Régulation des gènes de l'hôte par les microARN dérivés de l'élément TAR du VIH-1

Les microARN (miARN) sont des acides ribonucléiques (ARN) endogènes, d’environ 19 à 24 nucléotides (nt), produits lors du clivage d’une structure d’ARN en forme de tige-boucle par la ribonucléase III (ARNase III) Dicer. Il a été rapporté que le virus de l’immunodéficience humaine de type 1 (VIH-1), en période de latence dans les lymphocytes T CD4+, produit un court transcrit d’ARN appelé « Trans-Activation Response element » (TAR) et que celui-ci est sujet au clivage par Dicer, ce qui génère deux miARN fonctionnels, soit miR-TAR-5p et miR-TAR-3p. Il a donc été suggéré que les miARN dérivés de TAR pourraient jouer un rôle dans la latence du VIH-1. L’objectif, au cours de ma maîtrise, était de déterminer le rôle potentiel de ces miARN dans la régulation de l’expression des gènes de l’hôte en utilisant les cellules en culture J-lat et Jurkat exprimant miR-TAR-5p et miR-TAR-3p de manière stable. Suite à cela, une analyse protéomique grande échelle iTRAQ a été effectuée pour tenter de faire la lumière sur le rôle potentiel des miARN dérivés de l’ARN TAR du VIH-1. En conclusion, il a été montré que la majorité des protéines d’intérêts sont réfractaires à une régulation génique par les miARN. Par contre, ceci ne supporte pas l’idée que ces miARN ne possèdent aucun rôle, d’où l’importance des résultats protéomiques qui ont montré que plusieurs protéines sont potentiellement régulées par les miARN viraux

Muscle Plasticity and Intramuscular signaling in the Insulin-resistant Obese Zucker Rat

The ability to increase skeletal muscle mass may have important implications for the treatment of insulin resistance (IR) and diabetes [1-3]. Recent data suggest that IR muscle may adapt differently than normal muscle; however, molecular mechanism(s) responsible for this finding are not well understood [4]. Herein, we investigate the molecular mechanisms underlying the skeletal muscle remodeling in the IR Obese Zucker (OZ) rat. The OZ rat is characterized by skeletal muscle insulin resistance, hyperglycemia, and hyperlipidemia. Compared to LZ rats, our data demonstrate that soleus muscle hypertrophy was significantly attenuated in the OZ rats after 3-weeks of muscle overload and that these findings appear to be accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mammalian target of rapamycin (mTOR), 70 kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6) and protein kinase B (Akt). Recent in vitro and in vivo studies have suggested a role for AMP-activated protein kinase (AMPK) and dsRNA-dependent protein kinase (PKR) in skeletal muscle adaptation and their interactions with mTOR related signaling [5, 6]. Our data suggest that IR attenuates overload-induced skeletal muscle hypertrophy through the activation of AMPK and PKR, which appears to be associated with an inhibition of mTOR and eIF2α phosphorylation. This finding is consistent with the possible depression of protein synthesis. Other data demonstrate that IR resistance is associated with the PKR-mediated activation of p38 MAP kinase, which would be predicted to lead to increased protein degradation. Further, we demonstrated that the regulation of heat shock proteins (HSPs) and the mitogen-activated protein kinases (MAPKs) are altered during hypertrophy in OZ rat, which suggest that these molecules may play a role in explaining why IR may be associated with alterations in muscle plasticity. In addition to traditional biochemical signaling cascades, recent data have strongly suggested that muscle-specific miRNAs may participate in the regulation of load-induced skeletal muscle remodeling [7]. To this end, we demonstrate for the first time that miR-1 and miR133 expression levels are lower in IR muscle. Further, we also observed that overload decreased mir-1 expression in the LZ muscle to a greater extent to that measured in the OZ muscle. Combined, these results are the first to report evidence that overload-induced skeletal muscle remodeling in IR OZ rat is associated with multiple level decrements including changes in mTOR signaling, hyperphosphorylation of AMPK and PKR and altered regulation of muscle-specific miRNAs

Role of microRNAs in Jaagsiekte sheep retrovirus infection

Ovine pulmonary adenocarcinoma (OPA) is a lung cancer that affects sheep, caused by jaagsiekte sheep retrovirus (JSRV). OPA is present in most sheeprearing countries of the world, but, at present, there are no reliable early-stage tests to diagnose the disease, and OPA continues to pose an animal welfare threat and cause substantial economic losses. In addition, OPA is a valuable animal model to study early oncogenic events in human lung cancer. Specifically, OPA and some types of human lung cancer present similarities in activated signalling pathways (Ras-MEK-ERK1/2 and PI3K-AKT-mTOR) and their association with type II pneumocytes. Nevertheless, study of the molecular pathogenesis of OPA has been hindered due to the lack of a permissive cell line for JSRV replication. JSRV encodes an unusual envelope protein (Env) which is actively oncogenic and sufficient to drive transformation in vivo and in vitro. Despite the lack of a permissive cell line, early oncogenic events induced by JSRV can be studied by transfection of cell lines with plasmids encoding JSRV Env. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and essential cell processes such as cell proliferation and apoptosis. miRNAs are being extensively studied as biomarkers of several diseases, including cancer. The aims of this project were to investigate the role of miRNAs in the early oncogenic events induced by JSRV and to investigate their potential as OPA biomarkers. miRNA expression levels were investigated using small RNA sequencing in lung tissue from cases of experimentally induced OPA. No evidence of JSRV-encoded miRNAs was found, but levels of 40 miRNAs were found differentially expressed between affected and control sheep. Of those, upregulation of nine microRNAs (miR-135b, miR-182, miR-183, miR-21, miR-200b, miR-205, miR-31, miR-503 and miR-96) was confirmed by RT-qPCR in experimental and natural cases of OPA, suggesting that increased levels of these miRNAs were characteristic of OPA affected lung tissue. To investigate miRNAs as potential biomarkers, miRNA expression was measured in serum and bronchoalveolar lavage fluid (BALF) macrophages of OPA affected sheep. small RNA sequencing revealed 74 microRNAs and 85 miRNAs differentially expressed in serum and BALF macrophages, respectively. Interestingly, BALF macrophage microRNA expression was found to resemble more closely that of OPA affected sheep lungs. In addition, miRNA expression levels varied at different stages of the disease and no miRNAs were found to be consistently dysregulated in serum of OPA affected animals. Discordances in miRNA signatures in lung tissue and serum are not entirely unexpected. Lung tissue miRNAs might represent the tumour microenvironment and localised response to it, whereas miRNAs in serum may represent the global state of the animal, and tumour miRNAs might be released into circulation at low levels, making them difficult to detect. Expression of the nine upregulated miRNAs was then investigated in in vitro models to study their involvement in transformation. Lentiviral vectors encoding green fluorescent protein (GFP) or a GFP-2A-Env fusion protein were produced and used to transduce cell lines. Transformation was verified by immunocytochemical detection of the transformation markers P-Akt and PERK1/2. Nevertheless, miRNA expression levels in culture did not resemble those observed in lung tissue of OPA-affected sheep. These differences might be due to species variation, upregulation of miRNAs late in the transformation process, or involvement of other cell types in tissue besides the transformed cells. To study these questions further, JSRV and the GFP-2A-Env encoding lentiviral vectors were used to infect lung slices in culture. Expression levels of miRNAs did not, in any of the cases, resemble lung tissue findings. Fewer than 5% of cells in lung slices were found to be infected, suggesting that changes in miRNA expression could be masked by the background of normal cells. Nevertheless, increasing JSRV21 concentration did not yield higher infection levels, indicating that those might be more dependent on the availability of JSRV’s target cells, dividing type II pneumocytes, than viral concentration. Taken together, this study has revealed new information on miRNA expression in OPA-affected sheep, including expression patterns in lung and serum. Future work should focus on developing a permissive replication system to allow the study of miRNAs in early JSRV-induced transformation events

Novel methods for constructing, combining and comparing co-expression networks: Towards uncovering the molecular basis of human cognition

Network analyses, such as gene co-expression networks are an important approach for the systems-level study of biological data. For example, understanding patterns of \linebreak co-regulation in mental disorders can contribute to the development of new therapies and treatments. In a gene regulatory process a particular TF or ncRNA can up- or down-regulate other genes, therefore it is important to explicitly consider both positive and negative interactions. Although exists a variety of software and libraries for constructing and investigating such networks, none considers the sign of interaction. It is also required that the represented networks have high accuracy, where the interactions found have to be relevant and not found by chance or background noise. Another issue derived from building co-expression networks is the reproducibility of those. When constructing independent networks for the same phenotype, though, using different expression datasets, the output network can be remarkably distinct due to biological or technical noise in the data. However, most of the times the interest is not only to characterise a network but to compare its features to others. A series of questions arise from understanding phenotypes using co-expression networks: i) how to construct highly accurate networks; ii) how to combine multiple networks derived from different platforms; iii) how to compare multiple networks. For answering those questions, i) I improved the wTO method to construct highly accurate networks, where now each interaction in a network receives a probability. This method showed to be much more efficient in finding correct interactions than other well-known methods; ii) I developed a method that is able to combine multiple networks into one building a CN. This method enables the correction for background noise; iii) I developed a completely novel method for the comparison of multiple co-expression networks, CoDiNA. This method identifies genes specific to at least one network. It is natural that after associating genes to phenotypes, an inference whether those genes are enriched for a particular disorder is needed. I also present here a tool, RichR, that enables enrichment analysis and background correction. I applied the methods proposed here in two important studies. In the first one, the aim was to understand the neurogenesis process and how certain genes would affect it. The combination of the methods shown here pointed one particular TF, ZN787, as playing an important role in this process. Moreover, the application of this toolset to networks derived from brain samples of individuals with cognitive disorders identified genes and network connections that are specific to certain disorders, but also found an overlap between neurodegenerative disorders and brain development and between evolutionary changes and psychological disorders. CoDiNA also pointed out that there are genes involved in those disorders that are not only human-specific

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

The cyanobacterial rhomboid protease is a regulator of the CCM

Cyanobacteria are aquatic photosynthetic bacteria and useful models for study of the chloroplast and photosynthesis. We are studying a ‘rhomboid’ membrane-located proteases in Synechocystis sp. PCC 6803, which appears to function as a previously undiscovered regulator of the carbon concentrating mechanism (CCM) of this phototroph. Rhomboids are almost ubiquitous across evolution, and are known to activate diverse cellular processes via proteolysis of their specific, membrane-sequestered substrates. Although this well-conserved family has solved crystal structures of bacterial enzymes such as Escherichia coli GlpG, ironically, most work has been carried out on eukaryotic representatives. Following our study of the Arabidopsis thaliana chloroplast RBL10 protease, we identified cyanobacterial orthologues with the aim of discovering if roles might be conserved between these and organellar rhomboids. Molecular biology and reverse-genetics studies were made on slr1461, a mutant in the single rhomboid protease of Synechocystis. When photosynthetic parameters were investigated, it could be seen that inactivation of slr1461 did not affect nonphotochemical quenching, unlike the chloroplast rbl10 mutant, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This reduction allows cyanobacteria to avoid expending energy on the uptake of CO2 when an organic carbon source can be utilised: as might be expected, therefore, Slr1461 transcription was linked with downregulation of genes encoding proteins facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. Quantitative RT-PCR of CCM network genes suggested that Slr1461 is located upstream of known regulators, including another membrane protease, the Slr0228 FtsH, and a central, controlling transcription factor NdhR

Unveiling adaptive mechanisms though experimental evolution: the role of duplicated genes and phenotypic plasticity in yeast, and the genetic variability in Coxsackievirus

Los seres vivos se enfrentan a condiciones ambientales cambiantes y habitualmente estresantes, agravadas por el cambio climático, que ponen a prueba su capacidad de supervivencia. El cambio en la composición genética de las poblaciones reside en las mutaciones, que son la fuente para la evolución y la adaptación a los cambios. La diversidad genética intrapoblacional está regulada por dos grandes fuerzas evolutivas que cambian la composición genética permitiendo así el acceso a nuevos fenotipos: la deriva genética y la selección natural. Por un lado, la deriva genética fija mutaciones en la población de manera aleatoria e independiente del efecto que suponga dicha mutación para la población. Por otro lado, la selección natural si bien favorece la fijación de mutaciones beneficiosas también elimina mutaciones perjudiciales en un determinado ambiente. Por lo tanto, el efecto de las mutaciones está fuertemente ligado al ambiente y, en consecuencia, aquellas poblaciones que exhiben una mayor diversidad genética serán capaces de evolucionar más rápido y adaptarse mejor. Además de la evolución por selección natural y deriva genética, la duplicación genética también es de especial importancia para la evolución, pues es la principal fuente de nuevo material genético y de innovaciones biológicas. Tanto es así que las grandes transiciones evolutivas, como la radiación de las plantas angiospermas o las grandes innovaciones morfológicas en animales se han relacionado con eventos de duplicación. Sin embargo, los mecanismos moleculares que permiten mantener los genes duplicados durante largos periodos de tiempo siguen siendo desconocidos. Para intentar ampliar el conocimiento respecto a estos mecanismos, y dado que los efectos de la evolución en la naturaleza tarda mucho tiempo en poder observarse, se necesitan sistemas biológicos que sean capaces de evolucionar rápido. En este contexto, te tiempo adecuado al experimentador, se han llevado a cabo estudios de evolución experimental con virus y microorganismos que han supuesto una herramienta muy valiosa en el estudio de la biología evolutiva. Por un lado, los virus presentan tasas de mutación extremadamente elevadas, especialmente los virus de ARN, lo que les confiere la capacidad de adaptarse de manera muy rápida a un cambio ambiental. Por otro lado, la levadura Saccharomyces cerevisiae, cuyo origen se debe a una duplicación genómica acaecida hace más de 100 millones de años, es un buen modelo para estudiar la duplicación genética y su papel en la adaptación. Además, más allá de ser útil para la evolución experimental y el estudio de la biología evolutiva, la elevada capacidad evolutiva de los virus de ARN supone un desafío importante para la medicina y en la prevención de enfermedades emergentes y, la levadura S.cerevisiae es una de las especies con mayor impacto económico en la industria biotecnológica. Por lo tanto, llevar a cabo un análisis exhaustivo del efecto de las mutaciones en poblaciones virales y estudiar en profundidad como la duplicación genética influye la adaptación y la innovación biológica en la levadura, es fundamental para generar un marco de conocimiento que permitirá maximizar el potencial biomédico y biotecnológico de los virus de ARN y de la levadura. Esta tesis doctoral trata de abordar dos cuestiones fundamentales en biología evolutiva: ¿Cuáles son los mecanismos moleculares que determinan la estabilidad de los genes duplicados en el genoma durante el tiempo suficiente para que sean capaces de adquirir relevancia evolutiva? Y, ¿Cómo contribuye la variabilidad genética a la evolución y a la adaptación a nuevos ambientes? Para intentar responder a estas preguntas hemos utilizado dos modelos experimentales diferentes: la levadura S. cerevisiae y el coxsackievirus B3 (CVB3). En la primera parte de esta tesis, con la levadura hemos visto que el nivel de expresión génica de los genes duplicados, así como la divergencia transcripcional y funcional, son fundamentales para la estabilidad de los genes duplicados en el genoma. Además, hemos observado que la plasticidad transcripcional de los genes duplicados juegan un papel clave en la adaptación a nuevos ambientes desfavorables, tales como condiciones de estrés oxidativo o altas concentraciones de etanol, glicerol o ácido láctico. Y en la segunda parte de la tesisi, utilizando el virus CVB3, hemos realizado una aproximación de Deep mutational scanning sobre las proteínas de la cápside viral y hemos generado poblaciones virales con una elevada variabilidad genética. Gracias a ello hemos podido evaluar el efecto de las mutaciones en la cápside caracterizando alrededor del 90% de los cambios de amino ácidos. Además, hemos empleado estas poblaciones virales altamente diversas y hemos estudiado como esta variabilidad genética contribuye a la adaptación contra la inactivación térmica. Nuestros resultados muestran que, incluso en virus de ARN con tasas de mutación extremadamente elevadas, un aumento de la diversidad genética de la población al inicio de la evolución experimental acelera el proceso evolutivo y facilita la adaptación al nuevo ambiente.Living beings face changing and usually stressful environmental conditions, aggravated by climate change, which tests their ability to survive. Changes in the genetic composition of the population, in the form of mutations, is the source for evolution and adaptation to environmental changes. This genetic diversity is driven by two major evolutionary forces that change the genetic composition in a population, allowing access to new phenotypes: genetic drift and natural selection. On one hand, genetic drift randomly fixes mutations in the population independent of their effect. On the other, natural selection either selects for beneficial mutations or purges deleterious mutations in a given environment. Hence, the effect of the mutations is closely linked to the environment and, as a consequence, populations exhibiting high genetic diversity can evolve faster and adapt better to environmental fluctuations. In addition to natural selection and genetic drift, gene duplication is also of great importance to evolution, as it is the main source of new genetic material. Not surprisingly, gene duplication has been related to major leaps in evolution, such as the radiation of angiosperm plants or large morphological innovations in animals. However, the molecular mechanisms that underlie the preservation of duplicated genes for long periods of time remain unknown. To better understand these mechanisms, experimental systems enabling rapid evolution are needed as the natural time scale for natural evolution can be extremely long. For this reason, experimental evolution approaches using viruses and microorganisms have become a valuable tool in evolutionary biology. On one hand, viruses show extremely high mutation rates, especially RNA viruses, conferring them the ability to rapidly adapt to a changing environment, thus representing an ideal model to study the effect of mutations. On the other hand, the yeast Saccharomyces cerevisiae, which has its origin in a whole genome duplication that occurred more than 100 million years ago, is a good model for studying gene duplication and its role in adaptation. Moreover, beyond their use as models for understanding evolutionary processes, the rapid evolutionary capacity of RNA viruses poses a challenge for treating and preventing infections and S. cerevisiae is currently one of the species with the largest biotechnological and economic impact. Therefore, a comprehensive analysis of the effect of mutations in large virus populations and, a deep knowledge about how genetic duplication influences adaptation and biological innovation is essential for gaining a better understanding of evolutionary processes and can help maximize our use of the full biomedical and biotechnological potential of RNA viruses and the budding yeast. This doctoral thesis aims to shed light on two fundamental evolutionary biology questions: What are the molecular mechanisms that determine the genomic stability of duplicated genes that are maintained in the genome for enough time to acquire evolutionary relevance? And, how does genetic variability contribute to evolution and adaptation? To address these questions we used two different models: the yeast S.cerevisiae and the coxsackievirus B3 (CVB3). In the first part of this thesis, we show that the level of gene expression of duplicated genes, as well as transcriptional and functional divergence, are key for the stability of duplicated genes. In addition, we find that the transcriptional plasticity of duplicated genes plays a key role in adaptation to new and stressful environments like high concentrations of ethanol, glycerol, and lactate or for oxidative stress conditions. In the second part, we performed a deep mutational scanning of the CVB3 capsid to generate highly genetically diverse populations and captured the mutational fitness effect of >90% of all possible single amino acid mutations in the viral capsid. We then used these highly diverse populations to study the contribution of genetic variability to adapt to thermal inactivation, observing that increasing the initial genetic variability in the population helps evolution even in RNA viruses with extremely high mutation rates

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

The molecular and genetic evolution of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) (Family: Picornaviridae, Genus: Aphthovirus) is a significant global pathogen with extensive economic impact. FMDV has a low fidelity RNA-dependent RNA polymerase and lacks proof reading capability. This coupled with its relatively short generation time and large population sizes means it exists in a swarm of genetically closely related variants. The reservoir of diversity contained within this mutant spectrum allows the virus to adapt rapidly to new environments. Much of the previous work looking at virus evolution has focused on the consensus level genetic sequence. The advent of next generation sequencing (NGS) technologies enables evolutionary studies of the entire viral swarm. This PhD project uses NGS technologies to interrogate the swarm structure by investigating factors affecting the viral swarm and the dynamics of variants within it. Furthermore, this work shows how analysis of the swarm can reveal fundamental information about virus biology. A PCR-free NGS methodology was developed to create deep sequencing data sets of all genomes present within an FMDV viral swarm. The elimination of the PCR step results in less errors being introduced in the sequencing process thereby improving the resolution and reliability of the identification of low level variants. This optimised method was then used to define and compare the FMDV swarms of several wildtype isolates. This revealed differences in swarm structure from isolate to isolate and produced evidence of within swarm selection. Not all proteins known to be under selection at the consensus level were also under selection within the swarm. The diversity of viruses within the swarm was found to be dependent upon the host from which a virus was sampled, with African buffalo potentially able to maintain multiple infections. Subconsensus variants in these mixed samples had mutations at positions previously associated with immune escape. Investigation of the evolution of swarm structure when adapting to new cell type in vitro indicated that two distinct population structures can exist relative to the existence of adaptive pressure. These two population structures have different distributions of variable nucleotides but comparative total levels of variation (as measured by Shannon's entropy). Deep sequencing of the virus swarm enabled the discovery of conserved novel stem loop structures, which were hypothesized to be required for packaging of the virus genome. Mutating these sites produced a virus with decreased packaging efficiency. This thesis includes novel analysis techniques for considering the viral swarm. It demonstrates how investigating the diversity in the swarm can help us to understand virus molecular biology, its evolution and the limits upon this. Understanding viral evolution at this scale has the capacity to improve our fundamental understanding of the biology and evolution of FMDV which can in turn inform vaccine design and disease control strategie