MicroRNAs (miRNAs) are endogenous non-coding small RNAs that posttranscriptionally regulate gene expression primarily through binding to the 3’ untranslated region (3’UTR) of target mRNAs, and are known to play important roles in various developmental and physiological processes. The work presented in this thesis was centered on understanding how Caenorhabditis elegans miRNAs are modulated by genetic, environmental, or physiological factors and how these small RNAs function to maintain the robustness of developmental processes under stressful conditions. To identify modulators of the miRNA pathway, I developed sensitized genetic backgrounds that consist of a panel of miRNA gene mutants and miRNA biogenesis factor mutants with partially penetrant phenotypes. First, I found that upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an opportunistic pathogen of diverse plants and animals, let-7 family miRNAs are engaged in reciprocal regulatory interactions with the p38 MAPK innate immune pathway to maintain robust developmental timing despite the stress of pathogen infection. These let-7 family miRNAs, along with other developmental timing regulators, are also integrated into innate immune regulatory networks to modulate immune responses. Next, I demonstrated that loss-of-function mutations of Staufen (stau-1), a double-stranded RNA-binding protein, increase miRNA activity for several miRNA families, and this negative modulation of Staufen on miRNA activity acts downstream of miRNA biogenesis, possibly by competing with miRNAs for binding to target mRNA 3’UTRs. In summary, these studies provide a better understanding on how miRNAs are modulated by various environmental and cellular components, and further support the role of the miRNA pathway in conferring robustness to developmental processes under these perturbations.

Though symptoms such as loss of vision, decline in cognition and memory are evident during aging, the underlying processes that affect neuronal function during aging are not well understood. Unlike changes in other tissues and organs, age-related changes in the nervous system affect the overall physical, mental as well as social state of human beings. To start elucidating the molecular mechanisms underlying normal age-dependent brain decline, we have characterized structural neuronal changes occurring during Caenorhabditis elegans aging. Our analysis reveals distinct neuronal alterations that arise with age and that the types of changes and their age of onset are neuronal-type specific, highlighting the differential susceptibility of neurons to the stresses of life. We also find that these age-dependent neuronal changes are largely uncoupled from lifespan. As a first step towards understanding the neuropathological conditions manifested during senescence, we have characterized the role of the neuronal maintenance gene sax-7/L1CAM in normal C. elegans aging. Our comparison of age-related structural changes in the wild-type nervous system with that of sax-7 mutants, indicates that loss of function of sax-7 results in accelerated neuronal deterioration that mimics alterations occurring during normal aging. Conversely, overexpressing wild-type copies of SAX-7 delays some of the neuronal changes that accompany normal aging, indicating that SAX-7 plays a neuroprotective role. Additionally we find that x mechanical stress from body movements impacts the neuronal changes during adulthood. Taken together, our results give an entry point into the mechanisms of age-related neuroanatomical changes and neuronal protection.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria. / Dissertation

L’ubiquitin-fold modifier (UFM1) fait partie de la classe 1 de la famille de protéine ubiquitin-like (Ubl). UFM1 et Ub ont très peu d’homologie de séquence, mais partagent des similarités remarquables au niveau de leur structure tertiaire. Tout comme l’Ub et la majorité des autres Ubls, UFM1 se lie de façon covalente à ses substrats par l’intermédiaire d’une cascade enzymatique. Il est de plus en plus fréquemment rapporté que les protéines Ubls sont impliquées dans des maladies humaines. Le gène Ufm1 est surexprimé chez des souris de type MCP développant une ischémie myocardique et dans les îlots de Langerhans de patients atteints du diabète de type 2. UFM1 et ses enzymes spécifiques, UBA5, UFL1 et UFC1, sont conservés chez les métazoaires et les plantes suggérant un rôle important pour les organismes multicellulaires. Le Caenorhabditis elegans est le modèle animal le plus simple utilisé en biologie. Sa morphologie, ses phénotypes visibles et ses lignées cellulaires ont été décrits de façon détaillée. De plus, son cycle de vie court permet de rapidement observer les effets de certains gènes sur la longévité. Ce modèle nous permet de facilement manipuler l’expression du gène Ufm1 et de mieux connaître ses fonctions. En diminuant l’expression du gène ufm-1 chez le C.elegans, par la technique de l’ARN interférence par alimentation, nous n’avons observé aucun problème morphologique grave. Les vers ressemblaient aux vers sauvages et possédaient un nombre de progéniture normal. Cependant, les vers sauvage exposés à l’ARNi d’ufm-1 vivent significativement moins longtemps que les contrôles et ce, de façon indépendante de la voie de signalisation de l’insuline/IGF. Chez le C. elegans la longévité et la résistance au stress cellulaire sont intimement liées. Nous n’avons remarqué aucun effet d’ufm-1 sur le stress thermal, osmotique ou oxydatif, mais il est requis pour la protection contre le stress protéotoxique. Il est également nécessaire au maintien de l’intégrité neuronale au cours du vieillissement des animaux. L’ensemble de nos données nous renseigne sur les fonctions putatives du gène Ufm1. / The ubiquitin-fold modifier (UFM1) is part of the type 1 class of the family of ubiquitin-like protein (Ubl). UFM1 and Ub have very little sequence homology but share remarkable similarities in their tertiary structure. Like Ub and most other UBLS, UFM1 binds covalently to its substrates through an enzymatic cascade. It is frequently reported that UBLs are involved in human diseases. UFM-1 is overexpressed in mice developing a myocardial ischemia and in the islets of patients suffering from type 2 diabetes. UFM1 and its specific enzymes, UBA5, UFL1, and UFC1 are conserved in metazoans and plants suggesting an important role in multicellular organisms. Caenorhabditis elegans is one of the the simplest animal models used in biology. Some features such as morphology, visible phenotypes and cell lineage have completely been described. The short lifecycle of C. elegans makes it easy to observe gene effects on longevity. This model allows us to easily manipulate the expression of the Ufm1 gene and learn more about its putative functions. To study putative functions of Ufm1, we decreased the expression of ufm-1 using RNA interference introduces through feeding. No gross morphological disturbances were observed; worms resembled wild type and had a normal brood size. However, worms exposed to ufm-1 RNAi had a significantly shorter lifespan than the controls. This effect is independent of the insulin/IGF pathway, which is a major axis of longevity genetics. In C. elegans longevity and cellular stress resistance are intimately linked. We have observed no effect of ufm-1 on thermal, osmotic or oxidative stress, but it is required for protection against proteotoxic stress. It is also necessary to maintain neuronal integrity during aging. Together, our results shed light on putative functions of Ufm1 gene.

Cette thèse est dédiée aux développements méthodologiques et applications de la métabolomique par Résonance Magnétique Nucléaire (RMN) à très hauts champs. La première partie de ce manuscrit est dédiée à une présentation générale de la métabolomique par RMN. Nous décrivons ensuite les résultats obtenus concernant l’introduction d’une technique à dimensionnalité réduite pour la caractérisation des mélanges complexes, dénommée spectroscopie RMN par projections ciblées. La seconde partie de ce manuscrit décrit les résultats de trois études métabolomiques portant sur des populations humaines. La première analyse démontre que les échantillons de sérum collectés dans le cadre de la cohorte européenne prospective internationale EPIC sont appropriés pour une étude métabolomique. Les deux études suivantes recherchent une signature métabolique dans le sérum du cancer du sein métastatique et une signature plasmatique potentielle pour différentes pathologies hépatiques comme le carcinome hépatocellulaire. La troisième partie de cette thèse est dédiée à l’étude d’organismes modèles. La première étude caractérise les différences métaboliques systémiques entre quatre souches de rats couramment utilisées comme contrôles en génétique. Dans la seconde analyse, nous étudions les effets du vieillissement physiologique chez Caenorhabditis elegans (C. elegans), observons que le processus de restriction alimentaire tamponne les modifications métaboliques associées au vieillissement et que des perturbations du métabolisme de la phosphocholine corrèlent avec l’espérance de vie. La troisième étude caractérise des modifications métaboliques importantes chez un mutant de C. elegans, pour le gène ahr-1, suggérant un rôle dans le développement et le vieillissement. Enfin, nous étudions les effets au niveau métabolique de l’interaction entre la protéine endogène E4F1 et la protéine virale HBx dans des cellules hépatiques infectées par le virus de l’hépatite B. / This thesis is dedicated to developments and applications of metabolomics, exploiting high field NMR spectroscopy. The first part is dedicated to a general presentation of metabolomics. We also report results about the introduction of reduced dimensionality techniques for the characterization of complex mixtures, coined targeted projection NMR spectroscopy. The second part of this manuscript reports results about three different metabolomic studies carried out in human populations. The first analysis demonstrates the suitability for metabolomics of serum samples collected in the framework of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. The second study investigates a serum metabolic signature of metastatic breast cancer. The last analysis establishes potential plasma metabolic signatures for different liver pathologies, like hepatocellular carcinoma. The third part of this thesis is dedicated to the characterization of various model organisms. The first study presents a characterization of plasma and urine metabolic differences between four rat strains commonly used as controls in genetic studies. In the second study, we investigate the effects of physiological aging in Caenorhabditis elegans (C. elegans) and observe that dietary restriction buffers metabolic changes associated with aging. We further identify that perturbations in phosphocholine metabolism correlate with life expectancy. The third analysis of this part characterizes the ahr-1 C. elegans mutant, showing strong metabolic changes in ahr-1 mutants, which suggest an involvement in development and aging processes. We finally investigate in the last study the effects at the metabolic level of the interaction between an endogenous protein E4F1 and a viral protein HBx in liver cells infected by hepatitis B virus.

L'évolution contemporaine des organismes vivants est de plus en plus dépendante des perturbations d'origine anthropique. En particulier, la pollution amplifie l'intensité des pressions de sélection auxquelles sont soumises les populations. Or ces changements peuvent avoir des effets négatifs sur la vie des individus, la démographie des populations, mais aussi, au cours des générations, sur leurs caractéristiques phénotypiques et génétiques. Ainsi en réponse aux pressions de sélection, des changements microévolutifs sont susceptibles de se manifester. Mais ces phénomènes entraînent en parallèle la mise en place de coûts adaptatifs qui fragilise le maintien des populations. Il est donc indispensable de développer nos connaissances sur l'évolution des populations en milieu pollué. Dans ce contexte, cette étude vise à déterminer les réponses microévolutives de populations de C. elegans, exposées à diverses pollutions et à mesurer les coûts adaptatifs de ces microévolutions. Ces populations ont été exposées expérimentalement, durant 22 générations, à une forte concentration en uranium et/ou en chlorure de sodium. Nous avons mis en évidence une différentiation génétique des populations exposées, associée à augmentation de la résistance, au cours du temps. La vitesse des réponses évolutives était dépendante des conditions d'exposition et de leurs effets sur l'expression de la structure génétique des traits d'histoire de vie. Ces phénomènes ont pu être reliés à des coûts adaptatifs, comme une réduction de la fertilité, dans de nouveaux environnements stressants (ex : augmentation de la température) ou en l'absence de stress. Ce projet nous a permis de mieux identifier comment une exposition à un ou deux polluants peut affecter la réponse évolutive de populations de C. elegans et d'évaluer les conséquences sur leur sensibilité. / The contemporary evolution of organisms is largely dependent on anthropogenic disturbances. In particular, the pollution amplifies the intensity of selection pressures suffered by populations. However, these changes may have negative effects on the life of individuals, the demographics of the populations, and its phenotypic and genetic characteristics over generations. Thus, microevolutionary changes are likely to occur in response to selection pressures. These phenomenon lead to collateral damages: adaptive costs. Populations can be more susceptible to many environmental changes. Hence, it is essential to expand our knowledge on the evolution of populations in polluted environment. In this context, our study aims to determine the microevolutionary response of C. elegans populations exposed to different pollutions, and to measure their costs of adaptation. Populations were experimentally exposed for 22 generations to a high concentration of uranium and/or sodium chloride. We confirmed the genetic differentiation between populations with an increase of resistance in populations exposed to different pollutions. The speed of evolutionary responses depended on exposure conditions and their effects on the expression of the genetic structure of life history traits. Microevolutionary changes were linked to costs of adaptation, such as reduced fertility, in stressful novel environments (e.g. fast temperature raise) or in the absence of stress. This project allowed us to better identify how exposure to one or two pollutants affects the evolutionary response of C. elegans populations and evaluate the impact on their sensitivity to environmental conditions.

Durante a anidrobiose (um estado ametabólico muito estável), o nematóide Panagrolaimus superbus tolera vários tipos de estresses físicos. A fim de melhor compreender essa extremotolerância, P. superbus foi submetido a regimes de hiperaceleração (RHA) de até 400.000 x g. Surpreendentemente, foi observado que esse verme tolera a exposição à RHA tanto dessecado (i.e., em anidrobiose) como hidratado. Para verificar se esse fenômeno era específico para essa espécie ou algo observável em outros organismos, os mesmos procedimentos experimentais foram realizados no organismo modelo Caenorhabditis elegans. Intrigantemente, C. elegans também mostrou o mesmo perfil de sobrevivência. Ademais, o desenvolvimento, comportamento, morfologia e crescimento populacional desse nematóide também foram analisados após a exposição ao RHA, não sendo observada quaisquer mudanças nesses parâmetros em função da exposição à hipergravidade. Em seguida, foram realizadas buscas (tBLASTn) no genoma de C. elegans por homólogos de genes relacionados ao gravitropismo que são naturalmente encontrados em plantas. Essa busca resultou nos genes dnj- 16 (homólogo ao ARG1), ipla-1 (homólogo ao SGR2) e uma sequência não caracterizada (homóloga a TWD1). Especial atenção foi despendida ao gene dnj-16, uma vez que é o mais conservado entre eles. As análises de RT-qPCR revelaram que o dnj-16 é ligeiramente regulado para baixo durante o RHA, o que não era esperado caso ele possuísse função semelhante ao seu homólogo em plantas. A análise do estado metabólico desse nematoide durante o RHA lançou luz sobre os dados de RT-qPCR, mostrando que a queda na expressão de dnj-16 é provavelmente devida à centrifugação. Posteriormente, diversas análises in silico foram realizadas a fim de caracterizar o gene dnj-16 e a sua respectiva proteína. Inicialmente, a análise comparativa dos domínios DnaJ, transmembrana e coiled coil das proteínas dnj-16, ARG1, ARL1 e ARL2 apontou para uma grande semelhança não apenas na sequência como na estrutura dessas proteínas. Essa grande similaridade motivou análises para desvendar o papel e a origem do gene dnj-16. Três hipóteses ((i) homologia, (ii) convergência e (iii) transferência gênica horizontal (TGH)) foram consideradas na investigação desse intrigante gene. Os resultados obtidos nas análises in silico apontaram para uma TGH mediada por RNA, potencialmente ocorrida a 1325 m.a., como a hipótese mais plausível para explicar a origem de dnj-16 e algumas espécies parasitas do gênero Phytophthora como prováveis mediadores dessa transferência. Dessa forma, os dados apresentados nessa tese mostram pela primeira vez que C. elegans é tolerante a RHA ordens de magnitude mais altas do que se pensava serem compatíveis com a vida multicelular. Além disso, os dados sugerem que dnj-16 foi transferido horizontalmente de plantas para nematoides e que a ultracentrifugação leva a uma redução no metabolismo de C. elegans, o que ajudaria a explicar a sua sobrevivência sob tal condição extrema. Por fim, o conjunto de dados desse trabalho representa contribuições originais para a compreenção da biologia, da genética e da evolução de C. elegans. / During anhydrobiosis (a very stable ametabolic state), the nematode Panagrolaimus superbus tolerates many types of physical stresses. In order to better understand this extremotolerance, P. superbus underwent hyperacceleration regimes (RHA) of up to 400,000 x g. Surprisingly, it was observed that this worm tolerated RHA exposure both dried (i.e., during anhydrobiosis) and hydrated. In order to verify if this phenomenon was specific for this species or something observable in other organisms, the same experimental procedures were performed in the model organism Caenorhabditis elegans. Intriguingly, C. elegans also showed the same survival profile. In addition, the development, behavior, morphology and population growth of this nematode were also analyzed after the exposure to RHA and no changes were observed in these parameters due to the hypergravity exposure. Thereafter, searches (tBLASTn) were performed on C. elegans genome by homologs of gravitropism related genes that are naturally found in plants. These searches resulted in the genes dnj-16 (homologous to ARG1), ipla-1 (homologous to SGR2) and an uncharacterized sequence (homologous to TWD1). Special attention was given to dnj-16 gene, since it is the most conserved among them. RT-qPCR analyzes revealed that dnj-16 is slightly down regulated during RHA, which was not expected if it had similar function to its homologue in plants. Analysis of the metabolic status of this nematode during RHA shed light on the RT-qPCR data, showing that the decrease in dnj-16 expression was probably due to centrifugation. Subsequently, several in silico analyzes were performed in order to characterize the dnj-16 gene and its respective protein. Initially, the comparative analyzis of the DnaJ, transmembrane and coiled coil domains of dnj-16, ARG1, ARL1 and ARL2 proteins pointed to a great similarity not only in the sequence as well as in the structure of these proteins. This great similarity motivated analyzes in order to uncover the real nature of the dnj-16 gene. Three hypotheses ((i) homology, (ii) convergence and (iii) horizontal gene transfer (HGT)) were considered in the investigation of this intriguing gene. The results obtained in the in silico analyzes indicated an RNA mediated TGH, potentially occurred 1325 my (millions of years), as the most plausible hypothesis to explain the origin of dnj-16 and some parasitic species of the Phytophthora genus as probable mediators of this transference. Therefore, data presented in this thesis show for the first time that C. elegans tolerates RHA of magnitude orders higher than it was thought to be compatible with multicellular life. In addition, the data suggest that dnj-16 was transferred horizontally from plants to nematodes and that ultracentrifugation leads to a reduction in C. elegans metabolism, which would help explain its survival under such extreme condition. Finally, the data set of this work represent original contributions to the understanding of the C. elegans\' biology, genetics and evolution.

Die Infektion mit Filarien (Nematoden) ruft massive Schädigungen beim Menschen hervor. Strategien zur Bekämpfung dieser Parasitose basieren auf einer Massenbehandlung mit Ivermectin und Derivaten. Allerdings ist die Behandlung der Patienten zeitaufwändig und teuer. In diesem Zusammenhang stellt Aufklärung molekularer Mechanismen, die die parasitische Lebensform dieser Würmer ermöglichen, einen neuen Ansatzpunkt für die Entwicklung von Therapeutika und Präventivmaßnahmen dar. Die Moleküle Cystatin und Chitinase wurden, auf Grund ihrer immunodulatorischen und katalytischen Eigenschaften, bei der Nager-Filarie Acanthocheilonema viteae als essentielle Proteine identifiziert. Ziel der vorliegenden Arbeit war daher die detaillierte, funktionale Charakterisierung dieser beiden Proteine. Um das räumliche Expressionsmuster und damit potentielle Funktionen des Sekretionsprotein Cystatins zu ermitteln, wurde der frei lebende Nematode Caenorhabditis elegans als heterologes Expressionssystem genutzt. Unter dem Einfluss des Cystatin-Promoters konnte GFP in pharyngealen und rektalen Zellen von C. elegans exprimiert werden. Möglicherweise ist das Cystatin damit bei A. viteae in den Häutungsprozess involviert, da bei C. elegans derartige Enzyme in den pharyngealen Zellen gespeichert werden. Des Weiteren wurde der Häutungsprozess der infektiösen L3 zum Stadium der L4 durch Ausschalten des Gens mittels RNAi um drei Tage verzögert. Allerdings war der Effekt transient und die Verzögerung des Häutungsprozesses beeinflusste weder die Viabilität noch die Infektiösität der Larven. Die Analyse der Regulation von Cystatin während der Entwicklung des Parasiten mittels der Real-Time PCR zeigte, dass das Gen im Stadium der Mikrofilarien, die der Immunantwort des Wirtes voll exponiert sind, maximal exprimiert wird. Für die Chitinase von A. viteae konnte eine essentielle Rolle im Häutungsprozess nachgewiesen werden. Das Ausschalten des Gens führte zu einer Hemmung der Häutung bei 90 % der L3 und damit zu ihrem Tod. Die maximale Expression im L3 Stadium des Parasiten ist ein weiterer Hinweis darauf, dass dieses Protein in den Häutungsprozess involviert ist. Mittels RNAi bei adulten Parasiten konnte die katalytische Rolle der Chitinase beim Abbau des Chitins im Ei bestätigt werden, da hier nur ungeschlüpfte Mikrofilarien ausgeschieden wurden. Die Ergebnisse dieser Arbeit liefern weitere Hinweise darauf, dass sowohl das Cystatin als auch die Chitinase von A. viteae auf Grund ihrer essentiellen endogenen Funtionen attraktive Zielmoleküle in der Bekämpfung von Filariosen darstellen. / Nematodes which cause filariasis have detrimental effect on humans. Strategies to eliminate this disease are based on mass treatment with drugs like ivermectin. However, they have several drawbacks such as long duration required for treatment and tenuous financial supports. Understanding the molecular mechanisms of genes required for parasitism will help to develop novel therapeutic and preventive strategies. The aim of this study was a detailed functional characterization of cystatin and chitinase of Acanthocheilonema viteae, a rodent filarial nematode. To this end, C. elegans was used as a heterologous system to determine the spatial expression pattern of A. viteae cystatin and chitinase and thereby their possible functions. The promoter of cystatin drove the expression of reporter, GFP, to the pharyngeal and rectal gland cells of transgenic C. elegans lines, this being compatible with the fact that cystatin is secreted in the parasites. This also suggests that cystatin in A. viteae is probably required for moulting since generally in C. elegans the enzymes required for moulting are stored in the pharyngeal gland cells. Moreover, knockdown of cystatin by RNAi delayed moulting of the infective L3 to the L4. However, the RNAi effect was transient and the delay in moulting did not affect the viability and infectivity of the larvae. Analyses of the developmental regulation of cystatin by real-time PCR showed that it is maximally expressed in the blood microfilarial stage, which are exposed to the full force of host immune responses. This is compatible to the fact that A. viteae cystatin immunomodulates the host immune responses. This study also determined that chitinase of A. viteae plays an essential role in the moulting of the L3 larvae since knockdown of chitinase inhibited moulting in 90% of the L3 larvae thereby killing them. Moreover, maximum expression of chitinase was observed by real-time PCR in the L3 stage supplementing that it is involved in moulting. RNAi of chitinase in adults led to the release of unhatched microfilariae confirming the essential catalytic role of chitinase in the degradation of the chitin egg shells. This study substantiates that cystatin and chitinase of A. viteae are attractive intervention targets due to their essential endogenous functions in the parasite.

Caenorhabditis elegans possui quatro genes de kpcs (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Em C. elegans, dois dos quatro polipeptídeos de vitelogenina encontrados dentro dos ovócitos, YP115 e YP88, se originam a partir de um precursor polipeptídico (VIT-6) clivado pós-traducionalmente após o motivo RGKR. Nematóides transgênicos foram produzidos com construções repórteres transcricionais de GFP. Foi verificada expressão de kpc-1 tanto em neurônios quanto em células musculares e intestinais. Esses dados, aliados aos dados da literatura para os outros genes kpc de C. elegans, sugerem o envolvimento de KPC-1 no processamento de VIT-6, que é secretada por células intestinais. Ensaios de Western-blot compararam o processamento de VIT-6 em nematóides selvagens, mutantes e knock-down por RNAi para os diferentes genes kpc. A análise de nematóides mutantes e knock-down por RNAi combinado para os outros três genes de convertase de C. elegans confirmou a redundância da atividade dessas enzimas no processamento de VIT-6. / Four kpc genes are found in the Caenorhabditis elegans genome: (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Two of the four vitellogenin polypeptides, YP115 and YP88, originate from a precursor, VIT-6. VIT-6 is cleaved post-translationally after the RGKR motif. Transgenic worms carrying GFP transcription reporter constructs were produced. Expression of kpc-1 has been localized to neurons as well as muscular and intestinal cells. These data, together with the ones available from the literature for the other kpc genes, suggest the involvement of KPC-1 in the processing of VIT-6, which is secreted from intestinal cells. Western-blot analysis compared the pattern of VIT-6 processing in wild-type, mutants and RNAi-treated worms for the other kpcs. Analysis of worms treated by combined RNAi confirmed the redundancy of KPCs in VIT-6 processing.