Made available in DSpace on 2014-06-11T19:33:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-22Bitstream added on 2014-06-13T20:45:28Z : No. of bitstreams: 1 silva_bms_dr_jabo.pdf: 2245286 bytes, checksum: dd4988388b8b4412932a2d90984e471f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Ormosia paraensis Ducke, possui sementes ornamentais usadas na confecção de biojóias, assim como, sua madeira é utilizada por moveleiros. Para a identificação florestal e tecnologia de sementes, as informações sobre a biologia e, por conseguinte, para a produção de mudas de tento são incipientes. Desta forma, o objetivo do presente trabalho foi descrever a morfo-anatomia e determinar os métodos mais adequados para superação de dormência, germinação e emergência de plântulas de tento. Para a descrição morfo-anatômica, os frutos, as sementes e as plântulas foram avaliadas por meio de microscopia óptica e eletrônica de varredura. Para a quebra de dormência das sementes, foi utilizada a abrasão com lixa e a imersão em ácido sulfúrico. Os frutos de tento são legumes nucóides, pseudo-septados, castanhos a pretos, deiscentes, portando uma ou duas sementes, de placentação lateral. As sementes são bitegumentadas, exalbuminosas e arredondadas. As plântulas de tento apresentam folhas simples e alternadas, com raiz pivotante e caule cilíndrico. A germinação é hipógea criptocotiledonar. A abrasão com lixa e por meio da imersão em ácido sulfúrico por 60 ou 120 minutos são adequadas para a superação de dormência de sementes de tento. A faixa de temperatura ótima para a germinação está entre 25 e 35 oC tanto entre areia quando entre papel. As semeaduras em areia e em vermiculita são mais adequadas em profundidades nunca superiores a 2cm / Ormosia paraensis Ducke, known as “tento”, possess seeds used to make handicrafts and wood worked by furniture makers. For the forest identification and seeds technology, the information on the biology and therefore for the production of “tento” seedlings are incipient. Thus, the aim of this study was the morpho-anatomical description as well as determining the most appropriate methods for overcoming dormancy, germination and seedling emergence of “tento”. For the morpho-anatomical description, the evaluations were examined by optical and scanning electron microscopy. For the dormancy break of the seeds, the methods were physical and chemical scarification. For the temperature, substrate and depth evaluation were used temperatures from 5 to 45 oC, the substrates sand, paper, vermiculite and PlantmaxR and the depths of 0, 2 and 4 cm. The fruit is a nutant legume, brown to black, dehiscent and with one or two seeds of lateral placentation. The seeds are bitegmic, exalbuminous and rounded. The seedlings have simple and alternate leaves, and their germination is hypogeal cryptocotyledonary. The mechanical scarification and the chemical by sulfuric acid immersion for 60 or 120 minutes are suitable for overcoming seed dormancy. The range of the optimum temperature for germination is between 25 and 35 oC between sand or paper. In the nursery are sand and vermiculite, sowings higher than 2 cm depths are unsuitable for seedling emergence

Made available in DSpace on 2014-06-11T19:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-05-29Bitstream added on 2014-06-13T19:57:56Z : No. of bitstreams: 1 galindo_cam_me_jabo.pdf: 862767 bytes, checksum: af26a620af6aa1461436f3ff8272fde7 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Com o objetivo de estudar a dormência tegumentar e caracterizar possíveis relações entre tamanho e qualidade fisiológica e permeabilidade dos tegumentos, qualidade fisiológica e permeabilidade dos tegumentos, teor de água inicial e qualidade fisiológica de sementes de mucuna preta, foram conduzidos os seguintes experimentos: determinação do teor inicial de água dos lotes (RAS); DIC, 4 repetições de 25 sementes cada; peso de 100 sementes, 4x100, DIC; teste de %G e IVG 4x25 cada, DIC; 12 tratamentos para superação de dormência; classificação dos lotes em três classes de tamanho; teor inicial de água das sementes de diferentes tamanhos (RAS), DBC, repetições 4x25, fatorial 6 lotes x 3 tamanhos; curvas de embebição com dados em dispersão e uso de função polinomial de quarto grau, dados obtidos pela razão peso final(PF)/peso inicial(PI); teste de CE com repetições 4x25, DIC, fatorial 6 lotes x 3 tamanhos, substrato solo/areis. Os lotes apresentaram relação positiva inversa para teor de água inicial em relação à germinação; resultados dos tratamentos para superação de dormência com interações para lotes e para tamanhos dentro dos lotes; os testes demonstraram haver maior interação entre lotes, ocorrendo o contrário para a variável tamanho de sementes. Concluiu-se que: sementes pequenas são mais permeáveis em lotes não dormentes; entre lotes de germinações semelhantes o de menor teor de água inicial é o de maior vigor; sementes grandes produzem mais fitomassa e o tratamento mais eficiente para quebra de dormência foi escarificação com ácido sulfúrico concentrado (98%) por 7 minutos. / Aiming to study damage and characterize possible relationships between size and physiological quality; size and coat permeability; physiological quality and tegument permeability; initial content of water and physiological quality of mucuna preta sedes (Stizolobium aterrimum Pierce & Tracy) the following experiments were conducted: análisis of water content of seed lots by oven meted at 105°C during 24 hours using with four replications of 100 seeds each; germination test (%G) and speed of germination index (SGI) with four replications of 25 seeds in complete randomized design; twelve treatments to break dormancy The seed lots presented inverse positive relationship for water content related to germination; treatment to break dormancy with interaction between lotsand seed sizes within lots; the tests demonstrated higher interaction between lots, occuring the inverse for the variable seed size; the imbibition curve proved to be na important tool in studies related with tegument permeabillity and levels of vigor among lots. The following conclusion can be withdrawn form data: small sedes are more permeable in nondormant lots; seed lots with similar germination or with lower water content, higher is the vigor; large sedes produce higher amount of phytomass. The best treatment to break seed dormancy of mucuna preta seed was acid escarification during seven minutes.

Orientador: Teresinha de Jesus Deléo Rodrigues / Banca: Maria Lidia Stipp Paterniani / Banca: Ana Regina Pimentel de Almeida / Resumo: Com o objetivo de estudar a dormência tegumentar e caracterizar possíveis relações entre tamanho e qualidade fisiológica e permeabilidade dos tegumentos, qualidade fisiológica e permeabilidade dos tegumentos, teor de água inicial e qualidade fisiológica de sementes de mucuna preta, foram conduzidos os seguintes experimentos: determinação do teor inicial de água dos lotes (RAS); DIC, 4 repetições de 25 sementes cada; peso de 100 sementes, 4x100, DIC; teste de %G e IVG 4x25 cada, DIC; 12 tratamentos para superação de dormência; classificação dos lotes em três classes de tamanho; teor inicial de água das sementes de diferentes tamanhos (RAS), DBC, repetições 4x25, fatorial 6 lotes x 3 tamanhos; curvas de embebição com dados em dispersão e uso de função polinomial de quarto grau, dados obtidos pela razão peso final(PF)/peso inicial(PI); teste de CE com repetições 4x25, DIC, fatorial 6 lotes x 3 tamanhos, substrato solo/areis. Os lotes apresentaram relação positiva inversa para teor de água inicial em relação à germinação; resultados dos tratamentos para superação de dormência com interações para lotes e para tamanhos dentro dos lotes; os testes demonstraram haver maior interação entre lotes, ocorrendo o contrário para a variável tamanho de sementes. Concluiu-se que: sementes pequenas são mais permeáveis em lotes não dormentes; entre lotes de germinações semelhantes o de menor teor de água inicial é o de maior vigor; sementes grandes produzem mais fitomassa e o tratamento mais eficiente para quebra de dormência foi escarificação com ácido sulfúrico concentrado (98%) por 7 minutos. / Abstract: Aiming to study damage and characterize possible relationships between size and physiological quality; size and coat permeability; physiological quality and tegument permeability; initial content of water and physiological quality of mucuna preta sedes (Stizolobium aterrimum Pierce & Tracy) the following experiments were conducted: análisis of water content of seed lots by oven meted at 105°C during 24 hours using with four replications of 100 seeds each; germination test (%G) and speed of germination index (SGI) with four replications of 25 seeds in complete randomized design; twelve treatments to break dormancy The seed lots presented inverse positive relationship for water content related to germination; treatment to break dormancy with interaction between lotsand seed sizes within lots; the tests demonstrated higher interaction between lots, occuring the inverse for the variable seed size; the imbibition curve proved to be na important tool in studies related with tegument permeabillity and levels of vigor among lots. The following conclusion can be withdrawn form data: small sedes are more permeable in nondormant lots; seed lots with similar germination or with lower water content, higher is the vigor; large sedes produce higher amount of phytomass. The best treatment to break seed dormancy of mucuna preta seed was acid escarification during seven minutes. / Mestre

Resumo: Ormosia paraensis Ducke, possui sementes ornamentais usadas na confecção de biojóias, assim como, sua madeira é utilizada por moveleiros. Para a identificação florestal e tecnologia de sementes, as informações sobre a biologia e, por conseguinte, para a produção de mudas de tento são incipientes. Desta forma, o objetivo do presente trabalho foi descrever a morfo-anatomia e determinar os métodos mais adequados para superação de dormência, germinação e emergência de plântulas de tento. Para a descrição morfo-anatômica, os frutos, as sementes e as plântulas foram avaliadas por meio de microscopia óptica e eletrônica de varredura. Para a quebra de dormência das sementes, foi utilizada a abrasão com lixa e a imersão em ácido sulfúrico. Os frutos de tento são legumes nucóides, pseudo-septados, castanhos a pretos, deiscentes, portando uma ou duas sementes, de placentação lateral. As sementes são bitegumentadas, exalbuminosas e arredondadas. As plântulas de tento apresentam folhas simples e alternadas, com raiz pivotante e caule cilíndrico. A germinação é hipógea criptocotiledonar. A abrasão com lixa e por meio da imersão em ácido sulfúrico por 60 ou 120 minutos são adequadas para a superação de dormência de sementes de tento. A faixa de temperatura ótima para a germinação está entre 25 e 35 oC tanto entre areia quando entre papel. As semeaduras em areia e em vermiculita são mais adequadas em profundidades nunca superiores a 2cm / Abstract: Ormosia paraensis Ducke, known as "tento", possess seeds used to make handicrafts and wood worked by furniture makers. For the forest identification and seeds technology, the information on the biology and therefore for the production of "tento" seedlings are incipient. Thus, the aim of this study was the morpho-anatomical description as well as determining the most appropriate methods for overcoming dormancy, germination and seedling emergence of "tento". For the morpho-anatomical description, the evaluations were examined by optical and scanning electron microscopy. For the dormancy break of the seeds, the methods were physical and chemical scarification. For the temperature, substrate and depth evaluation were used temperatures from 5 to 45 oC, the substrates sand, paper, vermiculite and PlantmaxR and the depths of 0, 2 and 4 cm. The fruit is a nutant legume, brown to black, dehiscent and with one or two seeds of lateral placentation. The seeds are bitegmic, exalbuminous and rounded. The seedlings have simple and alternate leaves, and their germination is hypogeal cryptocotyledonary. The mechanical scarification and the chemical by sulfuric acid immersion for 60 or 120 minutes are suitable for overcoming seed dormancy. The range of the optimum temperature for germination is between 25 and 35 oC between sand or paper. In the nursery are sand and vermiculite, sowings higher than 2 cm depths are unsuitable for seedling emergence / Orientador: Fabíola Vitti Môro / Coorientador: Roberval Daiton Vieira / Banca: Silvelena Vanzolini Segato / Banca: Raquel Silva Costa / Banca: Rita de Cássia Panizzi / Banca: Emerson Iossi / Doutor

Made available in DSpace on 2014-08-20T13:44:24Z (GMT). No. of bitstreams: 1 dissertacao_daisy_leticia-ramirez_monzon.pdf: 495372 bytes, checksum: 4109f5d12e44f58ba6cd6d0affc3d675 (MD5) Previous issue date: 2012-02-07 / The soybean (Glycine max L. Max) is one of the main crops grown in Brazil, the second largest world producer. Its evolution has been strongly supported by the development of technologies that allowed the acreage increase. Currently, the use of biotechnology tools aims to contribute to the development of new genotypes with characteristics that can continue increase the production. The identification of genes responsible for characters that can assist in developing varieties with improved seed quality may constitute something innovative. The seed tegument has an important function as for the seeds resistance to deterioration. In this sense, the partial characterization of the protein expression FLA 14 (Arabinogalactan), directly related to the cell wall synthesis in the seed tegument may contribute to the advancement of these studies. The aim of this work was to generate and validate primers capable of quantifying the gene expression of the FLA 14 protein in four soybean genotypes, two with black tegument (TP and IAC 222) and higher lignin content and two with yellow tegument (CD 202 and Potência) and lower lignin content. The plant tissue (tegument) was collected 50 days after the anthesis. In order to obtain the gene expression among genotypes, it was used the qRT-PCR technique, where it was tested four combinations of primers, constructed from sequence models of soybeans ESTs. With the validation results, the primers that were found within the standards were the FLA 14p1, FLA 14p2 and β-Actina. They were all used at the expression studies. The results of the relative expression showed that soybean genotypes with black tegument and higher lignin content express the FLA 14 protein quantitatively superior to the genotypes with yellow tegument and lower lignin content. / A soja (Glycine max L. Merr.) é uma das principais culturas produzidas no Brasil, sendo considerado o segundo maior produtor mundial. Sua evolução foi fortemente amparada pelo desenvolvimento de tecnologias que possibilitaram o aumento da área cultivada. Atualmente o emprego de ferramentas da biotecnologia busca contribuir para o desenvolvimento de novos genótipos com características que possam continuar incrementando a produção. A identificação de genes responsáveis por caracteres que possam auxiliar no desenvolvimento de variedades com melhor qualidade da semente, pode se constituir em algo inovador. O tegumento apresenta uma função importante quanto à resistência das sementes à deterioração. Nesse sentido, a caracterização parcial da expressão do gene da proteína FLA 14 (Arabinogalactano), relacionadas diretamente com a síntese da parede celular no tegumento da semente pode contribuir para o avanço dos estudos. O objetivo do trabalho foi gerar e validar primers capazes de quantificar a expressão gênica da proteína FLA 14 em quatro genótipos de soja, dois desses com tegumento preto (TP e IAC 222) e maior conteúdo de lignina, e outros dois com tegumento amarelo (CD 202 e Potência) e menor conteúdo de lignina. O tecido vegetal (tegumentos) foi coletado aos 50 dias após a antese. Para a análise da expressão gênica entre os genótipos, utilizou-se a técnica de qRT-PCR, onde foram testadas quatro combinações de primers construídos a partir de modelos de sequência de ESTs da soja. Com os resultados da validação, os primers que se encontraram dentro dos padrões foram os FLA 14p1, FLA 14p2 e β-Actina utilizando-se no estudo de expressão. Os resultados da expressão relativa permitiram concluir a expressão do gene da proteína FLA 14 é quantitativamente superior nos genótipos de soja com tegumento preto e conteúdo superior de lignina em relação aos genótipos com tegumento amarelo e menos teor de lignina.

Une étude parasitaire de l'anguille d'Europe a été menée dans les lagunes de Biguglia et d'Urbino en Corse. La composition des communautés de parasites a été décrite. Treize espèces parasites ont été identifiées parmi lesquelles: trois digènes, Bucephalus anguillae, Deropristis inflata, Lecithochirium musculus; un monogène, Pseudodactylogyrus anguillae; trois cestodes, Bothriocephalus claviceps, Proteocephalus macrocephalus, Myzophyllobothrium sp. (larve); trois nématodes, Anguillicoloides crassus, Contracaecum sp. (larve enkystée), Goezia anguillae; un acanthocéphale, Acanthocephaloides incrassatus; un copépode, Ergasilus gibbus; et un myxozoaire, Myxobolus portucalensis. La présence d'espèces invasives, notamment le parasite branchial P. anguillae et le nématode parasite A. crassus, dans les lagunes corses est confirmée. Ces espèces, et particulièrement le monogène, présentent des valeurs épidémiologiques croissantes depuis les dernières études menées. Plusieurs espèces présentent des différences de prévalence significatives entre les deux lagunes. Des différences au niveau de la richesse spécifique et des valeurs de diversité, plus élevées pour les parasites des anguilles de la lagune d'Urbino au niveau intestinal métacommunautaire et infracommunautaire, ont été démontrées. Cependant les valeurs les plus élevées de diversité spécifique et les valeurs de dominance les plus basses ont été calculées pour les communautés parasitaires des anguilles de la lagune de Biguglia. Nous avons également mis en avant une diversité parasitaire spécifique plutôt faible chez les anguilles des lagunes corses par rapport aux autres lagunes d'Europe. Les communautés parasitaires de l'anguille d'Europe dans les lagunes de Biguglia et d'Urbino en Corse sont marquées par l'environnement de leur hôte. Une dépendance vis-à-vis de la salinité de la lagune a ainsi été démontrée. Les valeurs d'infestation les plus élevées ont été observées durant les saisons les plus chaudes de l'année pour la majorité des espèces parasites observées (B. anguillae, D. inflata, L. musculus, P. anguillae, P. macrocephalus, A. crassus, les kystes de Contracaecum sp., A. incrassatus et E. gibbus). Nous avons également démontré que l'état d'argenture et la taille ont une influence significative sur les taux d'infestation de sept espèces parasites (D. inflata, L. musculus, P. anguillae, P. macrocephalus, les kystes de Contracaecum sp., A. incrassatus et E. gibbus). La méthode de l'espèce indicatrice a confirmé que le site d'étude, la saison, l'état d'argenture ou la taille de l'anguille pouvait influer sur la présence de certaines espèces parasites. Le tégument de trois digènes parasites de l'anguille d'Europe, B. anguillae, L. musculus et D. inflata, a été étudié en microscopie électronique à balayage et à transmission. Nous avons démontré la présence de structures caractéristiques de l'organisation tégumentaire des digènes ainsi que de formations spécifiques, notamment au niveau de la structure des récepteurs sensoriels et des écailles. / A survey of the parasitic fauna of the European eel has been conducted in the Biguglia and Urbino lagoons in Corsica. The composition of the parasite communities was determined. Thirteen parasite species were identified namely: three digeneans, Bucephalus anguillae, Deropristis inflata, Lecithochirium musculus; one monogenean, Pseudodactylogyrus anguillae; three cestodes, Bothriocephalus claviceps, Proteocephalus macrocephalus, larvae of Myzophyllobothrium sp.; three nematodes, Anguillicoloides crassus, encysted larvae of Contracaecum sp., Goezia anguillae; one acanthocephalan, Acanthocephaloides incrassatus; one copepod, Ergasilus gibbus; and plasmodia of one myxozoan, Myxobolus portucalensis. The presence of invasive species in lagoons from Corsica, namely the gill monogenean P. anguillae and the swimbladder nematode A. crassus, was confirmed. These species, particularly the monogenean, exhibit increasing infection rates since the last studies conducted. Many species showed significant differences in prevalence between the two lagoons. Differences in the species richness and higher values of diversity for the intestinal parasite component communities and infracommunities of eels from the Urbino lagoon were demonstrated. However, highest values of richness and lowest dominance values were observed for the parasite communities of eels from the Biguglia lagoon. We also demonstrated lower values of diversity for the parasite communities of eels from Corsica in comparison to eels from other European lagoons. The environment of the host (in particular the salinity range) has been demonstrated to have a significant influence on the composition of the parasite communities of eels from the Biguglia and Urbino lagoons. Highest values of infestation were observed for the warmer seasons of the year for the majority of the parasite species (B. anguillae, D. inflata, L. musculus, P. anguillae, P. macrocephalus, A. crassus, encysted larvae of Contracaecum sp., A. incrassatus, and E. gibbus). We also demonstrated that silvering stage and length have a significant influence on the rates of infestation by seven parasite species (D. inflata, L. musculus, P. anguillae, P. macrocephalus, cysts of Contracaecum sp., A. incrassatus et E. gibbus). The indicator species method confirmed the assumption that site sampling, season, silvering stage and length of the eel could have an influence on the presence of parasite species. The teguments of three digeneans (B. anguillae, L. musculus and D. inflata) recovered within the European eel were studied using scanning and transmission electron microscopy. We showed the presence of structures characteristic of the tegumental organization of digeneans but also the presence of specific structures such as various types of sensory receptors and spines.

Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope and a proteinaceous tegument layer. There are over fifty viral proteins and many host proteins identified in HSV-1 virions. The final formation of mature virus particles requires the membrane wrapping of tegumented capsids in the cytoplasm, a process termed secondary envelopment. This process involves the coordination of numerous viral and cellular proteins and results in double-membrane structures with enveloped virions contained within cellular vesicles. Mature viruses are then released through the fusion of these virion-containing vesicles and plasma membranes. This thesis describes investigation into the functions of viral glycoprotein M (gM) and the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) in secondary envelopment. Firstly, it has been reported that gH/L can be efficiently internalised and targeted to the TGN by the co-expression of gM in transfection assays. In order to examine the role of gM in guiding the localisation of viral proteins in infected cells, a HSV-1 gM deletion virus (∆gM), and its revertant virus were constructed. The major phenotype demonstrated was that the absence of gM caused the internalisation of cell surface gH/L to be inhibited and higher levels of gH/L to be observed on the cell surface. Further, lower levels of gH/L were detected in purified ∆gM virions, which was in agreement with the delayed entry kinetics, smaller plaque sizes and greater replication deficits at low multiplicity of infection observed in ∆gM infected cells. Over all the results presented in this thesis demonstrate that in infected cells the efficient incorporation of gH/L into virions relies on the function of gM in HSV-1. Secondly, during HSV-1 secondary envelopment the budding and scission of the viral envelope from the host membrane share topological similarities with the formation of intraluminal vesicle in multivesicular bodies, retrovirus budding, and abscission at the end of cytokinesis, processes that require the cellular ESCRT machinery. There are four multiprotein ESCRT complexes and many associated proteins involved in their regulation. It has been previously shown that the ESCRT-III complex and a functional ATPase VPS4 are required for HSV-1 secondary envelopment, but different from the strategy utilised by HIV-1, the recruitment of ESCRT during HSV-1 infection is independent of TSG101 and/or ALIX. Data presented in this thesis demonstrate that CHMP4A/B/C proteins of the ESCRT-III complex are specifically crucial for HSV-1 secondary envelopment. Simultaneous depletion of CHMP4A/B/C proteins significantly inhibited HSV-1 replication. Ultrastructure analysis revealed that there were virtually no extracellular virions in CHMP4A/B/C depleted samples while more free capsids were observed in the cytoplasm, although the nuclear capsids and primary envelopment events appeared to be normal. In order to identify interactions between HSV-1 and ESCRT proteins, 22 HSV-1 tegument proteins were cloned and tested against a panel of ESCRT and ESCRT-associated proteins in yeast two-hydrid assays. Analysis of positive hits from yeast two-hybrid interaction screens using GST pull-down, co-immunoprecipitation and protein co-localisation assays have validated interactions of pUL47 with CC2D1A/1B, CIN85, CHMP6 and ALIX, pUL46 and pUL49 with CC2D1A/1B and CIN85, and pUL16 with CC2D1A/1B. Furthermore, the newly identified ESCRT associated proteins CC2D1A and CC2D1B have been detected in purified virions. The role of the identified ESCRT proteins in HSV-1 replication has been investigated using siRNA depletion. Unfortunately siRNA depletions of the various ESCRT candidates individually or in combinations did not show any significant effect on HSV-1 replication. Overall these data suggest that unlike HIV and other retroviruses, HSV-1 has evolved multiple parallel pathways to hijack the ESCRT machinery to facilitate its replication, particularly, through the interactions that lead directly to the recruitment of CHMP4A/B/C proteins. Disruption of some of these pathways did not prevent HSV-1 replication in tissue culture, suggesting any one potential pathway is sufficient for ESCRT recruitment to sites of HSV-1 assembly.

Le virus herpès simplex de type 1 (HSV 1) affecte la majorité de la population mondiale. HSV 1 cause de multiples symptômes délétères dont les plus communs sont les lésions orofaciales usuellement appelées feux sauvages. Le virus peut aussi causer des effets plus sérieux comme la cécité ou des troubles neurologiques. Le virus réside de façon permanente dans le corps de son hôte. Malgré l’existence de nombreux traitements pour atténuer les symptômes causés par HSV 1, aucun médicament ne peut éliminer le virus. Dans le but d’améliorer les connaissances concernant le cycle viral de HSV 1, ce projet cible l’étude du transport du virus dans la cellule hôte. Ce projet aura permis la collecte d’informations concernant le modus operandi de HSV 1 pour sortir des compartiments cellulaires où il séjourne. Les différentes expérimentations ont permis de publier 3 articles dont un article qui a été choisi parmi les meilleurs papiers par les éditeurs de « Journal of Virology » ainsi qu’un 4e article qui a été soumis. Premièrement, un essai in vitro reproduisant la sortie de HSV 1 du noyau a été mis sur pied, via l’isolation de noyaux issus de cellules infectées. Nous avons démontré que tout comme dans les cellules entières, les capsides s’évadent des noyaux isolés dans l’essai in vitro en bourgeonnant avec la membrane nucléaire interne, puis en s’accumulant sous forme de capsides enveloppées entre les deux membranes nucléaires pour finalement être relâchées dans le cytoplasme exclusivement sous une forme non enveloppée. Ces observations appuient le modèle de transport de dé-enveloppement/ré-enveloppement. Deuxièmement, dans le but d’identifier des joueurs clefs viraux impliqués dans la sortie nucléaire du virus, les protéines virales associées aux capsides relâchées par le noyau ont été examinées. La morphologie multicouche du virus HSV 1 comprend un génome d’ADN, une capside, le tégument et une enveloppe. Le tégument est un ensemble de protéines virales qui sont ajoutées séquentiellement sur la particule virale. La séquence d’ajout des téguments de même que les sites intracellulaires où a lieu la tégumentation sont l’objet d’intenses recherches. L’essai in vitro a été utilisé pour étudier cette tégumentation. Les données recueillies suggèrent un processus séquentiel qui implique l’acquisition des protéines UL36, UL37, ICP0, ICP8, UL41, UL42, US3 et possiblement ICP4 sur les capsides relâchées par le noyau. Troisièmement, pour obtenir davantage d’informations concernant la sortie de HSV 1 des compartiments membranaires de la cellule hôte, la sortie de HSV 1 du réseau trans golgien (TGN) a aussi été étudiée. L’étude a révélé l’implication de la protéine kinase D cellulaire (PKD) dans le transport post-TGN de HSV 1. PKD est connue pour réguler le transport de petits cargos et son implication dans le transport de HSV 1 met en lumière l’utilisation d’une machinerie commune pour le transport des petits et gros cargos en aval du TGN. Le TGN n’est donc pas seulement une station de triage, mais est aussi un point de rencontre pour différentes voies de transport intracellulaire. Tous ces résultats contribuent à une meilleure compréhension du processus complexe de maturation du virus HSV 1, ce qui pourrait mener au développement de meilleurs traitements pour combattre le virus. Les données amassées concernant le virus HSV 1 pourraient aussi être appliquées à d’autres virus. En plus de leur pertinence dans le domaine de la virologie, les découvertes issues de ce projet apportent également de nouveaux détails au niveau du transport intracellulaire. / Herpes simplex virus type 1 (HSV 1) affects the majority of the world population. HSV 1 causes various deleterious symptoms with the most common being facial mucosal lesions usually named cold sores. The virus can also contribute to more serious effects such as corneal blindness and neurological problems. The virus is permanently residing in the host body. Despite the existence of several treatments against HSV 1 symptoms, no drug is able to eliminate the virus. In order to improve knowledge of the viral cycle of HSV 1, this project focuses on the transport of the virus in the host cell. During this project we collect data to detail the modus operandi used by HSV 1 to leave cellular compartments such as the nucleus and the TGN. The different experimentations achieved during this PhD allowed the publication of three articles, including one selected as worthy of note by the editors of “Journal of virology” and a fourth article that has been submitted. Firstly, an in vitro assay that reproduces the exit of HSV 1 virus from nuclei was established via the isolation of nuclei from infected cells. We found that, as in intact cells, capsids escaped the isolated nuclei in the in vitro assay by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytoplasm exclusively as unenveloped capsids. These observations support the de-envelopment / re-envelopment model of transport. Secondly, to identify viral players implicated in the nuclear egress of HSV 1, viral proteins associated with nuclear released capsids were investigated. HSV 1 has a multilayered morphology that includes a DNA genome, a capsid, a tegument and an envelope. The tegument represents viral proteins added sequentially on the viral particle. The sequential order and intracellular compartments where the tegument is added are the subject of intense research. The in vitro assay was used to investigate this tegumentation process. The acquired data suggest a sequential process that involved the acquisition of viral proteins UL36, UL37, ICP0, ICP8, UL41, UL42, US3 and possibly ICP4 on capsids released by the nucleus. Thirdly, to obtain information regarding another process of egress of HSV 1 from a membranous cellular organelle, the egress of HSV 1 from the TGN was also studied. The study revealed the implication of the cellular protein kinase D (PKD) in HSV 1 post-TGN transport. The involvement of this kinase, known to regulate the transport of small cargos, highlights the post TGN trafficking of both small and large entities (such as HSV 1) by a common machinery, in sharp contrast to earlier steps of transport. This indicates that the TGN is not only a sorting station but also a meeting point where different intracellular routes can meet. All these outcomes contribute to a better understanding of the complex maturation process of HSV 1 that could lead to the development of better tools to fight the virus. Results acquired concerning HSV 1 could also be applied to other viruses. Besides their relevance in the virology field, findings provided by this project also supply new details about cellular biology concerning intracellular transport.

Le virus Herpès simplex de type 1 (HSV-1), agent étiologique des feux sauvages, possède une structure multicouche comprenant une capside icosaédrale qui protège le génome viral d’ADN, une couche protéique très structurée appelée tégument et une enveloppe lipidique dérivant de la cellule hôte et parsemée de glycoprotéines virales. Tous ces constituants sont acquis séquentiellement à partir du noyau, du cytoplasme et du réseau trans-golgien. Cette structure multicouche confère à HSV-1 un potentiel considérable pour incorporer des protéines virales et cellulaires. Toutefois, l’ensemble des protéines qui composent ce virus n’a pas encore été élucidé. De plus, malgré son rôle critique à différentes étapes de l’infection, le tégument demeure encore mal défini et ce, tant dans sa composition que dans la séquence d’addition des protéines qui le composent. Toutes ces incertitudes quant aux mécanismes impliqués dans la morphogenèse du virus nous amènent à l’objectif de ce projet, soit la caractérisation du processus de maturation d’HSV-1. Le premier article présenté dans cette thèse et publié dans Journal of Virology s’attarde à la caractérisation protéique des virus extracellulaires matures. Grâce à l’élaboration d’un protocole d’isolation et de purification de ces virions, une étude protéomique a pu être effectuée. Celle-ci nous a permis de réaliser une cartographie de la composition globale en protéines virales des virus matures (8 protéines de la capside, 23 protéines du tégument et 13 glycoprotéines) qui a fait la page couverture de Journal of Virology. De plus, l’incorporation potentielle de 49 protéines cellulaires différentes a été révélée. Lors de cette étude protéomique, nous avons aussi relevé la présence de nouveaux composants du virion dont UL7, UL23, ICP0 et ICP4. Le deuxième article publié dans Journal of General Virology focalise sur ces protéines via une analyse biochimique afin de mieux comprendre les interactions et la dynamique du tégument. Ces résultats nous révèlent que, contrairement aux protéines ICP0 et ICP4, UL7 et UL23 peuvent être relâchées de la capside en présence de sels et que les cystéines libres jouent un rôle dans cette relâche. De plus, cet article met en évidence la présence d’ICP0 et d’ICP4 sur les capsides nucléaires suggérant une acquisition possible du tégument au noyau. La complexité du processus de morphogenèse du virus ainsi que la mise en évidence d’acquisition de protéines du tégument au noyau nous ont incités à poursuivre nos recherches sur la composition du virus à un stade précoce de son cycle viral. Les capsides C matures, prémisses des virus extracellulaires, ont donc été isolées et purifiées grâce à un protocole innovateur basé sur le tri par cytométrie en flux. L’analyse préliminaire de ces capsides par protéomique a permis d’identifier 28 protéines virales et 39 protéines cellulaires. Les données recueilles, comparées à celles obtenues avec les virus extracellulaires, suggèrent clairement un processus séquentiel d’acquisition des protéines du tégument débutant dans le noyau, site d’assemblage des capsides. Finalement, tous ces résultats contribuent à une meilleure compréhension du processus complexe de maturation d’HSV-1 via l’utilisation de techniques variées et innovatrices, telles que la protéomique et la cytométrie en flux, pouvant être appliquées à d’autres virus mais aussi permettre le développement de meilleurs traitements pour vaincre l’HSV-1. / Herpes simplex virus type 1 (HSV-1), the etiological agent of cold sores, has a multilayered structure that includes an icosahedral capsid that protects the viral DNA genome, a highly structured proteinaceous layer called tegument and a host-derived lipid envelope studded with viral glycoproteins. All these constituents are sequentially acquired from the nucleus, the cytoplasm and the trans-Golgi network. This multilayered structure confers to HSV-1 a considerable potential to incorporate viral and cellular proteins; however, all the proteins that compose this virus have not yet been elucidated. Moreover, despite its critical role at different stages of infection, the tegument is still poorly defined both in its composition and its sequence of addition of proteins. All these uncertainties about the mechanisms involved in the morphogenesis of the virus lead us to the goal of this project, which is the characterization of the maturation process of HSV-1. The first article presented in this thesis and published in Journal of Virology focuses on the protein characterization of extracellular mature virus. After developing a protocol for the isolation and purification of these virions, a proteomics study was performed. It allowed us to map the global viral protein composition of mature virions (8 capsid proteins, 23 tegument proteins and 13 glycoproteins), which made the cover page of Journal of Virology. Moreover, the potential incorporation of 49 cellular proteins was revealed. During this proteomics study, we confirmed the incorporation of novel virion components including UL7, UL23, ICP0 and ICP4. The second article published in Journal of General Virology focuses on these viral proteins by using a biochemical analysis to better understand the interactions and dynamic of the tegument. Our results revealed that, unlike ICP0 and ICP4 proteins, UL7 and UL23 can be released from the capsid in the presence of salts and that free cysteines play a role in this release. Moreover, this article highlights the presence of ICP0 and ICP4 on the nuclear capsids suggesting a potential acquisition of tegument proteins in the nucleus. The complexity of the viral morphogenesis process and the discovery of the tegument acquisition in the nucleus led us to pursue our research on the virus composition at an early stage of its viral cycle. The nuclear C capsids, precursors to the extracellular virus, were isolated and purified with an innovative protocol based on fluorescence activated cell sorting (FACS). The preliminary analysis of these capsids by proteomics allows us to identify 28 viral proteins and 39 cellular proteins. The collected data, compared to those obtained with extracellular viruses, clearly suggest a sequential process of tegument proteins acquisition starting in the nucleus, the assembly site of HSV-1 capsids. Finally, all these results contribute to a better understanding of the complex process of HSV-1 maturation by using varied and innovative techniques such as proteomic and FACS, which can be applied to other viruses and allow the development of better treatments to fight HSV-1.

Le virus herpès simplex de type 1 (HSV 1) affecte la majorité de la population mondiale. HSV 1 cause de multiples symptômes délétères dont les plus communs sont les lésions orofaciales usuellement appelées feux sauvages. Le virus peut aussi causer des effets plus sérieux comme la cécité ou des troubles neurologiques. Le virus réside de façon permanente dans le corps de son hôte. Malgré l’existence de nombreux traitements pour atténuer les symptômes causés par HSV 1, aucun médicament ne peut éliminer le virus. Dans le but d’améliorer les connaissances concernant le cycle viral de HSV 1, ce projet cible l’étude du transport du virus dans la cellule hôte. Ce projet aura permis la collecte d’informations concernant le modus operandi de HSV 1 pour sortir des compartiments cellulaires où il séjourne. Les différentes expérimentations ont permis de publier 3 articles dont un article qui a été choisi parmi les meilleurs papiers par les éditeurs de « Journal of Virology » ainsi qu’un 4e article qui a été soumis. Premièrement, un essai in vitro reproduisant la sortie de HSV 1 du noyau a été mis sur pied, via l’isolation de noyaux issus de cellules infectées. Nous avons démontré que tout comme dans les cellules entières, les capsides s’évadent des noyaux isolés dans l’essai in vitro en bourgeonnant avec la membrane nucléaire interne, puis en s’accumulant sous forme de capsides enveloppées entre les deux membranes nucléaires pour finalement être relâchées dans le cytoplasme exclusivement sous une forme non enveloppée. Ces observations appuient le modèle de transport de dé-enveloppement/ré-enveloppement. Deuxièmement, dans le but d’identifier des joueurs clefs viraux impliqués dans la sortie nucléaire du virus, les protéines virales associées aux capsides relâchées par le noyau ont été examinées. La morphologie multicouche du virus HSV 1 comprend un génome d’ADN, une capside, le tégument et une enveloppe. Le tégument est un ensemble de protéines virales qui sont ajoutées séquentiellement sur la particule virale. La séquence d’ajout des téguments de même que les sites intracellulaires où a lieu la tégumentation sont l’objet d’intenses recherches. L’essai in vitro a été utilisé pour étudier cette tégumentation. Les données recueillies suggèrent un processus séquentiel qui implique l’acquisition des protéines UL36, UL37, ICP0, ICP8, UL41, UL42, US3 et possiblement ICP4 sur les capsides relâchées par le noyau. Troisièmement, pour obtenir davantage d’informations concernant la sortie de HSV 1 des compartiments membranaires de la cellule hôte, la sortie de HSV 1 du réseau trans golgien (TGN) a aussi été étudiée. L’étude a révélé l’implication de la protéine kinase D cellulaire (PKD) dans le transport post-TGN de HSV 1. PKD est connue pour réguler le transport de petits cargos et son implication dans le transport de HSV 1 met en lumière l’utilisation d’une machinerie commune pour le transport des petits et gros cargos en aval du TGN. Le TGN n’est donc pas seulement une station de triage, mais est aussi un point de rencontre pour différentes voies de transport intracellulaire. Tous ces résultats contribuent à une meilleure compréhension du processus complexe de maturation du virus HSV 1, ce qui pourrait mener au développement de meilleurs traitements pour combattre le virus. Les données amassées concernant le virus HSV 1 pourraient aussi être appliquées à d’autres virus. En plus de leur pertinence dans le domaine de la virologie, les découvertes issues de ce projet apportent également de nouveaux détails au niveau du transport intracellulaire. / Herpes simplex virus type 1 (HSV 1) affects the majority of the world population. HSV 1 causes various deleterious symptoms with the most common being facial mucosal lesions usually named cold sores. The virus can also contribute to more serious effects such as corneal blindness and neurological problems. The virus is permanently residing in the host body. Despite the existence of several treatments against HSV 1 symptoms, no drug is able to eliminate the virus. In order to improve knowledge of the viral cycle of HSV 1, this project focuses on the transport of the virus in the host cell. During this project we collect data to detail the modus operandi used by HSV 1 to leave cellular compartments such as the nucleus and the TGN. The different experimentations achieved during this PhD allowed the publication of three articles, including one selected as worthy of note by the editors of “Journal of virology” and a fourth article that has been submitted. Firstly, an in vitro assay that reproduces the exit of HSV 1 virus from nuclei was established via the isolation of nuclei from infected cells. We found that, as in intact cells, capsids escaped the isolated nuclei in the in vitro assay by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytoplasm exclusively as unenveloped capsids. These observations support the de-envelopment / re-envelopment model of transport. Secondly, to identify viral players implicated in the nuclear egress of HSV 1, viral proteins associated with nuclear released capsids were investigated. HSV 1 has a multilayered morphology that includes a DNA genome, a capsid, a tegument and an envelope. The tegument represents viral proteins added sequentially on the viral particle. The sequential order and intracellular compartments where the tegument is added are the subject of intense research. The in vitro assay was used to investigate this tegumentation process. The acquired data suggest a sequential process that involved the acquisition of viral proteins UL36, UL37, ICP0, ICP8, UL41, UL42, US3 and possibly ICP4 on capsids released by the nucleus. Thirdly, to obtain information regarding another process of egress of HSV 1 from a membranous cellular organelle, the egress of HSV 1 from the TGN was also studied. The study revealed the implication of the cellular protein kinase D (PKD) in HSV 1 post-TGN transport. The involvement of this kinase, known to regulate the transport of small cargos, highlights the post TGN trafficking of both small and large entities (such as HSV 1) by a common machinery, in sharp contrast to earlier steps of transport. This indicates that the TGN is not only a sorting station but also a meeting point where different intracellular routes can meet. All these outcomes contribute to a better understanding of the complex maturation process of HSV 1 that could lead to the development of better tools to fight the virus. Results acquired concerning HSV 1 could also be applied to other viruses. Besides their relevance in the virology field, findings provided by this project also supply new details about cellular biology concerning intracellular transport.