Os parâmetros de avaliação de segurança de alimentos geneticamente modificados fundamentam-se na comparação de equivalência substancial entre as variedades e pela inocuidade de proteínas da planta GM com as proteínas encontradas nas plantas convencionais. O objetivo deste trabalho foi avaliar a segurança alimentar de três cultivares de sojas geneticamente modificadas para tolerarem o herbicida glifosato através da determinação da equivalência substancial e do potencial alergênico das mesmas quando comparadas às suas respectivas parentais isogênicas. Seis amostras de soja foram analisadas, sendo três convencionais parentais e três GM, referentes ao cultivo de 2004-2005, em Goiás. Para a composição química foram realizadas análises em triplicata de proteínas, lipídeos, umidade, minerais e fibra alimentar. Análises complementares para determinação de aminoácidos, ácidos graxos, isoflavonas e ácido fítico também foram realizadas. O potencial de alergenicidade foi avaliado em extratos protéicos brutos de três cultivares convencionais e suas correspondentes GM. Os mesmos extratos protéicos foram fracionados para obter as globulinas 7S e 11S por precipitação e posterior purificação em coluna de bioafinidade Sepharose 4B. A glicoproteína 7S foi obtida a partir da eluição com tampão contendo -D-mannopyranoside. A resistência à proteólise foi realizada a partir de dois fluidos gástricos simulados com pepsina nas proporções 2,5:100 e 13:1 de enzima/substrato. As amostras foram submetidas à eletroforese dissociante para se estimar a resistência à ação da pepsina em função da concentração da enzima e do tempo de incubação. Extratos brutos protéicos e frações hidrolisadas foram testados contra soros de pacientes comprovadamente alérgicos e não alérgicos à soja nas concentrações de 1/20 em ensaios imunoquímicos do tipo ELISA e 1/10 em ensaios do tipo Western blotting. Os resultados da composição básica de nutrientes mostraram dispersões normais esperadas entre as amostras de mesma origem, com tendências de níveis superiores de proteínas de 9 a 16% nas amostras GM. As análises de fibras insolúveis e isoflavonas revelaram valores de decréscimo das amostras GMs em relação aos teores das variedades convencionais, contrastando com o acréscimo de valores de ácido fítico nas mesmas cultivares. Quanto à proteólise, foi possível observar estabilidade de bandas protéicas com pesos moleculares em torno de 50 e 18 KDa nos extratos brutos, 10 KDa nos extratos de 11S e 50 KDa nos extratos de 7S, em geral similares entre as parentais isogênicas e GMs. Os testes de reatividade dos soros de pacientes alérgicos e não alérgicos em extratos brutos protéicos das cultivares GM demonstraram reatividade similar quando comparados às suas respectivas parentais isogênicas. Com a observação dos resultados pode-se concluir que as diferenças significativas apresentadas pelas amostras GM não as tornam inseguras para o consumo humano e animal. Da mesma forma que não foram observadas alterações na alergenicidade das amostras GM em relação às amostras parentais isogênicas por apresentarem perfis semelhantes de proteólise frente à pepsina e aos testes imunológicos contra soros de pacientes alérgicos. / The parameters of security evaluation of genetically modified foods are based on the substantial equivalence among varieties and the innocuity of GM vegetal proteins compared with conventional vegetal proteins. The aim of this work was to evaluate the food safety of three genetically modified soybean cultivars to tolerate glyphosate herbicide through substantial equivalence determination and allergenic potential when compared to their respective isogenic parental. Six samples analyzed were three parental soybean (conventional) and the others GM, regarding to the crop of 2004-2005, grown in Goiás state. The chemical composition was performed in triplicate and the content of moisture, minerals, proteins, lipids and fiber were determined. Additional compounds like aminoacids, fatty acids, isoflavons and phytates were analyzed. The potential of allergenic reactions was evaluated in crude protein extracts of three conventional and their corresponding GM cultivars. The same protein extracts were fractionated to obtain 7S and 11S globulins by precipitation and posterior purification on Sepharose 4B bioaffinity column. The 7S glycoprotein was obtained by elution with -D-mannopyranoside buffer. The resistance to proteolysis was performed by two simulated gastric fluids with pepsin at the proportions 2,5:100 and 13:1 enzyme/substrate. The samples were run by SDS electrophoresis to estimate the resistance to pepsin action according to enzyme concentration and incubation time. Crude protein extracts and hydrolyzed fractions were tested against serum of allergic and non-allergic patients through ELISA immunochemistry essays at concentrations of 1/20 and Western blotting, 1/10. The results of chemical composition of nutrients showed an expected normal dispersion among samples from the same origin, with tendencies for superior levels of proteins from 9 to 16% by GM samples. The analyses of insoluble fibers and isoflavons revealed decreased values at GM samples regarding to the contents on conventional varieties, contrasting with the increase of the phytic acid values in the same cultivars. After the proteolysis, some protein bands remained apparently stable, that correspond to molecular weights around 50 and 18 KDa for crude extracts, 10 KDa for 11S extracts and 50 KDa for 7S extracts. The undigested proteins are similar in both set of samples, the parental isogenic and GM soybeans. Immuno-reactivity of proteins from crude extracts with serum from allergic and non allergic patients were also similar for isogenic and GM cultivars. These results allow us to state that the significant differences observed in the composition of GM samples will neither affect the nutrient levels nor the safety of their consumption as food or feed. As well as there were no observed changes at the potential allergenicity of GM samples regarding to parental isogenic samples by exhibit similar proteolysis profile related to pepsin and immunological essays against allergic patient serums.

Les macrophages associés aux tumeurs (TAM) orchestrent l'inflammation nécessaire à la croissance tumorale (propriétés de type M1) et favorisent les métastases et l'angiogenèse (caractéristiques de type M2). Cependant, la nature des facteurs capables de conférer des propriétés M1 et M2 aux macrophages humains demeure inconnue. L'acide lactique (AL) est un métabolite produit par les cellules tumorales connu pour moduler les fonctions des cellules présentes dans le microenvironnement tumoral. Dans cette étude, nous avons analysé son impact sur la différenciation des monocytes humains. Les résultats montrent que l’AL induit la différentiation des monocytes (cultivées en présence de GM-CSF) en macrophages présentant un phénotype atypique (CD14high CD163high IL-10low IL-12low) et, de manière intéressante, des caractéristiques phénotypiques M1 (production de cytokines inflammatoires) et M2 (production de facteurs de croissance, expression de gènes prototypiques M2). Un profil similaire est obtenu lorsque les monocytes sont cultivés avec des cellules cancéreuses primaires glycolytiques. Ces effets de l'AL sur la polarisation des macrophages nécessitent l'entrée du lactate dans les cellules (via le transporteur MCT-1) et son oxydation en pyruvate et sont médiés par une stabilisation de HIF-1α et une consommation autocrine de M-CSF.Ces résultats (i) identifient l'AL comme un médiateur induisant la génération de macrophages humains présentant des caractéristiques M2 et des propriétés inflammatoires et (ii) renforcent l'intérêt de l’utilisation des inhibiteurs de la glycolyse aérobie pour moduler les fonctions des TAM. / In established tumors, tumor-associated macrophages (TAM) orchestrate unresolving cancer-related inflammation (M1-related properties) and favor tumor development, metastasis and angiogenesis (M2-like properties). However, to date, the nature of the polarization factor(s) able to confer M1 and M2 functional properties to human macrophages remains unknown.Lactic acid (LA), a metabolite produced at high levels in most established tumors, can impact the phenotype and functions of cells present in the tumor microenvironment. In this study, we analyzed the impact of LA on the human monocyte differentiation. Results showed that LA skews monocytes (differentiated in the presence of GM-CSF) into macrophages (GM+LA-Mφ) exhibiting an atypical CD14high CD163high IL-10low IL-12low phenotype. Interestingly they harbor M1 and M2 phenotypic features, as assessed the production of a wide variety of inflammatory and growth factors and the expression of prototypic M2-like genes. A similar profile is induced by culturing monocytes with glycolytic human primary cancer cells. These effects of LA on macrophage polarization require the entry of lactate into the cells (via the monocarboxylate transporter 1) and its oxidation into pyruvate and are mediated via HIF-1α stabilization and autocrine M-CSF consumption by differentiating cells. These results identify tumor-derived LA as a missing link reconciling the M2-like features of TAM with their inflammatory properties. They also reinforce the interest of aerobic glycolysis inhibitors to modulate the functions of TAM.

The purpose of the research was to explore the experiences of educators regarding the training for the implementation of inclusive education in a Full Service school. A qualitative research design was chosen, using a case study. Three methods of gathering data were used, namely individual interviews, focus group interviews and observations. The study was conducted in a primary schools in the North West province that was converted into a fullservice school in 2008. The findings indicated that educators demonstrated misunderstanding of the Screening, Identification, Assessment and Support strategy. The misunderstanding can be ascribed to the kind of training educators received. The training lacked in–depth content and practical demonstration. Recommendations on the content and the dynamics of the training process are made. The overarching recommendation on the dynamics of the training indicated that the training should be revisited for improved methods of training. / Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

The purpose of the research was to explore the experiences of educators regarding the training for the implementation of inclusive education in a Full Service school. A qualitative research design was chosen, using a case study. Three methods of gathering data were used, namely individual interviews, focus group interviews and observations. The study was conducted in a primary schools in the North West province that was converted into a fullservice school in 2008. The findings indicated that educators demonstrated misunderstanding of the Screening, Identification, Assessment and Support strategy. The misunderstanding can be ascribed to the kind of training educators received. The training lacked in–depth content and practical demonstration. Recommendations on the content and the dynamics of the training process are made. The overarching recommendation on the dynamics of the training indicated that the training should be revisited for improved methods of training. / Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Presented thesis examines possible environmental impact of the genetically modified (GM) maize expressing insecticidal Cry proteins. The impact was assessed from differences in the communities of the ground and above-ground arthropods in plots sown with the standard and the GM maize, respectively. The results revealed that neither the abundance nor the species richness of arthropods was affected. Laboratory experiments were used to study effect of maize expressing several types of Cry proteins on the arthropod predators. Despite the proven exposure of the predators to Cry proteins in the food, no Cry proteins accumulation and deleterious effects on predators were observed. The results confirm the importance of predators in insect resistance management using the GM maize seed blends method.

Abstract The efforts to improve forest trees could be accelerated by means of genetic engineering. Thus, the performance and effects of genetically modified (GM) trees have been investigated in numerous studies, which have generally concluded that GM trees have similar effects on environment and/or other organisms as do conventionally bred trees. In the present study, GM silver birch (Betula pendula Roth) and hybrid aspen (Populus tremula L. × tremuloides Michx.) lines were utilized to study the influence of transgenes to the transcription of related endogenous genes and to the production of soluble phenolic compounds in relation to ectomycorrhizal symbiosis or herbivory. The GM silver birch lines had altered lignin composition, whereas the hybrid aspen lines produced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated using biolistic transformation and monitored under greenhouse conditions for three growing seasons. The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had also reduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. This indicates that these members of the 4CL and COMT multigene families are likely to contribute to the monolignol biosynthesis pathway of silver birch. No unintended effects were detected in the PtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae. Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acid derivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, the responses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were found to be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alike when fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, no unintended changes were found in the GM silver birch lines with altered lignin composition or in the VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that were conducted under controlled conditions have certain limitations. / Tiivistelmä Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perinteistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin ja vuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskien arvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen (GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaan perinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuksia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremuloides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksi työssä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasymbioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavien hybridihaapalinjojen lehtien laatua perhostoukkien ravintona. Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi -geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- ja guaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivun Bp4CL1-geenituotteiden määrän vähenemisestä. Myös kaffeaatti/5-hydroksylaatti O-metyylitransferaasi -geeniä (PtCOMT) ilmentävissä, ligniinirakenteeltaan muunnetuissa rauduskoivulinjoissa havaittiin endogeenisen BpCOMT-geenin tuotteiden määrän väheneminen. Tulokset viittaavat siihen, että Bp4CL1- ja BpCOMT-geenien tuottamat entsyymit toimivat rauduskoivun monolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoisista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvät läheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkien kasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviä eroja ei todettu myöskään hybridihaapalinjojen herbivoria-vasteissa. On kuitenkin otettava huomioon, että kaikki tutkimuksen kokeet suoritettiin kasvihuoneissa käyttäen vasta juveniilivaiheessa olevia kasveja. Jotta abioottisten ja bioottisten ympäristötekijöiden sekä GM-puiden vuorovaikutusta olisi mahdollista arvioida kokonaisvaltaisesti, puita pitäisi tutkia pitkäaikaisissa kenttäkokeissa.

Os parâmetros de avaliação de segurança de alimentos geneticamente modificados fundamentam-se na comparação de equivalência substancial entre as variedades e pela inocuidade de proteínas da planta GM com as proteínas encontradas nas plantas convencionais. O objetivo deste trabalho foi avaliar a segurança alimentar de três cultivares de sojas geneticamente modificadas para tolerarem o herbicida glifosato através da determinação da equivalência substancial e do potencial alergênico das mesmas quando comparadas às suas respectivas parentais isogênicas. Seis amostras de soja foram analisadas, sendo três convencionais parentais e três GM, referentes ao cultivo de 2004-2005, em Goiás. Para a composição química foram realizadas análises em triplicata de proteínas, lipídeos, umidade, minerais e fibra alimentar. Análises complementares para determinação de aminoácidos, ácidos graxos, isoflavonas e ácido fítico também foram realizadas. O potencial de alergenicidade foi avaliado em extratos protéicos brutos de três cultivares convencionais e suas correspondentes GM. Os mesmos extratos protéicos foram fracionados para obter as globulinas 7S e 11S por precipitação e posterior purificação em coluna de bioafinidade Sepharose 4B. A glicoproteína 7S foi obtida a partir da eluição com tampão contendo -D-mannopyranoside. A resistência à proteólise foi realizada a partir de dois fluidos gástricos simulados com pepsina nas proporções 2,5:100 e 13:1 de enzima/substrato. As amostras foram submetidas à eletroforese dissociante para se estimar a resistência à ação da pepsina em função da concentração da enzima e do tempo de incubação. Extratos brutos protéicos e frações hidrolisadas foram testados contra soros de pacientes comprovadamente alérgicos e não alérgicos à soja nas concentrações de 1/20 em ensaios imunoquímicos do tipo ELISA e 1/10 em ensaios do tipo Western blotting. Os resultados da composição básica de nutrientes mostraram dispersões normais esperadas entre as amostras de mesma origem, com tendências de níveis superiores de proteínas de 9 a 16% nas amostras GM. As análises de fibras insolúveis e isoflavonas revelaram valores de decréscimo das amostras GMs em relação aos teores das variedades convencionais, contrastando com o acréscimo de valores de ácido fítico nas mesmas cultivares. Quanto à proteólise, foi possível observar estabilidade de bandas protéicas com pesos moleculares em torno de 50 e 18 KDa nos extratos brutos, 10 KDa nos extratos de 11S e 50 KDa nos extratos de 7S, em geral similares entre as parentais isogênicas e GMs. Os testes de reatividade dos soros de pacientes alérgicos e não alérgicos em extratos brutos protéicos das cultivares GM demonstraram reatividade similar quando comparados às suas respectivas parentais isogênicas. Com a observação dos resultados pode-se concluir que as diferenças significativas apresentadas pelas amostras GM não as tornam inseguras para o consumo humano e animal. Da mesma forma que não foram observadas alterações na alergenicidade das amostras GM em relação às amostras parentais isogênicas por apresentarem perfis semelhantes de proteólise frente à pepsina e aos testes imunológicos contra soros de pacientes alérgicos. / The parameters of security evaluation of genetically modified foods are based on the substantial equivalence among varieties and the innocuity of GM vegetal proteins compared with conventional vegetal proteins. The aim of this work was to evaluate the food safety of three genetically modified soybean cultivars to tolerate glyphosate herbicide through substantial equivalence determination and allergenic potential when compared to their respective isogenic parental. Six samples analyzed were three parental soybean (conventional) and the others GM, regarding to the crop of 2004-2005, grown in Goiás state. The chemical composition was performed in triplicate and the content of moisture, minerals, proteins, lipids and fiber were determined. Additional compounds like aminoacids, fatty acids, isoflavons and phytates were analyzed. The potential of allergenic reactions was evaluated in crude protein extracts of three conventional and their corresponding GM cultivars. The same protein extracts were fractionated to obtain 7S and 11S globulins by precipitation and posterior purification on Sepharose 4B bioaffinity column. The 7S glycoprotein was obtained by elution with -D-mannopyranoside buffer. The resistance to proteolysis was performed by two simulated gastric fluids with pepsin at the proportions 2,5:100 and 13:1 enzyme/substrate. The samples were run by SDS electrophoresis to estimate the resistance to pepsin action according to enzyme concentration and incubation time. Crude protein extracts and hydrolyzed fractions were tested against serum of allergic and non-allergic patients through ELISA immunochemistry essays at concentrations of 1/20 and Western blotting, 1/10. The results of chemical composition of nutrients showed an expected normal dispersion among samples from the same origin, with tendencies for superior levels of proteins from 9 to 16% by GM samples. The analyses of insoluble fibers and isoflavons revealed decreased values at GM samples regarding to the contents on conventional varieties, contrasting with the increase of the phytic acid values in the same cultivars. After the proteolysis, some protein bands remained apparently stable, that correspond to molecular weights around 50 and 18 KDa for crude extracts, 10 KDa for 11S extracts and 50 KDa for 7S extracts. The undigested proteins are similar in both set of samples, the parental isogenic and GM soybeans. Immuno-reactivity of proteins from crude extracts with serum from allergic and non allergic patients were also similar for isogenic and GM cultivars. These results allow us to state that the significant differences observed in the composition of GM samples will neither affect the nutrient levels nor the safety of their consumption as food or feed. As well as there were no observed changes at the potential allergenicity of GM samples regarding to parental isogenic samples by exhibit similar proteolysis profile related to pepsin and immunological essays against allergic patient serums.

Le Virus Herpès Simplex de type 1 (HSV-1) est un agent infectieux qui cause l’herpès chez une grande proportion de la population mondiale. L’herpès est généralement considéré comme une maladie bénigne dont la forme la plus commune est l'herpès labial (communément appelé « bouton de fièvre »), mais elle peut se révéler très sérieuse et causer la cécité et l’encéphalite, voir létale dans certain cas. Le virus persiste toute la vie dans le corps de son hôte. Jusqu'à présent, aucun traitement ne peut éliminer le virus et aucun vaccin n’a été prouvé efficace pour contrôler l’infection herpétique. HSV-1 est un virus avec un génome d’ADN bicaténaire contenu dans une capside icosaèdrale entourée d’une enveloppe lipidique. Treize glycoprotéines virales se trouvent dans cette enveloppe et sont connues ou supposées jouer des rôles distincts dans différentes étapes du cycle de réplication viral, incluant l'attachement, l'entrée, l’assemblage, et la propagation des virus. La glycoprotéine M (gM) qui figure parmi ces glycoprotéines d’enveloppe, est la seule glycoprotéine non essentielle mais est conservée dans toute la famille herpesviridae. Récemment, l’homologue de gM dans le Pseudorabies virus (PRV), un autre herpesvirus, a été impliqué dans la phase finale de l’assemblage (i.e. l’enveloppement cytoplasmique) au niveau du réseau trans-Golgi (TGN) en reconnaissant spécifiquement des protéines tégumentaires et d’autres glycoprotéines d’enveloppe ([1]). Toutefois, il a été proposé que cette hypothèse ne s’applique pas pour le HSV-1 ([2]). De plus, contrairement à la localisation au TGN dans les cellules transfectées, HSV-1 gM se localise dans la membrane nucléaire et sur les virions périnucléaires durant une infection. L’objectif du projet présenté ici était d’éclaircir la relation de la localisation et la fonction de HSV-1 gM dans le contexte d’une infection. Dans les résultats rapportés ici, nous décrivons tout abord un mécanisme spécifique de ciblage nucléaire de HSV-1 gM. En phase précoce d’une infection, gM est ciblée à la membrane nucléaire d'une manière virus ii dépendante. Cela se produit avant la réorganisation du TGN normalement induite par l’infection et avant que gM n’entre dans la voie de sécrétion. Ce ciblage nucléaire actif et spécifique de gM ne semble pas dépendre des plusieurs des partenaires d’interaction proposés dans la littérature. Ces données suggèrent que la forme nucléaire de gM pourrait avoir un nouveau rôle indépendant de l’enveloppement final dans le cytoplasme. Dans la deuxième partie du travail présenté ici, nous avons concentré nos efforts sur le rôle de gM dans l’assemblage du virus en phase tardive de l’infection et en identifiant un domaine critique de gM. Nos résultats mettent en valeur l’importance du domaine carboxyl-terminal cytoplasmique de gM dans le transport de gM du réticulum endoplasmique (RE) à l’appareil de Golgi, dans l’enveloppement cytoplasmique et la propagation intercellulaire du virus. Ainsi, l’export du RE de gM a été complètement compromis dans les cellules transfectées exprimant un mutant de gM dépourvu de sa région C-terminale. La délétion la queue cytoplasmique de gM cause une réduction légère du titre viral et de la taille des plaques. L'analyse de ces mutants par microscopie électronique a démontré une accumulation des nucléocapsides sans enveloppe dans le cytoplasme par rapport aux virus de type sauvage. Étrangement, ce phénotype était apparent dans les cellules BHK mais absent dans les cellules 143B, suggérant que la fonction de gM dépende du type cellulaire. Finalement, le criblage de partenaires d’interaction du domaine C-terminal de gM identifiés par le système de double-hybride nous a permis de proposer plusieurs candidats susceptibles de réguler la fonction de gM dans la morphogénèse et la propagation de virus. / Herpes Simplex Virus type 1 (HSV-1) is an infectious agent causing herpes, which affects a large population worldwide. Herpes is generally considered a benign disease whose most common form is oral herpes (commonly called "cold sores"), but it can be very serious and cause herpetic blindness and encephalitis, and even be lethal in some cases. The virus can persist throughout life in the body of its host. So far, no treatment can eliminate the virus and no vaccine has proven effective in controlling herpes infections. HSV-1 has a double-stranded DNA genome embedded in an icosahedral capsid surrounded by a lipid envelope. Thirteen viral glycoproteins are located in the envelope and are known or believed to play different roles in different stages of the viral replication cycle, including attachment, entry, assembly, and viral propagation. Among these envelope glycoproteins, glycoprotein M (gM) is the only nonessential glycoprotein but is conserved in all the herpesviridae family. Recently, the homologue of gM in Pseudorabies virus (PRV), another herpesvirus, has been implicated in the final phase of assembly (e.g. the cytoplasmic envelopment) at the trans-Golgi network (TGN) ([1]). However, it was suggested that this does not apply to HSV-1 ([2]). Moreover, unlike its TGN localization in transfected cells, HSV-1 gM localizes to the nuclear membrane and on the perinuclear virions during infection. The objective of the project presented here was to clarify the relationship of the location and function of HSV-1 gM in the context of an infection. In the results reported here, we first describe a specific and active mechanism of nuclear targeting of HSV-1 gM. In early phase of infection, gM is targeted to the nuclear membrane in a virus dependent manner. This occurs before the known reorganization of the TGN induced by the virus and before gM enters the secretory pathway. This active and specific nuclear targeting of gM seemingly does not depend on the functional interaction partners proposed in the literature. These data suggest that nuclear gM could have a new role independent of that in the final envelopment in the cytoplasm. In the second part of the work presented here, we focused iv our efforts on the role of gM in virus assembly in the late phase of infection and define an important functional domain within gM. Our results highlight the importance of the carboxyl-terminal domain of gM in the intracellular transport of gM from endoplasmic reticulum (ER) to Golgi apparatus, in the cytoplasmic envelopment of the capsids and the intercellular spread of the virus. Hence, gM ER export was completely compromised in transfected cells after deletion of its C-terminal tail. Deletion of the gM cytoplasmic tail in mutant viruses resulted in a slight reduction in viral titer and plaque size. The analysis of these mutants by electron microscopy showed an accumulation of nucleocapsids without envelope in the cytoplasm compared to wild-type virus. Interestingly, this phenotype is apparent in BHK cells but not in 143B cells, hinting that the importance of gM may be cell type specific. Finally, screening of interaction partners of C-terminal domain of gM identified by the two-hybrid system allowed us to propose several interesting candidates that may regulate the function of gM in the virus morphogenesis and propagation.

Le Virus Herpès Simplex de type 1 (HSV-1) est un agent infectieux qui cause l’herpès chez une grande proportion de la population mondiale. L’herpès est généralement considéré comme une maladie bénigne dont la forme la plus commune est l'herpès labial (communément appelé « bouton de fièvre »), mais elle peut se révéler très sérieuse et causer la cécité et l’encéphalite, voir létale dans certain cas. Le virus persiste toute la vie dans le corps de son hôte. Jusqu'à présent, aucun traitement ne peut éliminer le virus et aucun vaccin n’a été prouvé efficace pour contrôler l’infection herpétique. HSV-1 est un virus avec un génome d’ADN bicaténaire contenu dans une capside icosaèdrale entourée d’une enveloppe lipidique. Treize glycoprotéines virales se trouvent dans cette enveloppe et sont connues ou supposées jouer des rôles distincts dans différentes étapes du cycle de réplication viral, incluant l'attachement, l'entrée, l’assemblage, et la propagation des virus. La glycoprotéine M (gM) qui figure parmi ces glycoprotéines d’enveloppe, est la seule glycoprotéine non essentielle mais est conservée dans toute la famille herpesviridae. Récemment, l’homologue de gM dans le Pseudorabies virus (PRV), un autre herpesvirus, a été impliqué dans la phase finale de l’assemblage (i.e. l’enveloppement cytoplasmique) au niveau du réseau trans-Golgi (TGN) en reconnaissant spécifiquement des protéines tégumentaires et d’autres glycoprotéines d’enveloppe ([1]). Toutefois, il a été proposé que cette hypothèse ne s’applique pas pour le HSV-1 ([2]). De plus, contrairement à la localisation au TGN dans les cellules transfectées, HSV-1 gM se localise dans la membrane nucléaire et sur les virions périnucléaires durant une infection. L’objectif du projet présenté ici était d’éclaircir la relation de la localisation et la fonction de HSV-1 gM dans le contexte d’une infection. Dans les résultats rapportés ici, nous décrivons tout abord un mécanisme spécifique de ciblage nucléaire de HSV-1 gM. En phase précoce d’une infection, gM est ciblée à la membrane nucléaire d'une manière virus ii dépendante. Cela se produit avant la réorganisation du TGN normalement induite par l’infection et avant que gM n’entre dans la voie de sécrétion. Ce ciblage nucléaire actif et spécifique de gM ne semble pas dépendre des plusieurs des partenaires d’interaction proposés dans la littérature. Ces données suggèrent que la forme nucléaire de gM pourrait avoir un nouveau rôle indépendant de l’enveloppement final dans le cytoplasme. Dans la deuxième partie du travail présenté ici, nous avons concentré nos efforts sur le rôle de gM dans l’assemblage du virus en phase tardive de l’infection et en identifiant un domaine critique de gM. Nos résultats mettent en valeur l’importance du domaine carboxyl-terminal cytoplasmique de gM dans le transport de gM du réticulum endoplasmique (RE) à l’appareil de Golgi, dans l’enveloppement cytoplasmique et la propagation intercellulaire du virus. Ainsi, l’export du RE de gM a été complètement compromis dans les cellules transfectées exprimant un mutant de gM dépourvu de sa région C-terminale. La délétion la queue cytoplasmique de gM cause une réduction légère du titre viral et de la taille des plaques. L'analyse de ces mutants par microscopie électronique a démontré une accumulation des nucléocapsides sans enveloppe dans le cytoplasme par rapport aux virus de type sauvage. Étrangement, ce phénotype était apparent dans les cellules BHK mais absent dans les cellules 143B, suggérant que la fonction de gM dépende du type cellulaire. Finalement, le criblage de partenaires d’interaction du domaine C-terminal de gM identifiés par le système de double-hybride nous a permis de proposer plusieurs candidats susceptibles de réguler la fonction de gM dans la morphogénèse et la propagation de virus. / Herpes Simplex Virus type 1 (HSV-1) is an infectious agent causing herpes, which affects a large population worldwide. Herpes is generally considered a benign disease whose most common form is oral herpes (commonly called "cold sores"), but it can be very serious and cause herpetic blindness and encephalitis, and even be lethal in some cases. The virus can persist throughout life in the body of its host. So far, no treatment can eliminate the virus and no vaccine has proven effective in controlling herpes infections. HSV-1 has a double-stranded DNA genome embedded in an icosahedral capsid surrounded by a lipid envelope. Thirteen viral glycoproteins are located in the envelope and are known or believed to play different roles in different stages of the viral replication cycle, including attachment, entry, assembly, and viral propagation. Among these envelope glycoproteins, glycoprotein M (gM) is the only nonessential glycoprotein but is conserved in all the herpesviridae family. Recently, the homologue of gM in Pseudorabies virus (PRV), another herpesvirus, has been implicated in the final phase of assembly (e.g. the cytoplasmic envelopment) at the trans-Golgi network (TGN) ([1]). However, it was suggested that this does not apply to HSV-1 ([2]). Moreover, unlike its TGN localization in transfected cells, HSV-1 gM localizes to the nuclear membrane and on the perinuclear virions during infection. The objective of the project presented here was to clarify the relationship of the location and function of HSV-1 gM in the context of an infection. In the results reported here, we first describe a specific and active mechanism of nuclear targeting of HSV-1 gM. In early phase of infection, gM is targeted to the nuclear membrane in a virus dependent manner. This occurs before the known reorganization of the TGN induced by the virus and before gM enters the secretory pathway. This active and specific nuclear targeting of gM seemingly does not depend on the functional interaction partners proposed in the literature. These data suggest that nuclear gM could have a new role independent of that in the final envelopment in the cytoplasm. In the second part of the work presented here, we focused iv our efforts on the role of gM in virus assembly in the late phase of infection and define an important functional domain within gM. Our results highlight the importance of the carboxyl-terminal domain of gM in the intracellular transport of gM from endoplasmic reticulum (ER) to Golgi apparatus, in the cytoplasmic envelopment of the capsids and the intercellular spread of the virus. Hence, gM ER export was completely compromised in transfected cells after deletion of its C-terminal tail. Deletion of the gM cytoplasmic tail in mutant viruses resulted in a slight reduction in viral titer and plaque size. The analysis of these mutants by electron microscopy showed an accumulation of nucleocapsids without envelope in the cytoplasm compared to wild-type virus. Interestingly, this phenotype is apparent in BHK cells but not in 143B cells, hinting that the importance of gM may be cell type specific. Finally, screening of interaction partners of C-terminal domain of gM identified by the two-hybrid system allowed us to propose several interesting candidates that may regulate the function of gM in the virus morphogenesis and propagation.

Entre le XIème et le XVIème siècle, la Mer des Indes fut le théâtre de nombreux mouvements populationnels aux fins essentiellement commerciales ou coloniales. Madagascar se trouve à la croisée des mondes asiatiques et africains. La côte sud-est malgache a vu l'arrivée de plusieurs migrations : la dernière, probablement vers la fin du XVème siècle, serait celle des Antemoro dont une partie d'entre eux se réclame d'une origine arabe et se rattache à La Mecque. L'éthnie des Antemoro a fait l'objet de nombreuses études anthropologiques et linguistiques. Néanmoins, le débat sur l'origine des migrants fait toujours l'objet d'hypothèses contradictoires. Leurs origines génétiques pourraient ainsi être l'Arabie, l'Afrique de l'Est, l'Inde ou encore l'Asie du Sud-Est à une époque où ces régions étaient déjà islamisées. Ce travail a consisté à étudier la diversité génétique d'une population Antemoro afin d'apporter des éléments de réponse à la question de leur origine biologique. Ce projet interdisciplinaire a pour objectif de mettre en relation l'anthropologie culturelle et sociale avec l'anthropologie biologique. Le polymorphisme du chromosome Y a été étudié afin de rechercher les origines des lignées paternelles par l'analyse de 17 marqueurs microsatellites ainsi que des mutations ponctuelles de l'ADN de la partie non recombinante du chromosome Y. De même, la variabilité génétique des lignées maternelles a été analysée par séquençage des régions hypervariables I et II de l'ADN mitochondrial, et par la définition de polymorphismes bialléliques dans sa région codante. Nous avons mis en évidence la présence de deux haplogroupes du chromosome Y chez certains groupes Antemoro, qui les différencient de la diversité habituellement rencontrée dans les populations malgaches. Bien que la majeure partie des Antemoro entre dans la diversité observée en Afrique sub-Saharienne et en Asie du Sud-Est, quelques haplotypes, des lignées paternelles, les lieraient au Moyen-Orient. Les lignées maternelles, quant à elles, ne les différencient pas de celles des autres populations malgaches. L'isolat génétique formé par certaines « pseudo-castes » Antemoro confirme bien l'isolat culturel. Ce travail apporte une nouvelle vision de la diversité génétique humaine à Madagascar. / Between the 11th and 16th century, the Indian Ocean was the scene of many population movements notably for commercial and colonial purposes. Madagascar is located at the crossroads of the Asian and African continents. Several migrations have occurred in this region; the last one during the late 15th century involved the Antemoro population who claimed an Arabian origin in Mecca. Many anthropological and linguistic studies have been carried out on this ethnic group, but the origin of these migrants remains contentious. It is uncertain whether their origins were in Arabia, East Africa, India or Southeast Asia, when these regions were Islamized. In this study we assessed the genetic diversity of an Antemoro population from villages between Manakara and Vohipeno, to determine their biological origin. The aim of our interdisciplinary study was to link cultural and social anthropology with biological anthropology. Y-chromosome polymorphisms were studied by analyzing 17 microsatellites markers and some SNPs in the non-recombining region of the Y-chromosome to determine the biological origins of the paternal lineages. In addition, genetic variability of maternal lineages was analyzed by sequencing hypervariables regions I and II, and by defining bi-allelic polymorphisms in the coding region of mitochondrial DNA. We found two Y-chromosome haplogroups in some Antemoro groups that differentiated them from the typical genetic variability found in other Malagasy populations. Although most of the Antemoro showed a genetic diversity similar to that observed in sub-Saharan Africa and Southeast Asia, few haplotypes associated to paternal lineages linked them to the Middle East. Maternal lineages did not differ from those found in other Malagasy populations. The genetic isolate formed by some Antemoro groups confirmed their cultural isolation. This study provides a new view of the human genetic diversity in Madagascar.