The importance of arenavirus glycoprotein processing has only been understood within the past decade, with the majority of work focused on the Old World arenaviruses. Evidence has shown that SKI-1/S1P (subtilisin kexin isozyme-1/site 1 protease) is the cellular protease responsible for glycoprotein cleavage in Old and New World arenaviruses. Furthermore, glycoprotein cleavage is shown to be necessary for the production of infectious virus particles in Lassa and Junín viruses. In this thesis, evidence is provided that the recently emerged Chapare virus (New World) is also processed by SKI-1/S1P. Additionally, novel serpin-based SKI-1 inhibitors were shown to effectively prevent SKI-1 mediated cleavage. Using a wide panel of recombinant vesicular stomatitis viruses expressing New World arenavirus glycoproteins, these inhibitors were capable of significantly reducing viral titres. This provides strong evidence that SKI-1 inhibitors can be used as an effective treatment against the majority of New World Clade B arenaviruses and LASV in vivo.

The importance of arenavirus glycoprotein processing has only been understood within the past decade, with the majority of work focused on the Old World arenaviruses. Evidence has shown that SKI-1/S1P (subtilisin kexin isozyme-1/site 1 protease) is the cellular protease responsible for glycoprotein cleavage in Old and New World arenaviruses. Furthermore, glycoprotein cleavage is shown to be necessary for the production of infectious virus particles in Lassa and Junín viruses. In this thesis, evidence is provided that the recently emerged Chapare virus (New World) is also processed by SKI-1/S1P. Additionally, novel serpin-based SKI-1 inhibitors were shown to effectively prevent SKI-1 mediated cleavage. Using a wide panel of recombinant vesicular stomatitis viruses expressing New World arenavirus glycoproteins, these inhibitors were capable of significantly reducing viral titres. This provides strong evidence that SKI-1 inhibitors can be used as an effective treatment against the majority of New World Clade B arenaviruses and LASV in vivo.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

Les inhibiteurs des protéases à sérine (Serpins) constituent une classe d'enzymes très peu étudiée chez les bactéries. Dans ce travail de thèse nous nous sommes intéressés à l'étude des serpins provenant du microbiote intestinal et l'investigation de leur potentiel anti-inflammatoire pour le traitement des maladies inflammatoires chroniques de l'intestin (MICI) chez l'homme. Pour cela nous avons identifié les serpins provenant du microbiote intestinal humain et analysé leur diversité ainsi que leur distribution entre les individus malades et sains. Ces données nous ont permis d'isoler les serpins significativement associées aux MICI. La purification de quarte d'entre elles nous a amené à démontrer qu'elles inhibent les protéases humaines impliquées dans les MICI. L'analyse biochimique et cinétique approfondie de ces protéines a montré qu'elles possèdent des propriétés originales notamment leur efficacité d'inhibition élevée. L'étude de l'effet protecteur de trois serpins chez un modèle animal de colite a démontré pour la première fois l'efficacité des serpins in vivo démontrant ainsi leur potentiel thérapeutique. / Serine protease inhibitors (Serpins) are a class of proteins that reamin poorly studied in bacteria. In this thesis we are interested in the study of serpins originating from the intestinal microbiota and the investigation of their anti-inflammatory potential for the treatment of inflammatory bowel diseases (IBD) in humans. For this we have identified serpins from the human gut microbiota and analyzed their diversity as well as their distribution between healthy and IBD patients. These data allowed isolating serpins significantly associated with IBD. The purification of four of them led us to demonstrate that they inhibit human proteases involved in IBD. Biochemical and kinetic analysis of these proteins showed that they exhibit original properties, in particular their high inhibition efficiency. The study of the protective effect of three serpins in an animal model of colitis demonstrated for the first time the efficacy of serpins in vivo demonstrating thus their therapeutic potential.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFNγ), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFNγ, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFNγ do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFNγ. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFNγ e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFNγ), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFNγ, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFNγ gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFNγ gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFNγ and Stratifin genes.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFNγ), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFNγ, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFNγ do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFNγ. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFNγ e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFNγ), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFNγ, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFNγ gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFNγ gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFNγ and Stratifin genes.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.