augmentation de l'espérance de vie, les pathologies ostéo-articulaires comme l'arthrose ou la polyarthrite rhumatoïde, caractérisées par la dégradation du cartilage articulaire, deviennent de réels problèmes de santé publique. Les traitements actuels sont essentiellement symptomatiques et aboutissent en ultime recours à la pose de prothèses. En absence de réparation spontanée du tissu et de traitement efficace, des approches d'ingénierie tissulaire du cartilage sont envisagées. Les techniques actuelles reposent sur la transplantation de chondrocytes autologues mais dans la majorité des cas, cette approche n'apporte pas de résultats supérieurs aux techniques chirurgicales utilisées actuellement. Grâce à leurs propriétés de différenciation, les cellules souches mésenchymateuses (CSM) représentent une nouvelle source de cellules ayant des potentiels thérapeutiques intéressants. Cependant, la complexité du processus de différenciation des CSMs vers des chondrocytes articulaires matures rend difficile l'obtention de cartilage fonctionnel après implantation. Il est donc important de mieux comprendre le processus de différenciation de ces cellules afin de mieux contrôler leur devenir in vivo. C'est pourquoi, le laboratoire s'intéresse au rôle des micro-ARNs (miARNs) dans la régulation du processus de différenciation des CSMs. L'objectif de mon projet de thèse a consisté à identifier des miARNs modulés dans la différenciation chondrocytaire des CSM humaines primaires et à étudier leur rôle et leur régulation au cours de la chondrogenèse. Nous avons identifié deux miARNs : miR-29a dont l'expression diminue progressivement au cours de la différenciation et miR-574-3p dont l'expression augmente rapidement puis est maintenue jusqu'à la fin de la différenciation. Ces deux miARNs sont régulés par le facteur de transcription SOX9 mais de manière opposée : SOX9 inhibe miR-29a et induit miR-574-3p. Nous montrons que SOX9 interagit avec YY1 pour réguler miR-29a mais pas miR-574-3p, ce qui pourrait expliquer les effets opposés de SOX9 sur l'expression des deux miARNs. Nous montrons également que ces miARNs sont des inhibiteurs de la différenciation chondrocytaire et avons identifié FOXO3A et RXRα comme cibles respectives de miR-29a et miR-574-3p. L'inhibition de FOXO3A ou RXRα avant l'induction de la différenciation, en utilisant des siARNs spécifiques ou en sur-exprimant les miARNs correspondants, bloque la différenciation des CSM. Ces résultats confirment sur des CSMs adultes, que ces protéines jouent un rôle important dans la chondrogenèse et que miR-29a et miR-574-3p participent aux processus de régulation de la différenciation chondrocytaire. En conclusion, nous avons identifié deux nouveaux miARNs contrôlés par SOX9 et régulant négativement la chondrogenèse grâce à la modulation de deux gènes cibles, dont l'expression est nécessaire avant d'induire la différenciation chondrocytaire. / Roles of miR-29a and miR-574-3p during the chondrogenic differentiation of mesenchymal stem cells. With the constant increase of the lifespan, osteoarticular pathologies such as osteoarthritis or rheumatoid arthritis, characterized by articular cartilage degradation, are important public health problems. In absence of spontaneous regeneration, cartilage engineering approaches are being considered. Current techniques rely on autologous chondrocyte transplantation but in the majority of cases, this approach gives similar results as current surgeries. Due to their capacity of differentiation toward chondrocytes, mesenchymal stem cells (MSC) represent a new source of cells with therapeutic potential. However, production of a functional cartilage in vivo after implantation of expanded MSC is hampered by the difficulty to reproduce the complexity of the differentiation process to get mature chondrocytes from MSC. The objective of my Ph.D thesis aimed to identify micro-RNAs (miRNAs) modulated during chondrogenic differentiation of primary human MSCs and to study their role as well as their regulation in this process. We identified two miRNAs: miR-29a whose expression decreases progressively during the differentiation and miR-574-3p whose expression rapidly increases and stays constant until the end of the differentiation. Both miRNAs are regulated by the transcription factor Sox9 but in an opposite manner: Sox9 inhibits miR-29a and induces miR-574-3p. We show that YY1 directly interact with Sox9 to regulate miR-29a but not miR-574-3p; this interaction likely explaining the opposite effects of Sox9 on miR-29a and miR-574-3p expression. Moreover we showed that miR-29a and miR-574-3p are both inhibitors of chondrogenesis and we identified FOXO3A and RXRα as their respective targets. In conclusion, we identified two new miRNAs which are regulated by Sox9 and inhibitors of chondrogenesis. They act through the modulation of two target genes, whose role during chondrogenic differentiation of adult MSC was previously not characterized.

Les micro-ARNs (miRNAs) sont de petits ARNs (20-25 nt) qui ont un rôle important dans les mécanismes d'interférence ARN. Les miRNAs sont des inhibiteurs de l'expression génique qui interviennent au niveau post-traductionnel en s'hybridant à des sites spécifiques de leurs ARNm cibles. Ce mécanisme induit la dégradation de l'ARNm ou l'inhibition de sa traduction. Puisque l'hybridation partielle du miRNA est suffisante pour induire une inhibition, chaque miRNA peut avoir des centaines de cibles. Les miRNAs sont impliqués dans de nombreuses fonctions biologiques et en particulier dans processus neuronaux. Plus de la moitié des miRNAs connus sont exprimés dans le cerveau de mammifère avec une distribution spécifique du miRNA considéré. A l'échelle sub-cellulaire il y a également une distribution hétérogène des miRNAs. De plus, il a été montré récemment une implication des miRNAs dans la régulation de la traduction locale dans les neurones. En effet, des miRNAs et des protyeines impliquées dans la biogenèse et la fonction des miRNAs ont été retrouvés dans le soma, les dendrites et les axones. Il a été montré que la dérégulation des miRNAs été impliquée dans de nombreux mécanismes pathologiques. Cette thèse a pour objectif de révéler le rôle des miRNAs dans la plasticité synaptique. Nous avons étudié l'implication des miRNAs dans les mécanismes de la plasticité synaptique homéostatique et dans la plasticité dysfonctionnelle rencontrée en condition de douleur cancéreuse.Notre hypothèse était que la régulation de la traduction locale des récepteurs AMPA dans les dendrites en condition d'homéostasie synaptique implique les miRNAs. Par bio-informatique, qRT-PCR et test luciférase, nous avons identifié le miRNA miR-92a comme régulateur de la traduction de l'ARNm de GluA1. Des immunomarquages des récepteurs AMPA et des enregistrements des courants miniatures AMPA montrent que miR-92a régule spécifiquement l'incorporation synaptique de nouveau récepteurs AMPA contenant GluA1 en réponse à un blocage de l'activité synaptique. La douleur est un symptôme très fréquemment associé au cancer et constitue un challenge pour les médecins puisque aucun traitement spécifique et efficace n'existe. C'est sans doute le résultat d'un manque de connaissances des mécanismes moléculaires responsables de la douleur cancéreuse. En combinant les screening des miRNA et des ARNm, nous avons mis en évidence une voie de régulation impliquant miR-124, un miRNA enrichi dans le système nerveux. Ainsi, dans un modèle de douleur cancéreuse chez la souris, la diminution de miR-124 est associée à une augmentation de ces cibles : calpain 1, synaptopodine et tropomyosine 4. Toutes ces protéines ont précédemment été identifiées comme des molécules clef de la fonction et de la plasticité synaptique. Des experiences in vitro ont confirmé que miR-124 exercait une inhibition multiple de calpain 1, synaptopodine et tropomyosine 4. La pertinence clinique de cette découverte a été vérifiée par le screening du liquide cérébro-spinal de patients souffrant de douleur cancéreuse qui montre également une diminution de miR-124. Ce résultat suggère un fort potentiel thérapeutique du ciblage de miR-124 dans les douleurs cancéreuses. Enfin, l'injection intrathécale de miR-124 dans des souris cancéreuses a permis de normaliser l'expression de la synaptopodine et de stopper la douleur cancéreuse lors de la phase initiale de la maladie. / MicroRNAs (miRNAs) are a type of small RNA molecules (21-25nt), with a central role in RNA silencing and interference. MiRNAs function as negative regulators of gene expression at the post-transcriptional level, by binding to specific sites on their targeted mRNAs. A process results in mRNA degradation or repression of productive translation. Because partial binding to target mRNA is enough to induce silencing, each miRNA has up to hundreds of targets. miRNAs have been shown to be involved in most, if not all, fundamental biological processes. Some of the most interesting examples of miRNA activity regulation are coming from neurons. Almost 50% of all identified miRNAs are expressed in the mammalian brain. Furthermore, miRNAs appear to be differentially distributed in distinct brain regions and neuron types. Importantly, miRNAs are reported to be differentially distributed at the sub-cellular level. Recently, miRNAs have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of miRNAs and the protein complexes involved in miRNA biogenesis and function in neuronal soma, dendrites, and axons. Deregulation of miRNAs has been shown to be implicated in pathological conditions. The present thesis aimed at deciphering the role of miRNA regulation in neuronal plasticity. Here we investigated the involvement of miRNA in synaptic plasticity, specifically in homeostatic synaptic plasticity mode. In addition, we investigated the involvement of miRNAs in the maladaptive nervous system state, specifically, in bone cancer pain condition.We hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic scaling may involve miRNAs. Using bioinformatics, qRT-PCR and luciferase reporter assays, we identified several brain-specific miRNAs including miR-92a, targeting the 3’UTR of GluA1 mRNA. Immunostaining of AMPA receptors and recordings of miniature AMPA currents in primary neurons showed that miR-92a selectively regulates the synaptic incorporation of new GluA1-containing AMPA receptors during activity blockade.Pain is a very common symptom associated with cancer and is still a challenge for clinicians due to the lack of specific and effective treatments. This reflects the crucial lack of knowledge regarding the molecular mechanisms responsible for cancer-related pain. Combining miRNA and mRNA screenings we were able to identify a regulatory pathway involving the nervous system-enriched miRNA, miR-124. Thus, miR-124 downregulation was associated with an upregulation of its predicted targets, Calpain 1, Synaptopodin and Tropomyosin 4 in a cancer-pain model in mice. All these targets have been previously identified as key proteins for the synapse function and plasticity. Clinical pertinence of this finding was assessed by the screening of cerebrospinal fluid from cancer patient suffering from pain who presented also a downregulation of miR-124, strongly suggesting miR-124 as a therapeutic target. In vitro experiments confirmed that miR-124 exerts a multi-target inhibition on Calpain 1, Synaptopodin and Tropomyosin 4. In addition, intrathecal injection of miR-124 was able to normalize the Synaptopodin expression and to alleviate the initial phase of cancer pain in mice.

PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...