Trypanosoma brucei and related parasites are causative agents of severe diseases that affect global health and economy. T. brucei is responsible for sleeping sickness in humans (African trypanosomiasis) and a wasting disease in livestock. More than 100 years after T. brucei was identified as the etiological agent for sleeping sickness, available treatments remain inadequate, complicated by toxicity, lengthy and expensive administration regiments, and drug-resistance. There is clear need for the development of a new antitrypanosomal drugs. Due to the unique evolutionary position of these early diverging eukaryotes, trypanosomes posses a number of biological properties unparalleled in other organisms, including humans, which could prove valuable for new drug targets. One of the most distinctive properties of trypanosomes is their mitochondrial DNA, called kinetoplast DNA (kDNA). kDNA is composed of over five thousand circular DNA molecules (minicircles and maxicircles) catenated into a topologically complex network. Replication of kDNA requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication and at least five DNA polymerases. Three of these (POLIB, POLIC, and POLID) are related to bacterial DNA polymerase I and are required for kDNA maintenance and growth. Each polymerase appears to make a specialized contribution to kDNA replication. The research described in this dissertation is a significant contribution to the field of kDNA replication and the advancement of kDNA replication proteins as putative drug targets for sleeping sickness. Functional characterization of POLIB indicated that it participates in minicircle replication but is likely not the only polymerase contributing to this process. Gene silencing of POLIB partially blocked minicircle replication and led to the production of a previously unidentified free minicircle species, fraction U. Characterization of fraction U confirmed its identity as a population of dimeric minicircles with non-uniform linking numbers. Fraction U was not produced in response to silencing numerous other previously studied kDNA replication proteins but, as we demonstrated here, is also produced in response to POLID silencing. This common phenotype led us to hypothesize that POLIB and POLID both participate in minicircle replication. Simultaneously silencing both polymerases completely blocked minicircle replication, supporting a model of minicircle replication requiring both POLIB and POLID. Finally, we demonstrate that disease-causing trypanosomes require kDNA and the kDNA replication proteins POLIB, POLIC, and POLID. These data provide novel insights into the fascinating mechanism of kDNA replication and support the pursuit of kDNA replication proteins as novel drug targets for combating African trypanosomiasis.

Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

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We have explored correlations between the measured efficiency of the RNAi process and several computed signatures that characterize equilibrium secondary structure of the participating mRNA, siRNA, and their complexes. A previously published data set of 609 experimental points was used for the analysis. While virtually no correlation with the computed structural signatures are observed for individual data points, several clear trends emerge when the data is averaged over 10 bins of N ~ 60 data points per bin. The strongest trend is a positive linear (r² = 0.87) correlation between ln(remaining mRNA) and ΔG_ms, the combined free energy cost of unraveling the siRNA and creating the break in the mRNA secondary structure at the complementary target strand region. At the same time, the free energy change ΔG_total of the entire process mRNA + siRNA → (mRNA – siRNA)_complex is not correlated with RNAi efficiency, even after averaging. These general findings appear to be robust to details of the computational protocols. The correlation between computed ΔG_ms and experimentally observed RNAi efficiency can be used to enhance the ability of a machine learning algorithm based on a support vector machine (SVM) to predict effective siRNA sequences for a given target mRNA. Specifically, we observe modest, 3 to 7%, but consistent improvement in the positive predictive value (PPV) when the SVM training set is pre- or post-filtered according to a ΔG_ms threshold. / Master of Science

<p>Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In <i>Drosophila melanogaster</i> research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite <i>Octosporea muscaedomesticae</i> and the <i>Drosophila</i> C virus as compared to natural infections of bacteria and fungi. By using Affymetrix <i>Drosophila</i> GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. </p><p>In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of <i>Hemolin</i> in pupae of <i>Hyalophora cecropia</i> led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, <i>Antheraea pernyi</i>, and discovered that hemolin was induced by dsRNA<i> per se</i>. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed <i>in vivo</i> experiments on baculovirus infected pupae. We could show that a low dose of ds<i>Hemolin</i> prolonged the period before the <i>A. pernyi</i> pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing <i>in silico</i> analysis of the hemolin sequence from <i>A. pernyi</i> in comparison with other<i> Hemolin</i> sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.</p>

La polyarthrite rhumatoïde (PR) est le plus fréquent des rhumatismes inflammatoires et représente un problème de santé publique majeur. A l'heure actuelle, les biothérapies anti-TNF sous forme de protéines recombinantes constituent une avancée considérable dans le traitement de la polyarthrite rhumatoïde (PR). Néanmoins, il convient de développer des approches thérapeutiques alternatives pour traiter les 40% de patients non-répondeurs ainsi queceux qui échappent à plusieurs années de traitement. La recherche de nouvelles cibles thérapeutiques est indispensable pour proposer des approches alternatives à ces biothérapies. Par ailleurs, les techniques de transfert de gène offrent une alternative thérapeutique possible pour pallier aux limitations des biothérapies actuelles, à condition de les adapter aux contraintes du tissu cible de la PR, les articulations. Les projets ont consisté à développer et valider dans des modèles expérimentaux d'arthrite de nouvelles stratégies anti-inflammatoires basées sur l'utilisation de l'ARN interférence comme outil thérapeutique. En effet, la possibilité d'interférer au niveau des mécanismes responsables de l'expression des protéines,la régulation de la stabilité des ARNm et de l'efficacité de la machinerie traductionnelle, présente un intérêt thérapeutique supérieur aux biothérapies actuelles basées sur l'inhibition des protéines sécrétées (anticorps ou récepteurs solubles) mais nécessite cependant de posséder un vecteur qui transduit efficacement les cellules productrices de la molécule ciblée. / Rheumatoid arthritis (RA) is the most frequent chronic inflammatory systemicautoimmune disease that remains a major medical challenge as the exact causes of the disease are not completely elucidated. The principal treatment strategies arebased on the inhibition of TNF-α, one of the major inflammatory cytokine in RA.Although risk and benefit analyses are in favour of the use of monoclonal antibodiesagainst TNF-α, the most currently used biotherapy, they are not devoid from multipleside effects. The search for new therapeutic targets is essential to proposealternative approaches to non responders to such biotherapies. The possibility to interfere in the mechanisms responsible for regulating mRNA stability andeffectiveness of the translational machinery also present a therapeutic benefitsuperior to current biologic therapies based on inhibition secreted proteins(antibodies or soluble receptors). Such approach however requires developingvectors that efficiently transduced the specific cell type producing the targeted gene.Projects of my PhD fellowship have included both the development of gene therapyvehicles for RNAi-based intervention in experimental mouse models of arthritis andevaluation of novel candidate genes for alternative anti-inflammatory therapy in RA.

Leishmania et Trypanosoma brucei sont des protozoaires parasites responsables de graves parasitoses de distribution mondiale. Aucun vaccin n'est disponible contre ces maladies dont le traitement reste basé sur un nombre limité de médicaments coûteux, souvent toxiques et peu efficaces, problème auquel s'ajoute celui des chimiorésistances. D'où l'urgente nécessité de trouver de nouvelles cibles pour le développement de nouveaux traitements qui soient à la fois efficaces, non ou moins toxiques et à un coût plus accessible. Les Trypanosomatidés, dont les génomes ont été entièrement séquencés, présentent de nombreuses originalités dans leur biologie cellulaire et moléculaire, par exemple un ADN mitochondrial unique et extrêmement complexe appelé kinétoplaste. Leur développement suit également un "double" cycle cellulaire répliquant, d'une part, classiquement le noyau et, d'autre part, l'ensemble "corps basal-ADN mitochondrial" dont la ségrégation correcte conditionne la cytodiérèse. Ils possèdent par ailleurs deux types de protéasomes, un classique (26S) et un de type procaryote, plus spécifique et absent chez l'homme, le complexe HslVU. Nous avons montré que HslVU est localisé exclusivement dans l'unique mitochondrie des parasites, et qu'il est, chez T. brucei, essentiel pour la survie de ces organismes. En effet, son inhibition par ARN interférence entraine un blocage de la cytodiérèse suivi par une mort cellulaire. Le premier objectif de cette thèse a été de tenter de mieux comprendre le rôle de HslVU dans le cycle cellulaire associé au kinétoplaste chez ces parasites possédant déjà un protéasome classique. Mettant un terme à plusieurs publications contradictoires, nous avons confirmé la localisation mitochondriale de ce complexe chez Leishmania et chez T. brucei. Nous montrons pour la première fois qu'il est tout aussi essentiel dans les formes sanguines, celles présentes chez l'hôte mammifère, que dans les formes procycliques. Nous montrons aussi un rôle différencié des différentes sous-unités du complexe dans le déroulement du cycle cellulaire associé au kinétoplaste. Le deuxième objectif de cette thèse a été d'identifier de nouvelles protéines mitochondriales régulatrices du cycle cellulaire associé au kinétoplaste. Pour ce faire, nous avons développé une approche de criblage par ARN interférence "semi-systématique" sur 104 protéines mitochondriales, principalement de fonction inconnue. Si l'inhibition de l'expression de la majorité de ces protéines (62) n'a aucun effet sur la croissance cellulaire, celle des 42 restantes induit une baisse moyenne ou sévère de cette croissance. De façon surprenante, cette inhibition modifie significativement et avec plus ou moins d'ampleur le déroulement du cycle cellulaire, suggérant qu'il est dépendant de multiples fonctions cellulaires. Finalement, ce travail valide le protéasome HslVU comme une cible thérapeutique pertinente tout particulièrement à l'adresse des formes sanguines de T. brucei. La différenciation fonctionnelle de HslU1 et HslU2 et l'activité indépendante de HslV donnent une image plus complexe sur le fonctionnement de ce protéasome. Les données d'ARN interférence pour leur part nous orientent vers une régulation du cycle cellulaire très intégrée à l'ensemble des activités cellulaires. / Leishmania and Trypanosoma brucei are protozoan parasites responsible for worldwide distributed severe diseases. No vaccine is available and the treatment relies upon a limited number of drugs, which are costly, often toxic and not highly efficient, and for which resistances are increasing. Hence the necessity to urgently discover novel drug targets with the aim of developing new drug treatments which would be more efficient, less toxic and if possible cheaper.Trypanosomatids, of which the genome has been entirely sequenced, exhibit numerous peculiarities in their cell and molecular biology, for example a single and complex mitochondrial DNA network termed kinetoplast. Also, their development follows a ‘double' cell cycle ensuring the replication of, on the one hand, the nucleus (classical mitosis) and on the other hand, the “basal body-kinetoplast” whole, of which the correct segregation conditions cytokinesis. They also possess tow types of proteasomes, one classical one (26S) and one of the prokaryotic type, more specific and absent in human, the HslVU complex. We have shown that HslVU is located exclusively in the single mitochondrion of these parasites and, in T. brucei, that it is essential to parasite's survival. Indeed, its RNAinterference-based knockdown leads to a cytokinesis block followed par cell death. The first aim of this work was to try to better understand the role of HslVU in the ‘kinetoplast-associated' cell cycle in these parasites that already possess a classical proteasome. Putting an end to several contradictory publications, we confirmed the mitochondrial location of this complex in Leishmania and T. brucei. For the first time, we also demonstrate that it is just as essential in bloodstream forms (those present in the mammalian host) than in procyclic forms. We finally show a differentiated role for the different subunits of the complex in the progress of the kinetoplast-associated cell cycle.The second aim of this work was to identify novel mitochondrial proteins which would participate in the regulation of the kinetoplast-associated cell cycle. To do this, we developed a ‘semi-systematic' screening approach using RNA interference for 104 mitochondrial proteins, most of them being of unknown function. If the inhibition of most of these proteins (64) had no effect on cell growth, that of the 42 remaining ones induced a moderate or severe growth defect. Surprisingly, this inhibition yielded significant and more or less visible modifications of the cell cycle progress, suggesting that the latter is dependent upon multiple cell functions.Finally, this study validates the HslVU proteasome as a pertinent drug target, particularly for the bloodstream forms of T. brucei. The functional differentiation of HslU1 and HslU2 and the independent activity of HslV are intriguing and give a complex picture of the functioning of this proteasome. On the other hand, the RNA interference data suggest a cell cycle regulation which would be highly integrated to the whole of the cell activities.