Les voies de régulation dépendant des courts ARN non-codants contrôlent plusieurs processus biologiques. Ces courts ARNs sont important dans le développement des cellules germinales de plusieurs espèces ainsi que dans la régulation génique et la résistance virale. Néanmoins, la contribution de la voie de régulation dépendant des microARNs dans la biogénèse des cellules germinales demeure peu comprise. Étant donné que les protéines Argonautes ALG-1 et ALG-2 sont exclusivement impliquées et indispensables à la voie des microARNs, nous avons décidé de manipuler génétiquement ces gènes afin de déterminer si la voie des microARNs est importante dans la prolifération et différentiation des cellules germinales des animaux en utilisant le nématode Caenorhabditis elegans comme modèle d’étude. La perte de fonction des gènes alg-1 et alg-2 rend les animaux stériles, ce qui est similaire aux phénotypes observés chez les nématodes mutant pour Drosha et Dicer (deux enzymes essentielles à la production des microARNs). Ainsi, ceci supporte que la voie de régulation des microARNs joue un rôle essentiel pour le maintien des cellules germinales. Pour définir le rôle précis de ALG-1 et ALG-2 dans les processus complexes de régulation des cellules germinales, nous avons tout d’abord établi la descendance des souches mutantes alg-1(gk214) et alg-2(ok304). Ces deux souches de nématodes ont un faible nombre de descendant qui peut s’expliquer par un problème dans la prolifération des cellules germinales, de méiose ou dans la formation de gamètes. Une analyse précise des gonades de ces animaux indique une plus petite région mitotique avec un nombre de cellules germinales en prolifération inférieur à celui retrouvé chez les animaux de type sauvage. Nous avons aussi observé que les cellules entrent en méiose de manière plus précoce dans les animaux alg-1 et alg-2 mutants, que ces mutants ont des défaut dans la formation de gamètes et qu’ils ont un nombre plus élevé de cellules germinales apoptotiques. En utilisant l’immunofluorescence et des rapporteurs d’expression, nous avons confirmé que ALG-1 et ALG-2 sont exprimées dans la DTC, une cellule spécialisée situé à l’extrémité distale des gonades de C. elegans qui est au cœur de la régulation de la transition mitose-méiose des cellules germinales. En utilisant une lignée transgénique qui exprime ALG-1 exclusivement dans la DTC, nous pouvons partiellement rétablir le nombre de descendants ainsi que rétablir totalement le nombre de cellules retrouvé dans la région mitotique. De façon intéressante, nous observons que la perte de cinq microARNs exprimés dans la DTC mène à des phénotypes similaires à ceux observés dans les mutants alg-1 et alg-2. Finalement, l’analyse de l’expression génique par micropuces des gonades de vers alg-1 mutant indique que la voie des microARNs contribue à la régulation de différentes voies moléculaires importantes pour la prolifération et la différentiation des cellules germinales. L’ensemble de ces études supporte l’implication de la voie des microARNs dans le contrôle de la biogénèse des cellules germinales chez C. elegans. / Small non-coding RNA pathways assume pleiotropic roles in the regulation of multitude of biological processes. These non-coding RNAs have been shown to be involved in germline development in diverse species, in addition to their well-known participation in gene regulation and viral resistance pathways. However, the contribution of the miRNA, one of the small non-coding RNA pathways in germline biogenesis has remained elusive. Since ALG-1 and ALG-2 are exclusively involved in the miRNA pathway and indispensible for miRNA mediated gene silencing, we decided to genetically manipulate these genes to address whether miRNA pathway plays an important role in germline proliferation and differentiation using C. elegans as animal model. As double knockout of alg-1 and alg-2 leads to sterility, which mirrors the phenotypes of Drosha and Dicer mutants, we reasoned that the miRNA pathway proteins are crucial in germline maintenance. To delineate the role of ALG-1 and ALG-2 in the complex processes of germline regulation, we first investigated the brood size of alg-1(gk214) and alg-2(ok304) animals. Both mutants had significantly decreased brood size, which could result from defects in germline proliferation, meiosis or gamete formation. An extensive analysis of the germline of these mutants revealed a smaller mitotic region with less number of proliferating germ cells compared to the wild type. We also observed early entry into meiosis in alg-1(gk214) and alg-2(ok304). Using immunofluorescence and transgenic reporters, we confirmed ALG-1 and ALG-2 expression in DTC, a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Using transgenic line with alg-1 expressed exclusively in the DTC, we were able to partially rescue the brood size defect and completely restored the number of cells in the mitotic region. These mutants also presented defects in gamete formation and an increase in germ cell apoptosis. Interestingly, we observed that the disruption of five miRNAs expressed in the DTC display similar phenotypes as observed in alg-1 and alg-2 mutants. Finally, gene expression analysis by microarray of alg-1 mutant gonads indicates that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Together, our data supports the role of miRNA pathway in controlling germline biogenesis in C. elegans.

NOTICE EN COURS DE TRAITEMENT / Les microARN (miARN) sont des courts ARN non codants qui régulent l'expression des gènes, au niveau post-transcriptionnel. Ces molécules d'environ 22 nucléotides de long s'associent aux protéines Argonautes (AGO) pour former un complexe appelé microRNA induced silencing complex (miRISC). Ensuite, les miARN recrutent le miRISC à des séquences partiellement complémentaires, dans les régions 3' non traduites d'ARN messagers (ARNm). Le miRISC peut ainsi réprimer la traduction d'ARNm spécifiques et souvent induire leur dégradation. Ce mécanisme est notamment important pour le développement animal et des défauts dans cette voie moléculaire sont liés à diverses pathologies chez l'humain. Des évidences récentes montrent que les interacteurs du miRISC et son mode d'action sur les ARNm peuvent diverger à différents moments du développement du nématode Caenorhabditis elegans. Nous avons donc posé l'hypothèse que des modifications post-traductionnelles pourraient expliquer certaines de ces différences moléculaires et fonctionnelles. Les objectifs de ce projet de recherche étaient donc d'identifier les événements de phosphorylation sur la protéine Argonaute ALG-1 de C. elegans et de déterminer leur fonction biologique au cours du développement animal. À cette fin, nous avons purifié la protéine Argonaute ALG-1 chez C. elegans avec un anticorps spécifique, ainsi que ses orthologues humains AGO 1-4, à partir de cellules humaines en culture. Nous avons déterminé par spectrométrie de masse les modifications post-traductionnelles sur ces protéines. En utilisant des méthodes de mutagenèse par édition du génome chez C. elegans, nous avons criblé l'importance de nombreux sites de phosphorylation en s'attardant aux phénotypes associés à la perte de fonction des miARN. Ceci nous a permis de mettre en évidence l'importance d'une région phosphorylable conservée de cinq résidus sérines/thréonine sur le domaine PIWI des Argonautes. La perte de phosphorylation de ALG-1, lorsque ces acides aminés sont mutés en alanines, produit des phénotypes développementaux beaucoup plus sévères que chez des animaux déplétés du gène alg-1. Au niveau moléculaire, nous avons montré, à partir de cellules humaines en culture, que l'hyperphosphorylation de ces acides aminés réduit l'association aux ARNm. De plus, nous avons montré que des mutants AGO2 qui ne sont pas en mesure de lier les miARN, ne sont pas hyperphosphorylés sur ces résidus dans les cellules humaines en culture. Ces résultats mettent en évidence un nouveau mécanisme de régulation de la voie de miARN, dans lequel l'hyperphosphorylation du domaine PIWI de l'Argonaute permet la dissociation du miRISC de sa cible. Nous proposons donc que la phosphorylation de cette région permettrait au miRISC d'être recyclé et de réprimer l'expression d'autres ARNm après sa déphosphorylation. En second lieu, notre crible a permis d'identifier une sérine phosphorylable sur le domaine MID de ALG-1 qui régule l'association de la protéine aux miARN, lors du développement du nématode. Nous avons montré que lorsque cette sérine est mutée en glutamate (phospho-mimétique) ALG-1 perd son association aux miARN. Par ailleurs, les animaux qui portent cette mutation présentent des niveaux de miARN moins élevés que chez les animaux sauvages, ainsi qu'une accumulation de brins passagers qui sont issus des duplex de miARN et normalement dissociés par AGO. Nous avons ensuite identifié l'enzyme qui produit la phosphorylation de cette sérine. Avec des expériences de phosphorylation in vitro, nous avons montré que cette phosphorylation pourrait être induite par la protéine kinase A (PKA). De surcroît, nos expériences soutiennent que alg-1 et PKA interagissent génétiquement. Précisément, le mutant non phosphorylable alg-1(S642A) supprime des phénotypes développementaux observés lors de la perte de fonction de la sous-unité régulatrice de PKA, kin-2. En somme, ce projet de recherche a permis de mettre en évidence un mécanisme conservé au cours de l'évolution qui régule l'association du miRISC aux ARNm par la phosphorylation des Argonautes, ainsi qu'un mécanisme qui régule l'association de ALG-1 aux miARN chez C. elegans. Notre étude indique d'ailleurs que le miRISC serait possiblement inhibé à des moments précis lors du développement animal, par exemple lors de la phosphorylation par PKA. Les études futures des voies signalétiques qui activent PKA chez le nématode nous permettra de mieux comprendre la fonction biologique et le contexte cellulaire qui requerrait l'inactivation du miRISC. / MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression in eukaryotes. These molecules are ~22 nucleotides in length and associate with Argonaute proteins (AGO) to guide them to mRNAs that contain sequences with partial complementarity, commonly found in the 3' untranslated region (UTR). The interaction between the miRISC (miRNA induced silencing complex) and the mRNA inhibits protein synthesis and often leads to degradation of the transcripts. While the function and importance of this gene regulation pathway has been studied in plant and animal models, mechanisms that modulate the miRISC gene silencing efficiency in different biological settings are still poorly understood. The hypothesis of my research project conveys the idea that post-translational modifications of Argonaute proteins modulate gene silencing during animal development. To test this hypothesis, we aimed to identify phosphorylation events on the Argonaute ALG-1 in the nematode C. elegans and uncover how these modifications affect its function during animal development. We purified ALG-1 protein from C. elegans extracts with a specific antibody and human Argonautes AGO1-4 from human cell cultures. We identified phosphorylated Argonaute peptides using mass spectrometry analysis and then we screened which modification affected ALG-1 function using gene editing. This led to the discovery of a highly conserved serine/threonine phosphorylation cluster on the PIWI domain of the Argonaute that when mutated into non-phosphorylatable amino-acids (alanine) caused phenotypes that were more severe than the loss of alg-1 in C. elegans. Molecular analysis of these phosphorylation sites showed that they modulate association to miRNA targets. Specifically, when using phospho-mimicking mutations on human AGO2, we showed that the hyperphosphorylation of this cluster causes the Argonaute to lose interaction with mRNAs. Furthermore, we showed that AGO mutants that are deficient for miRNA binding do not undergo hyperphosporylation. These results revealed a new mechanism that regulate miRNA-mediated gene silencing by which unphosphorylated AGO binds miRNA targets and following hyperphosporylation the miRISC is released from mRNAs. We proposed that this mechanism could be used by cells to recycle the complex and permit multiple rounds of silencing by the miRISC after dephosphorylation. Our forward genetic screen of ALG-1 phosphorylation sites identified a serine on the MID domain that modulates association to miRNAs. We showed that phospho-mimicking mutation of ALG-1 at this position impaired the ability of ALG-1 to bind most miRNAs. Furthermore, we found that this mutation led to accumulation of passenger strands miRNAs in the total RNA. Since the passenger strands are not bound by the phospho-mimicking mutant, we suggested that they accumulate as duplexes which would render them refractory to degradation by single stranded nucleases. Last we showed that the protein kinase A (PKA) phosphorylates this residue in vitro and interacts genetically with alg-1. Altogether, this research project uncovered new mechanisms that regulate the miRNA pathway through the phosphorylation of the Argonaute proteins. Our study also suggests that ALG-1 is inhibited at specific timing by PKA during C. elegans development, and further study of the biological settings that require this inactivation will be crucial to understand its function.

Under unfavorable environmental conditions Caenorhabditis elegans larvae enter a dauer stage which is a specialized non-feeding larval stage. In the dauer stage, worms display astonishingly low metabolism, which allows them to adapt themselves to environmental stress and to dwell without food for several months. Dauer larvae can enter into the reproductive larval stage, when environmental conditions become favorable. In this study, the metabolic transition of dauers into the reproductive larval stage is analyzed in detail: a. During the exit of dauers, several metabolic traits were examined. Primarily, dauer larva initiates the metabolic transition by activating feeding, which is followed by upregulated oxygen consumption and mitochondrial remodeling, as well as enhanced protein synthesis. b. To better understand the metabolic transition, inhibitors of the dauer exit were introduced. Lithium ions were shown to inhibit the transition of dauers to reproductive larvae and prevent the upregulation of metabolic activities required for this process. c. In liquid culture, the transition from the dauer to the reproductive larva is also inhibited, presumably because of the hypoxic character of the liquid culture. Thus, hypoxia has a negative effect on the metabolic transition. d. In the course of our investigation we discovered that the dauer larva is not a closed system but indeed, it can dwell on the externally available ethanol as a carbon source by incorporating it into the energy metabolism. This allows dauers to survive for longer periods in the absence of bacteria, the preferred food of worms. These findings clarify the nature of dauers, how they utilize distinct pathways during the metabolic transition and how they take advantage of the externally available carbon source. These results may in the future enable us to elucidate the complex pathways of metabolism, as well as the ways in which it can be regulated.

Depuis une vingtaine d’années, il est possible d’allonger la durée de vie génétiquement. Nombre d’études réalisées sur des espèces allant de la levure aux primates, ont permis d’identifier des cascades de signaux intracellulaires ayant un impact sur la longévité et la qualité du vieillissement. Il est important de noter que certaines de ces interventions réduisent considérablement l’incidence de cancers et de maladies liées au vieillissement chez les mammifères. Ceci témoigne des liens existant entre vieillissement et carcinogénèse et il probable que le développement de stratégies pharmacologiques ayant pour cible le vieillissement se révèlent efficaces contre les maladies du vieillissement comme le cancer, la maladie d’Alzheimer ou de Parkinson. Nous avons criblé la banque de mutants de Saccharomyces cerevisiae pour identifier des mutations génétiques qui augmentent la durée de vie. La plupart des gènes identifiés se sont révélés conservés puisqu’ils influencent aussi la longévité chez C. elegans. La protéine de liaison à l’acyl-CoA (ACBP) est une petite protéine (10 kDa) qui se lie avec une haute affinité aux chaîne d’acyl-CoA esters (moyennes et longues) et les transporte vers les sites de consommation de l'acyl-CoA. ACBP est hautement conservée parmi les espèces eucaryotes et joue un rôle important dans la biosynthèse des lipides et le trafic vésiculaire. Chez Saccharomyces cerevisiae, la délétion d’ACBP (ACB1) entraîne une augmentation de la longévité et favorise la résistance au stress. Pour tester si l’impact d’ACBP sur la longévité s'étend aux eucaryotes supérieurs, nous avons exploré le lien entre les gènes codant pour des ACBPs chez Caenorhabditis elegans et la longévité en utilisant l’ARN interfèrent. Chez C. elegans, sept paralogues ACBP ont été identifiés, qui sont exprimés dans différents tissus. Nous avons constaté que la réduction de l'expression de maa-1 (codant une ACBP associée aux membranes) prolonge la durée de vie des vers sauvages. Nos résultats démontrent que: 1) une perte de fonction de maa-1 entraîne une résistance au superoxyde, 2) et aux événements protéotoxiques telle que l'agrégation protéique associées aux maladies neurodégénératives comme la maladie de Huntington. Enfin, nous avons montré que l'activité du facteur de transcription HIF-1 (hypoxia inducible factor-1) contribue à la longévité causée par la mutation maa-1. En effet, la délétion du gène hif-1 annule complètement l’augmentation de la longévité causée par maa-1. / Understanding the aging process, its regulation, and how to delay it has become a priority for an increasing number of scientists worldwide. The principal reason for this is that it is becoming more and more evident that anti-aging interventions may be effective against age-related diseases such as cancer, cardiovascular, and neurodegenerative diseases. Simple model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae have been instrumental to identify the principal genes implicated in aging whose role has turned out to be conserved in mammals. The project presented here has originated from a genome-wide screen performed in S. cerevisiae that has led to discover several novel life span-regulatory genes whose deletion prevents aging. One of these genes encodes for Acyl-CoA binding protein (ACBP). ACBP is a small (10 kDa) protein that binds medium- and long-chain fatty acyl-CoA esters with high affinity and transports them to acyl-CoA consuming processes. ACBP is highly conserved among eukaryotic species and plays important roles in lipid biosynthesis and vesicle trafficking. In S. cerevisiae, lack of ACBP (Acb1) extends longevity and promotes stress resistance. To test whether the life span-regulatory role of ACBP extends to higher eukaryotes, we explored the link between the C. elegans ACBP genes and longevity by RNAi screening. In C. elegans, seven ACBP paralogs have been identified, which are expressed in different tissues. We found that reducing the expression of maa-1 (encoding a membrane associated ACBP) extended the longevity of wild-type worms. Our results show that 1) a loss of function maa-1 mutant is resistant to the superoxide-generating agent paraquat and 2) reduction of maa-1 expression increases resistance to the proteotoxicity associated with the aggregation of the Huntington's disease-associated polyQ peptide. The activity of the anti-aging transcription factor HIF-1 (hypoxia inducible factor-1) contributes to the extended longevity caused by lack of maa-1. The effect of MAA-1 loss on longevity was fully reverted by the deletion of the hif-1 gene.

L’adaptation cellulaire au stress dépend en partie de changements dans l’expression de gènes de réponse au stress, souvent accompagnés par des modifications dans la structure chromatinienne. Des facteurs chromatiniens pourraient être à l’origine de ces modifications mais leurs mécanismes d’action restent mal connus au cours du développement. La réponse aux protéines malconformées (UPR) est une réponse à des conditions de stress physiologique qui ciblent le réticulum endoplasmique (RE) ; l’UPR a été impliquée dans de nombreuses maladies humaines incluant le cancer et différents composants de cette réponse pourraient être de potentielles cibles pharmaceutiques. Nous avons démontré que HPL-2, l’homologue de la protéine HP1 chez Caenorhabditis elegans, est nécessaire pour la réponse au stress du RE. L’inactivation d’HPL-2 montre une résistance accrue au stress du RE qui dépend d’une part de la voie XBP-1 de l’UPR et d’autre part d’un flux autophagique augmenté. La résistance accrue des vers dépourvu d’HPL-2 est associée avec une augmentation de l’activation d’XBP-1 et de chaperonnes du RE en conditions physiologiques. L’expression d’HPL-2 est ubiquitaire et nous avons déterminé qu’HPL-2 joue un rôle antagoniste dans les cellules neuronales et intestinales pour influencer la réponse au stress du RE. Nous avons également montré qu’une modulation de l’état de la chromatine par une inhibition chimique d’histones déacétylases donnait le même phénotype que l’absence d’HPL-2. De plus, l’augmentation ou la diminution de la méthylation de la lysine 4 de l’histone 3 (H3K4me) joue également un rôle dans la réponse au stress du RE. Ces travaux contribuent ainsi à une meilleure compréhension du lien entre l’UPR, le stress du RE et la structure chromatinienne aussi bien dans un processus normal que dans certaines pathologies. / Cellular adaptation to environmental changes and stress relies on a wide range of regulatory mechanisms which are tightly controlled at several levels, including transcription. Chromatin structure and chromatin binding proteins are important factors contributing to the transcriptional response to stress. However, it remains largely unknown to what extent specific chromatin factors influence these distinct responses in a developmental context. One of the best characterized stress response pathways is the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). Here, we show that Caenorhabditis elegans HPL-2, the homologue of the HP1 chromatin associated protein, is required for the ER stress response. Inactivation of HPL-2 results in enhanced resistance to ER stress dependent on the XBP-1 branch of the UPR and the closely related process of autophagy. Increased resistance to ER stress in animals lacking HPL-2 is associated with increased basal levels of XBP-1 activation and ER chaperones under physiological conditions. Using tissue specific rescue experiments, we find that HPL-2 plays antagonistic roles in intestinal and neuronal cells to influence the ER stress response. We further show that chemical inhibition of histone deacetylase activity mimics the HPL-2 loss of function phenotype, and that increasing or decreasing histone H3 lysine 4 methylation (H3K4me) has antagonistic effects on animal survival in response to ER stress. Altogether our results point to an important function for specific chromatin factors and chromatin modifications in maintaining ER homeostasis in a developmental context.

No presente trabalho o gênero fóssil Bauruemys elegans é redescrito. Sua anatomia craniana e pós-craniana foi analisada em detalhes com base em novos exemplares coletados deste fóssil. Bauruemys elegans é um quelônio do Cretáceo Superior da Bacia Bauru, encontrado no Município de Pirapozinho, interior do estado de São Paulo e descrito originalmente por Suárez em 1969 como “Podocnemis elegans". Os materiais analisados foram comparados com a osteologia de representantes atuais dos clados Chelidae, Pelomedusidae e Podocnemididae, bem como com descrições de outros táxons disponíveis na literatura. As relações filogenéticas de Bauruemys elegans dentro do clado Podocnemidinura foram analisadas através da análise de parcimônia envolvendo 19 táxons e 56 caracteres. Nesta análise Bauruemys elegans constitui, de fato, um gênero distinto entre os podocnemidídeos, corroborando propostas já descritas na literatura. A análise também revelou que, através dos caracteres osteológicos, Bauruemys elegans possui afinidades filogenéticas próximas aos podocnemidídeos atuais, posicionando-se como grupo-irmão do clado formado pelo fóssil Cambaremys langertoni e pela Família Podocnemididae. / In this study, the fossil Bauruemys elegans is redescribed. Its cranial and postcranial anatomy was analysed in detail based on new specimens collected. Bauruemys elegans is a chelonian from Upper Cretaceous of the Bauru Basin founded in the municipality of Pirapozinho, São Paulo state and was originally described by Suarez (1969) as Podocnemis elegans. The specimens analysed was compared with some extant representatives of Chelidae, Pelomedusidae and Podocnemididae families and descriptions avaliable on the literature. The phylogenetic affinities of the Bauruemys elegans within Podocnemidinura was evaluated through a parsimony analysis envolving 19 taxa and 56 characters. The results of phylogenetic analysis shows that Bauruemys elegans is, in fact, a distinct genus within the clade Podocnemididae. This corroborated anterior hypothesis proposed. Adittionally, this analysis using osteological characters, indicate Bauruemys elegans as the sister group of Cambaremys langertoni and Podocnemididae.

Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process.

"We are using genetic and molecular techniques to study a simple model organism, C. elegans, to determine the cues involved in the formation of the nervous system. Two molecules currently being studied in the laboratory play roles in the formation of the IL2 neurons, a class of sensory neurons in C. elegans. The first gene, dig-1, influences the sensory process or dendrite and is involved in adhesion as well as potentially providing directional information during development. The second gene, mig-10, influences the axon and may be involved in a cell signal cascade. Genetic screens of C. elegans using Ethyl methyl sulfonate (EMS) as a mutagen resulted in the isolation of mutants with defects in the IL2 sensory map; sensory processes followed aberrant paths, appearing to be defasciculated. Complementation tests showed that the mutations failed to complement n1321, a known allele of dig-1; thus, these new mutations were alleles of dig-1 (Ryder unpub. results). Several of these new alleles of dig-1, including nu336 and n1480, have been further studied to elucidate the role of this gene in sensory map formation. A dig-1 candidate gene was identified that encodes a protein that is a member of the immunoglobulin super-family (IgSF). The candidate gene is predicted to be a large gene, with a transcript of approximately 45Kb. The encoded protein contains three distinct regions and is similar to the hyalectan family of proteoglycans. N terminal region 1 contains immunoglobulin and fibronectin-like domains. Central region 2 is an area that is highly repeated with a potential to have GAGs attached. C-terminal region 3 contains domains associated with adhesion. Polymerase chain reaction (PCR) products from alleles nu336 and n1480 were amplified and sequenced from the candidate gene. The DNA lesion present in the candidate gene from both alleles fit the method for how that mutation was generated. The point mutation in allele nu336 removes a potential glycosylation site. The large re-arrangement in allele n1480 truncates the transcript, suggesting that the protein is also truncated. The sequencing results along with rescuing data (R. Proenca, personal communication) showed that the candidate gene for dig-1 was the gene of interest. Each of the alleles was further studied to determine how severe that allele was by looking at the neuronal process aspect and the brood size as well as displacement of the gonad. In general, alleles with severe defects in the nervous system also had severe gonad displacement, suggesting the gene functions similarly in the two tissues. To determine if the gene was expressed at the RNA level, reverse transcriptase polymerase chain reaction (RT-PCR) was used. Most of the RT-PCRs amplified a cDNA of the appropriate size that showed dig-1 was expressed at the RNA level. RT-PCR further suggested that all three regions were in one transcript as well as confirming part of the predicted exon structure to be correct. In addition, northern analysis showed the presence of a large transcript in wildtype worms as well as a smaller truncated transcript from allele n1480. To investigate developmental differences mixed stage of RNA and embryonic RNA from wildtype animals were compared using gene specific primers. The initial RT-PCR showed potential alternative splicing occurring at the 5? end of the gene during development. To examine expression at the protein level, two recombinant proteins from dig-1 were successfully made by cloning cDNA products from the 5?and 3? end of dig-1. The constructs were sequenced and shown to be in frame. The recombinant proteins (Ant1Con1 and Ant3Con3) were mass produced and sent to a commercial source for injection into pre-screened rabbits. Western analysis showed the presence of an antibody in the serum from two of the rabbits. These antibodies should prove useful in future determination of correctness of our models of DIG-1 function. IgSF members have been shown to have many roles in nervous system development. DIG-1 could act in either an attractive or a repellent role to position sensory processes during development. DIG-1 might also change its function over time; early in development DIG-1 could be adhesive and later become repellant as more sugars are added. The gene mig-10 is involved in sensory map formation. To localize MIG-10 expression, several transgenic animals were generated by injection of two constructs that should recombine in the worm to create a MIG-10::GFP fusion protein. Ten transgenic lines were generated and screened by PCR for the presence of the correct recombinant construct. If this construct makes functional, rescuing protein, the GFP expression should reflect the expression pattern of the MIG-10 protein."

De acordo com dados da Organização Mundial da Saúde (OMS) estimam-se que os agrotóxicos causam anualmente 70 mil intoxicações agudas e crônicas que evoluem para óbito. Dentre eles, o paraquat (PQ) é o que apresenta maior taxa de mortalidade, sendo responsável por cerca de 13% de todos os casos registrados, principalmente devido a falta de um tratamento efetivo. O principal mecanismo de toxicidade proposto está associado ao ciclo redox do PQ, onde ocorre a formação de espécies reativas (ERs) de oxigênio e nitrogênio, levando ao estresse oxidativo (EO). Na literatura há relatos do uso de antioxidantes para casos de intoxicação do PQ. Dessa maneira, nesse trabalho avaliou-se o uso de melatonina associada a nanocápsulas de núcleo lipídico (Mel-LNC) na proteção contra os danos causados pelo PQ, uma vez que o uso da nanotecnologia melhorou a atividade antioxidante dessa molécula. Para tal utilizou-se o sistema in vitro, linhagem celular de adenocarcinoma pulmonar (A549), e o modelo alternativo in vivo, Caenorhabditis elegans. Mel-LNC e nanocápsulas de núcleo lipídico (LNC) foram preparadas de acordo com o método de deposição do polímero pré-formado. Ambas as formulações foram caracterizadas avaliando tamanho de partícula, potencial zeta e pH, e para Mel-LNC foram determinadas a concentração de melatonina e porcentagem de encapsulação. Os resultados encontrados estão de acordo com os parâmetros já validados para essas formulações. Foi possível verificar que as formulações MEL-LNC e LNC se mantiveram estáveis nos meios de cultura utilizados nos ensaios in vitro e in vivo. No estudo in vitro foi observado que o tratamento com ambas as formulações não causaram diminuição da viabilidade nem dano de DNA na linhagem celular utilizada. Além disso, foi verificado a internalização da Mel-LNC utilizando-se a formulação marcada com rodamina B, sendo possível verificar uma intensa fluorescência vermelha ao redor do núcleo da célula. O pré-tratamento com Mel-LNC foi capaz de aumentar a viabilidade celular e diminuir o dano oxidativo de DNA causado pelo paraquat após 24 horas de exposição, porém isso não ocorreu quando as células foram pré-tratadas com melatonina livre. No estudo com o modelo alternativo C. elegans, foi utilizada uma formulação de Mel-LNC marcada com rodamina B (Mel-LNC-RoB), a fim de verificar a absorção dessa formulação pelo nematoide. Foi possível observar que a internalização da Mel-LNC no C. elegans ocorre principalmente pela via oral, uma vez que se verificou uma intensa fluorescência no intestino do nematoide após o tratamento com a Mel-LNC-RoB e após três horas, essa fluorescência se distribuiu pelo restante do corpo, apresentando inúmeros pontos de fluorescência fora do intestino. Com relação à avaliação do efeito protetor nesse modelo alternativo in vivo, pode-se inferir que o pré-tratamento com Mel-LNC aumentou a sobrevida, diminuiu a produção de espécies reativas (ERs) e manteve o desenvolvimento normal dos nematoides após a exposição ao PQ, sendo que isso não foi verificado quando os mesmos foram pré-tratados com melatonina livre. Além disso, verificou-se que as nanocápsulas de núcleo lipídico (LNC) são seguras para o uso no modelo C. elegans, uma vez que apresentou alto valor para a dose letal 50 (DL50), e alterações no desenvolvimento e produção de ERs somente ocorreram em doses mais elevadas que as utilizadas em nossos experimentos. Dessa maneira, a formulação de Mel-LNC mostrou-se um promissor candidato para estudos futuros nos casos de intoxicação por paraquat. / According to estimations by World Health Organization (WHO), pesticides are responsible for 70 thousand acute intoxication cases that lead to death per year. Among these compounds, paraquat (PQ) presents the highest mortality rate, about 13% of all registered cases, especially for the lack of effective treatment. The major mechanism of toxicity proposed is associated to its redox cycle, in which oxygen and nitrogen reactive species (RS) are generated culminating in oxidative stress (OS). Some reports in the literature support the use of antioxidants for PQ intoxication cases. The present study aimed to evaluate the use of melatonin-loaded lipid-core nanocapsules (Mel-LNC) in the protection against PQ-induced damages, considering that nanotechnology has improved the antioxidant activity of this molecule. For this purpose, an in vitro system composed by lung adenocarcinoma (A549) cell line, and the in vivo alternative model of Caenorhabditis elegans have been utilized. Mel-LNC and unloaded lipid-core nanocapsules were prepared by self-assembly and characterized by particle sizing, zeta potential and pH, and for Mel-LNC formulation it was determined the drug content and encapsulation efficiency. The results are in agreement with the parameters already validated for these formulations. It was possible verify that Mel-LNC and LNC formulations remained stable in the culture medium utilized in in vitro and in vivo experiments. Results from in vitro studies showed that none of the formulations induced reduction in cell viability or DNA damage in treated cells. Besides, it was observed the internalization of Mel-LNC marked with rhodamine B, showing an intense red fluorescence around the cell nucleus. Pretreatment with Mel-LNC was able to enhance cell viability and diminish DNA oxidative damage caused by paraquat after 24h exposure, which could not be observed when cells were pretreated with Mel. In the study with the alternative model C. elegans, a rhodamine (Ro)-linked Mel-LNC formulation was prepared in order to assess the absorption of the formulation by the nematode. Mel-LNC uptake in C. elegans was found to occur mainly by the oral route, once an intense fluorescence was observed in the intestine after treatment with Mel-LNC-RoB, which after 3h distributed to the rest of the body, presenting numerous fluorescence dots outside the intestine. In relation to the evaluation of protection with the in vivo alternative model, results indicate that pretreatment with Mel-LNC increased survival rate, reduced the production of reactive species and maintained the normal development of nematodes after paraquat exposure, while the same observations were not found after pretreatment with free melatonin. In addition, the lipid-core nanocapsules (LNC) were found to be safe in the C. elegans model, due to its high lethal dose (LD50) value, and development alterations and RS production only occurred in the higher doses than those utilized in our experiments. Therefore, the Mel-LNC formulation demonstrated to be a promising candidate for future studies aiming treatment of paraquat intoxication cases.

As embryos proceed through development, they must undergo a series of cell fate decisions. At each division, potency is progressively restricted until a terminally differentiated, postmitotic cell is produced. An important part of that cell type determination is repression of alternative fate possibilities. In this thesis, I have explored the mechanisms by which a single transcription factor activates certain cell fates while inhibiting others, using the Caenorhabditis elegans ALM and BDU neurons as a model. ALM neuron identity is regulated by two interacting transcription factors: the POU homeobox gene unc-86 and the LIM homeobox gene mec-3. I investigated fate determination in BDU neurons, the sister cells of ALM. I found that BDU identity is broadly defined by a combination of unc-86 and the Zn finger transcription factor pag-3, while the neuropeptidergic subroutine of BDU is determined by the LIM homeobox gene ceh-14. In addition, I found that reciprocal homeotic transformations occur between ALM and BDU neurons upon loss of either mec-3 or pag-3. In mec-3 mutants, ALM neurons acquire the gene expression profile and morphological characteristics of BDU cells, while in pag-3 mutants, BDU neurons express genes normally found in ALM and change some aspects of their morphology to resemble ALM. While these fate switches appear to be a simple case of cross-repression, the mechanism is in fact more complicated, as pag-3 is expressed not just in BDU but also in ALM. In this thesis, I present evidence that MEC-3 inhibits execution of BDU identity in ALM by physically binding to UNC-86 and sequestering it away from the promoters of BDU genes. This work expands upon the literature examining simultaneous activation of one identity program and repression of alternate programs by introducing a novel mechanism by which a transcription factor competes to direct specific cell fates.