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Lors des pathologies articulaires, les chondrocytes sont soumis à différents processus qui concourent à la dégradation du cartilage articulaire soit en entraînant la mort par apoptose (nonrenouvellement des constituants de la matrice) soit par l’activation de protéases détruisant les constituants matriciels. Le potentiel chondroprotecteur des protéines de stress (Hsp70 / Hsp27) lors des pathologies dégénératives ayant été démontré, nous avons souhaité évaluer l'intérêt thérapeutique de l’induction de ces protéines par un inhibiteur réversible du protéasome (MG132) dans un modèle expérimental : modèle par section du ligament croisé antérieur (SLCA). Au cours de cette étude, nous avons du évaluer un nouveau système de vectorisation afin de surexprimer les protéines de stress dans le cartilage articulaire. Cette technique (électroporation biphasique) repose sur la dissociation des impulsions perméabilisantes et électrophorétiques. Nous avons donc développé des plasmides nous permettant de surexprimer ces protéines, fusionnées à une protéine traceur, facilitant ainsi leur détection dans le tissu ciblé. Puis, nous avons évalué l’innocuité et l’efficacité des impulsions sur des tissus sains et pathologiques (dégénératifs et inflammatoires). Nous avons constaté que cette technique, en plus de limiter les altérations du tissu articulaire ou les phénomènes de dissémination, offrait des propriétés d’adressage tissulaire de part la multiplicité des combinaisons d’impulsions (nombre, fréquence et intensité). Enfin, les effets de l’induction (MG132) des Hsps sur un modèle physiopathologique (SLCA) ont été évalués et nous avons pu montrer une diminution de la sévérité des atteintes articulaires, tant au niveau du cartilage que de la membrane synoviale. Cette molécule non seulement permet à la fois de renforcer la résistance des chondrocytes aux atteintes mais également de limiter l’amplitude de la réponse inflammatoire qui participe à la dégradation. / During articular diseases, chondrocytes suffer different mechanisms which take part in the degradation of the cartilage, either by generating cell death by apoptosis (without renewal of extracellular matrix components), or by protease activation which destroy matrix components. Based on the cytoprotective potential of Heat Shock proteins (Hsp70 and Hsp27) during degenerative diseases, we evaluated the therapeutic interest of these proteins induced by a transient proteasome inhibitor (MG132), in an experimental model, by transection of the anterior cruciate ligament (ACLT). During this study, we have evaluated a new electroporation system to overexpress HSPs in articular cartilage. This technique is based on two sets of electric pulses, wich have two roles, to permeabilize the target and to transport DNA across the permeabilized membrane. We have developed expression vectors to generate a fusion protein (Hsps linked to GFP). Effectively, GFP permit to detect simply the fusion protein in the targeted tissue by fluorescence. Besides, we have evaluated safety and efficiency of electric pulses on healthy and alterated tissues (degenerative and inflammatory). We have reported that this technique could limit articular tissue damages and, moreover, could offer the ability to target more specifically this tissue. Indeed, this apparatus allows a great number of electrics pulses combinations (number, frequency, intensity). Finally, the effects of the induction via MG132 of Hsps in a physiopathological ACLT model, have been evaluated and we have shown a decrease of severity of joint lesions, in cartilage and synovial tissues. This molecule has the advantage to reinforce the resistance of chondrocytes at stressful stimuli and moreover, to limite the amplitude of inflammatory response which contribute to the magnification of extracellular matrix destruction.

Made available in DSpace on 2016-01-26T18:56:17Z (GMT). No. of bitstreams: 1 dissertacao Danielle.pdf: 217929 bytes, checksum: 214e01390ff5f39b31c3b618d4cd1624 (MD5) Previous issue date: 2008-08-21 / Snap Beans (Phaseolus vulgaris L) are highly valuable nutritionally, and it is cultivated by small, medium and big farmers. It represents a significant parcel of Brazilian economy concerning to the society. Culture emergency is a critical point in the production process and it is affected mainly by the water deficiency at this phase. The objective of this work was to simulate water deficiency in the germination beginning at the laboratory on seeds of snap beans ´Pérola´, using mannitol, CaCl2, MgCl2 and NaCl as osmotic in the potential of 0; -0.3; -0.6; -0.9 and -1.2MPa calculated with the aim of Van´t Hoff s equation and to evaluate the electrophorectical protein patterns of total soluble proteins by SDS-PAGE. Germination, vigour classification, roots and shoot dry weight and differential protein expression response was evaluated as parameters. The experimental design was completely randomized. Data was analysed by F test (ANOVA) and polynomial regression for the osmotic potential for each parameter evaluated. Banding pattern was evaluated by gel image. Simulation of deficiency, in laboratory, allowed the perception of the stress originated by NaCl in all parameter evaluated, validating the harsh of the NaCl and the lack of expression of low molecular weight proteins in this osmotic. 110 and 30kDa proteins were indicative of water stress, but not of salinity. / O feijão (Phaseolus vulgaris L) é uma cultura de grande expressão alimentícia. A emergência da cultura é dependente de água, sendo considerada a fase mais crítica. O objetivo deste foi simular deficiência de água no início da germinação em laboratório, em sementes de feijão Pérola , utilizando-se: manitol, CaCl2, MgCl2 e NaCl em potenciais de 0; -0,3; -0,6; -0,9 e -1,2MPa estabelecidos pela equação de Van´t Hoff e avaliar o perfil eletroforético de proteínas totais solúveis através de SDS-PAGE. Foram avaliados: germinação, classificação de vigor, massa seca de raiz e de parte aérea e resposta diferencial de expressão de proteínas. O delineamento experimental foi inteiramente casualizado. Os dados foram analisados através da aplicação do teste F, para análise de variância, regressão polinomial para os níveis de potencial osmóticos para cada uma das variáveis fisiológicas estudadas. O bandeamento eletroforético foi avaliado visualmente através da imagem dos géis. A simulação do estresse permitiu avaliar a drasticidade do NaCl em todos os parâmetros avaliados e a ausência de proteínas de baixo peso molecular neste osmótico. As proteínas de 110 e 30kDa foram indicativas de estresse hídrico, mas não do salino.

Made available in DSpace on 2016-07-18T17:51:02Z (GMT). No. of bitstreams: 1 dissertacao Danielle.pdf: 217929 bytes, checksum: 214e01390ff5f39b31c3b618d4cd1624 (MD5) Previous issue date: 2008-08-21 / Snap Beans (Phaseolus vulgaris L) are highly valuable nutritionally, and it is cultivated by small, medium and big farmers. It represents a significant parcel of Brazilian economy concerning to the society. Culture emergency is a critical point in the production process and it is affected mainly by the water deficiency at this phase. The objective of this work was to simulate water deficiency in the germination beginning at the laboratory on seeds of snap beans ´Pérola´, using mannitol, CaCl2, MgCl2 and NaCl as osmotic in the potential of 0; -0.3; -0.6; -0.9 and -1.2MPa calculated with the aim of Van´t Hoff s equation and to evaluate the electrophorectical protein patterns of total soluble proteins by SDS-PAGE. Germination, vigour classification, roots and shoot dry weight and differential protein expression response was evaluated as parameters. The experimental design was completely randomized. Data was analysed by F test (ANOVA) and polynomial regression for the osmotic potential for each parameter evaluated. Banding pattern was evaluated by gel image. Simulation of deficiency, in laboratory, allowed the perception of the stress originated by NaCl in all parameter evaluated, validating the harsh of the NaCl and the lack of expression of low molecular weight proteins in this osmotic. 110 and 30kDa proteins were indicative of water stress, but not of salinity. / O feijão (Phaseolus vulgaris L) é uma cultura de grande expressão alimentícia. A emergência da cultura é dependente de água, sendo considerada a fase mais crítica. O objetivo deste foi simular deficiência de água no início da germinação em laboratório, em sementes de feijão Pérola , utilizando-se: manitol, CaCl2, MgCl2 e NaCl em potenciais de 0; -0,3; -0,6; -0,9 e -1,2MPa estabelecidos pela equação de Van´t Hoff e avaliar o perfil eletroforético de proteínas totais solúveis através de SDS-PAGE. Foram avaliados: germinação, classificação de vigor, massa seca de raiz e de parte aérea e resposta diferencial de expressão de proteínas. O delineamento experimental foi inteiramente casualizado. Os dados foram analisados através da aplicação do teste F, para análise de variância, regressão polinomial para os níveis de potencial osmóticos para cada uma das variáveis fisiológicas estudadas. O bandeamento eletroforético foi avaliado visualmente através da imagem dos géis. A simulação do estresse permitiu avaliar a drasticidade do NaCl em todos os parâmetros avaliados e a ausência de proteínas de baixo peso molecular neste osmótico. As proteínas de 110 e 30kDa foram indicativas de estresse hídrico, mas não do salino.

La synthèse des protéines de choc thermique (HSPs) est un moyen de défense développé par la cellule pour faire face aux diverses agressions auxquelles elle peut être soumise. En tant que chaperons, les HSPs participent aux mouvements intracellulaires des protéines, préviennent l'agrégation des protéines altérées, éliminent les protéines anormales et contribuent à la conformation correcte des peptides nouvellement synthétisées. Mon équipe d’accueil s’intéresse aux rôles des HSPs dans des processus cellulaires tels que l’apoptose et la différenciation cellulaire. Le but de mon travail de thèse consiste à étudier le rôle des protéines de choc thermique HSP90 et HSP70 au cours de la différenciation des monocytes en macrophages. J’ai dans un premier temps étudié l’implication de HSP90 dans la différenciation macrophagique. c-IAP1 est un membre de la famille des protéines inhibitrices de l’apoptose impliqué dans la régulation de l’apoptose, dans le cycle cellulaire et dans la signalisation cellulaire. Nous avons précédemment montré que c-IAP1 migre du noyau vers le cytoplasme au cours de la différenciation cellulaire. Nous démontrons dans ce travail que c-IAP1 est une protéine cliente de la protéine de choc thermique HSP90β. Dans trois différents modèles de différenciation, ces protéines interagissent et migrent ensemble du noyau vers le cytoplasme au cours de la différenciation cellulaire. L’inhibition de HSP90 ou la déplétion spécifique de l’isoforme β par des siRNA conduisent à sa dégradation par le protéasome. La fonction de chaperon moléculaire de HSP90 envers c-IAP1 est spécifique de l’isoforme β car la déplétion de l’isoforme α n’a pas d’effets sur c-IAP1. De plus l’inhibition de HSP90 ou la déplétion de HSP90β bloquent la différenciation cellulaire tout comme la déplétion de c-IAP1 par siRNA. La deuxième partie de montre travail a consisté à étudier le rôle de HSP70 dans la différenciation macrophagique. Nous montrons que cette protéine est fortement induite après stimulation des cellules par le facteur de croissance M-CSF et que son inhibition bloque la différenciation des monocytes en macrophage. HSP70 interagit avec la protéine Spi-1/Pu.1, facteur de transcription clé de la différenciation macrophagique. L’expression de Spi-1/Pu.1 augmente également au cours de la différenciation macrophagique et ce de manière similaire à celle de HSP70. Ceci suggère l’implication des facteurs de transcription responsables de l’induction des HSPs, les Heat Shock Factor (HSF). L’étude du promoteur de Spi-1/Pu.1 a révélé la présence d’une séquence ressemblant fortement aux éléments de réponse classiques sur lesquels se fixe HSF1. HSF1 est capable de se fixer sur le promoteur de Spi-1/Pu.1 et l’inhibition de HSF1 bloque l’expression de Spi-1/Pu.1. HSF1 participe donc au contrôle de l’expression de Spi-1/Pu.1 lors de la différenciation macrophagique. HSP90 et HSP70 sont donc essentielles à la différenciation macrophagique. Comprendre les mécanismes cellulaires impliqués dans les voies de différenciation se révèle extrêmement important puisque des altérations des mécanismes de l’hématopoïèse sont retrouvées dans plusieurs types de leucémies (leucémies aiguës myéloblastiques et leucémies myélo-monocytaires chroniques). Connaître le rôle des HSPs dans la différenciation cellulaire permettrait donc de développer de nouvelles stratégies thérapeutiques pour le traitement de ces pathologies. / Heat shock proteins (HSPs) are molecular chaperones whose expression is increased after many different stresses. They have a protective function helping the cell to cope with lethal conditions. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins and by preventing their aggregation. My team is interested in understanding the roles of HSPs in two physiological related processes: apoptosis and cell differentiation. The aim of my work is to study the functions of HSP90 and HSP70 in macrophagic differentiation. I first studied the role of HSP90 in macrophagic differentiation. We previously reported that cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. c-IAP1 is a member of the inhibitor of apoptosis protein (IAP) family which has demonstrated functions in cell death, cell signaling and mitosis. Here, we show that c-IAP1 is a client protein of the stress protein HSP90β. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm during the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90isoform by siRNA both leads to c-IAP1 degradation by the proteasomal machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its β isoform as specific depletion of HSP90α isoform does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation. The second part of my work consisted in the study of the role of HSP70 in macrophagic differentiation. We used two models of differentiation: human peripheral monocytes exposed to M-CSF and THP1 cells induced to differentiate by TPA. We found that in both models, HSP70 expression was induced during differentiation. Interestingly, the expression of Spi-1/Pu.1, a transcription factor essential for monocytes to differentiate, was similarly induced. Upon differentiation, both proteins co-localized in the nucleus and associated. Inhibition or down regulation of HSP70 induced Spi-1/Pu.1 degradation and blocks the differentiation process, indicating that the necessity of Spi-1/Pu.1 to be chaperoned by HSP70 during differentiation. Since Spi-1/Pu.1 promoter has a HSE-like, we studied whether transcription factors responsible for HSPs induction, HSF, could be involved. We show that although HSF2 do not seem involved, HSF1 binds to Spi-1/Pu.1 promoter and its inhibition blocks Spi-1/Pu.1 expression and monocytes differentiation. So HSP90 and HSP70 are essentials for macrophagic differentiation. Understanding monocyte differentiation regulation is important since defects of the differentiation process can lead to the development of leukemias (acute myeloid leukemia, chronic myelomonocytic leukemia). A better understanding of the roles of HSPs may provide new therapeutic strategies for the treatment of these pathologies.

Made available in DSpace on 2016-12-08T16:24:09Z (GMT). No. of bitstreams: 1 PGCA10MA080.pdf: 1326677 bytes, checksum: 58057ce0c8eb68eed4a42da002d7ab29 (MD5) Previous issue date: 2010-10-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The trypanosomiasis is enzootic caused by several species of the genus Trypanosoma. It is a hemoflagellate widely distributed and of great veterinary importance, because infects a wide variety of mammals. Trypanosoma evansi is the causative agent of disease popularly known as surra , which has great economic importance in Africa, Asia and South America. These protozoa possess digenetic life cycles. When the parasites move from vector to host they suffer a heat shock. The HSPs are found in several pathogens, where the heat shock is a natural event of their biology. The heat shock response is a homeostatic mechanism that protects cells from the deleterious effects of environmental stress. The HSPs have a key role in intracellular work such as DNA replication, cell division, transcription, translation, functions in membrane transport proteins as well as providing assistance to correct protein folding nascent and unfolded by stress accumulation. This work aimed to amplify the HSP10 gene, to be able, afterwards, to observe its expression in trypanosomatids. Rats Wistar were infected with T. evansi, the parasites were purified, the DNA and RNA extraction was made, beyond the reverse transcriptase, PCR, transformation into E. coli DH5α and sequencing of clones. The bands that corresponded to the size of HSP10 gene were selected for clone. The constructions of the inserts selected were subjected to sequencing to verify the correct construction of the clones. The sequencing analysis showed that the sequence obtained has a homology of 40% corresponding to HSP10 hypothetical T. brucei. This is one of the first papers that attempted to identify a gene family of HSPs in T. evansi, that should be used as a chemotherapeutic target / A tripanossomíase é uma enzootia ocasionada por diversas espécies do gênero Trypanosoma. Este é um hemoflagelado amplamente distribuído e de grande importância veterinária, pois infecta uma grande variedade de mamíferos. O Trypanosoma evansi é o agente causador da doença denominada popularmente como surra, que possui grande importância econômica na África, Ásia e América do Sul. Estes protozoários possuem ciclos de vida digenéticos. Quando os parasitos passam do vetor para o hospedeiro estes acabam sofrendo um choque térmico. As HSPs são encontradas em uma variedade de patógenos, onde o choque térmico é um evento natural de sua biologia. A resposta de choque térmico é um mecanismo homeostático que protege as células dos efeitos deletérios do estresse ambiental. As HSPs possuem um papel fundamental no trabalho intracelular como replicação de DNA, divisão celular, transcrição, tradução, funções nas membranas, transporte de proteínas, além de darem assistência correta ao enovelamento de proteínas nascentes e desenoveladas pelo acúmulo de estresse. Este trabalho teve como intuito amplificar o gene da HSP10, para que seja possível futuramente observar sua expressão nos tripanosomatídeos. Ratos Wistar foram infectados com T. evansi, os parasitos foram purificados, efetuou-se a extração de DNA e RNA, além da transcrição reversa, PCR, transformação em E. coli DH5α e o seqüenciamento dos clones. As bandas que corresponderam ao tamanho do gene HSP10 foram selecionadas para clonagem. As construções dos insertos selecionados foram submetidos ao sequenciamento a fim de verificar a construção correta dos clones. A análise do seqüenciamento demonstrou que a sequência obtida possui uma homologia de 40% correspondente a HSP10 hipotética de T. brucei. Este é um dos primeiros trabalhos que buscam identificar um gene da família das HSPs em T. evansi, que poderá ser usado como alvo quimioterápico

The aim of the present study was to investigate how sensory processing sensitivity is related to the personality traits extraversion, agreeableness, emotional stability, conscientiousness and autonomy of the five-factor model. The samples for the study were members of the Association for the Highly Sensitive in Sweden and a Facebook community for highly sensitive individuals. The participants in the present study responded on a web-based questionnaire to participate. To answer the purpose of the study The Highly Sensitive Person scale (HSPS) were used to measure the degree of a person’s sensitivity. The Five Factor Personality Inventory (FFPI) was used to measure the Big Five personality traits. The data were analyzed in the statistics program SPSS with Pearson’s correlations coefficient and a multiple regression analysis. The result of the multiple regression analysis showed that personality traits of neuroticism and agreeableness predicted sensory processing sensitivity. Further on results showed that the personality traits of extraversion, conscientiousness and autonomy not predicted sensory processing sensitivity. The results of the study conclude that participants of the study are much likely to have the personality traits neuroticism and introversion. Furthermore, results indicated that the participants of the study had the personality traits agreeableness, conscientiousness and autonomy. Continued studies with other methodological starting points are needed to achieve greater knowledge about the personality trait sensory processing sensitivity. / Studiens syfte var att undersöka om det fanns en relation mellan högkänslighet och personlighetsdragen extraversion, vänlighet, samvetsgrannhet, öppenhet samt emotionell stabilitet enligt femfaktormodellen. Urvalet bestod av medlemmar från Sveriges Förening för Högkänsliga och en Facebookgrupp som riktar sig till högkänsliga personer. För att besvara frågeställningen användes en webbaserad enkät som mailades ut till medlemmarna i Sveriges Förening för Högkänsliga samt publicerades i Facebookgruppen. Beroendevariabeln högkänslighet mättes med mätinstrumentet The Highly Sensitive Person Scale (HSPS). Oberoendevariablerna extraversion, vänlighet, samvetsgrannhet, emotionell stabilitet och öppenhet mättes med mätinstrumentet The Five Factor Personality Inventory (FFPI). Dataanalyserna som genomfördes i studien var Pearsons korrelationskoefficient och multipel regressionsanalys. Regressionsanalysen visade att personlighetsdragen emotionell stabilitet och vänlighet var prediktorer för högkänslighet. Personlighetsdragen extraversion, samvetsgrannhet och öppenhet var inte prediktorer för högkänslighet. Resultatet indikerade att studiens deltagare i högre grad hade personlighetsdragen neuroticism och introversion. Vidare visade resultatet att studiens deltagare hade grad av personlighetsdragen samvetsgrannhet, vänlighet och öppenhet. Studien gav en indikation på hur hög grad av högkänslighet var i relation till andra personlighetsdrag. Resultatet kan således öka kunskapen om högkänslighet och vad det medför. Fortsatta studier med andra metodologiska utgångspunkter krävs för att få ökad kunskap om personlighetsdraget högkänslighet.

O dermatófito Trichophyton rubrum é um fungo filamentoso, queratinofílico e antropofílico, sendo o principal agente etiológico de micoses cutâneas em humanos. Sua distribuição cosmopolita e seu acometimento grave em pacientes imunocomprometidos fazem dele um dos desafios a ser enfrentado pelos serviços de saúde pública mundial. As interações patógeno-hospedeiro envolvem diferentes processos relacionados com a degradação de queratina e com modificações metabólicas que permitem sua adesão e posterior penetração nos tecidos infectados. Essas alterações são importantes para o sucesso do processo infeccioso e envolvem mecanismos que modulam a expressão gênica, a secreção de proteínas específicas, a adaptação metabólica e as alterações no pH cutâneo, fundamentais para o estabelecimento da infecção. Dentre as proteínas que participam do processo de interação patógeno-hospedeiro estão as proteínas de choque térmico HSPs (Heat Shock Proteins), relacionadas aos mais diversificados processos celulares. Nesse sentido, a hipótese desse trabalho foi avaliar se os genes hsps de T. rubrum, bem como seu principal fator de transcrição (Hsf1), estão envolvidos nos processos de resposta a situações adversas e na interação com o microambiente hospedeiro, e se estes genes são modulados pelo fator de transcrição pacC, um regulador envolvido na sinalização do pH ambiente. Para tanto, a expressão dos genes hsps foi analisada em resposta ao cultivo de T. rubrum em diferentes meios de cultura, durante a exposição a antifúngicos, estresse térmico e interação com fragmentos de unha e pele humanas. O envolvimento da Hsp90 na modulação da expressão gênica, na suscetibilidade a antifúngicos e na interação de T. rubrum com fragmentos de unha humana foi avaliado utilizando-se um inibidor químico específico para esta proteína. Os resultados indicam uma expressão variável dentre os genes hsps e até mesmo dentro de cada família HSP, em resposta a cada condição ambiental ou interação a que o fungo foi exposto. Além disto, temos indício de que a expressão dos genes hsps seja modulada pelo fator de transcrição PacC, através da modulação do fator de transcrição Hsf1. Constatamos ainda, a influência de Hsp90 na susceptibilidade de T. rubrum às drogas Itraconazol e Micafungina, e no desenvolvimento de T. rubrum em fragmentos de unha humana. Esses resultados revelam a participação das proteínas HSPs em diversos aspectos do metabolismo de T. rubrum, e sugerem a participação de Hsp90 na patogenicicade e na suscetibilidade a drogas deste dermatófito / The dermatophyte Trichophyton rubrum is a filamentous, keratinophilic, and anthrophophilic fungi, being the major etiologic agent of cutaneous mycoses in humans. Its cosmopolitan distribution and the severe infection in immunocompromised patients make it one of the challenges to be faced by public health agencies worldwide. Hostpathogen interactions involve different processes related to keratin degradation and metabolic changes that allow adhesion and subsequent penetration of the infected tissue. These changes are important to the success of the infectious process and involve mechanisms that modulate gene expression, secretion of specific proteins, and metabolic adaptation, and cutaneous pH changes, essential to the establishment of the infection. Among the proteins that participate in the host-pathogen interaction are the heat shoch proteins (HSPs), related to diverse cellular processes. Thus, the hypothesis of this work was to evaluate whether T. rubrum hsp genes, as well as their major transcription factor (Hsf1), are involved in the response to adverse situations and in the interaction with the host microenvironment, and if these genes are regulated by the transcription fator PacC, a regulator of the pH signaling pathway. The expression of the hsp genes was evaluated in response to the cultivation of T. rubrum in different culture medium, during exposure to antifungal drugs, heat stress, and interaction with human nail and skin. The involvement of T. rubrum Hsp90 in the modulation of gene expression, susceptibility to antifungal drugs, and interaction with human nails was evaluated by using a chemical inhibitor, specific to this protein. Our results indicate a variable expression of the hsp genes, even among members of the same HSP family, in response to each environmental condition or interaction, to which the fungus was exposed. Furthermore, we have evidence that the hsp gene expression is modulated by the PacC transcription factor, by modulating the expression of the Hsf1 coding gene. We also found that Hsp90 is involved in T. rubrum susceptibility to the drugs Itraconazole and Micafungin, and in the development of this dermatophyte in human nails. These results reveal the involvement of HSPs in several aspects of T. rubrum metabolism, suggesting a role for Hsp90 in the pathogenicity and drug susceptibility in this dermatophyte

O dermatófito Trichophyton rubrum é um fungo filamentoso, queratinofílico e antropofílico, sendo o principal agente etiológico de micoses cutâneas em humanos. Sua distribuição cosmopolita e seu acometimento grave em pacientes imunocomprometidos fazem dele um dos desafios a ser enfrentado pelos serviços de saúde pública mundial. As interações patógeno-hospedeiro envolvem diferentes processos relacionados com a degradação de queratina e com modificações metabólicas que permitem sua adesão e posterior penetração nos tecidos infectados. Essas alterações são importantes para o sucesso do processo infeccioso e envolvem mecanismos que modulam a expressão gênica, a secreção de proteínas específicas, a adaptação metabólica e as alterações no pH cutâneo, fundamentais para o estabelecimento da infecção. Dentre as proteínas que participam do processo de interação patógeno-hospedeiro estão as proteínas de choque térmico HSPs (Heat Shock Proteins), relacionadas aos mais diversificados processos celulares. Nesse sentido, a hipótese desse trabalho foi avaliar se os genes hsps de T. rubrum, bem como seu principal fator de transcrição (Hsf1), estão envolvidos nos processos de resposta a situações adversas e na interação com o microambiente hospedeiro, e se estes genes são modulados pelo fator de transcrição pacC, um regulador envolvido na sinalização do pH ambiente. Para tanto, a expressão dos genes hsps foi analisada em resposta ao cultivo de T. rubrum em diferentes meios de cultura, durante a exposição a antifúngicos, estresse térmico e interação com fragmentos de unha e pele humanas. O envolvimento da Hsp90 na modulação da expressão gênica, na suscetibilidade a antifúngicos e na interação de T. rubrum com fragmentos de unha humana foi avaliado utilizando-se um inibidor químico específico para esta proteína. Os resultados indicam uma expressão variável dentre os genes hsps e até mesmo dentro de cada família HSP, em resposta a cada condição ambiental ou interação a que o fungo foi exposto. Além disto, temos indício de que a expressão dos genes hsps seja modulada pelo fator de transcrição PacC, através da modulação do fator de transcrição Hsf1. Constatamos ainda, a influência de Hsp90 na susceptibilidade de T. rubrum às drogas Itraconazol e Micafungina, e no desenvolvimento de T. rubrum em fragmentos de unha humana. Esses resultados revelam a participação das proteínas HSPs em diversos aspectos do metabolismo de T. rubrum, e sugerem a participação de Hsp90 na patogenicicade e na suscetibilidade a drogas deste dermatófito / The dermatophyte Trichophyton rubrum is a filamentous, keratinophilic, and anthrophophilic fungi, being the major etiologic agent of cutaneous mycoses in humans. Its cosmopolitan distribution and the severe infection in immunocompromised patients make it one of the challenges to be faced by public health agencies worldwide. Hostpathogen interactions involve different processes related to keratin degradation and metabolic changes that allow adhesion and subsequent penetration of the infected tissue. These changes are important to the success of the infectious process and involve mechanisms that modulate gene expression, secretion of specific proteins, and metabolic adaptation, and cutaneous pH changes, essential to the establishment of the infection. Among the proteins that participate in the host-pathogen interaction are the heat shoch proteins (HSPs), related to diverse cellular processes. Thus, the hypothesis of this work was to evaluate whether T. rubrum hsp genes, as well as their major transcription factor (Hsf1), are involved in the response to adverse situations and in the interaction with the host microenvironment, and if these genes are regulated by the transcription fator PacC, a regulator of the pH signaling pathway. The expression of the hsp genes was evaluated in response to the cultivation of T. rubrum in different culture medium, during exposure to antifungal drugs, heat stress, and interaction with human nail and skin. The involvement of T. rubrum Hsp90 in the modulation of gene expression, susceptibility to antifungal drugs, and interaction with human nails was evaluated by using a chemical inhibitor, specific to this protein. Our results indicate a variable expression of the hsp genes, even among members of the same HSP family, in response to each environmental condition or interaction, to which the fungus was exposed. Furthermore, we have evidence that the hsp gene expression is modulated by the PacC transcription factor, by modulating the expression of the Hsf1 coding gene. We also found that Hsp90 is involved in T. rubrum susceptibility to the drugs Itraconazole and Micafungin, and in the development of this dermatophyte in human nails. These results reveal the involvement of HSPs in several aspects of T. rubrum metabolism, suggesting a role for Hsp90 in the pathogenicity and drug susceptibility in this dermatophyte

La synthèse des protéines de choc thermique (HSPs) est un moyen de défense développé par la cellule pour faire face aux diverses agressions auxquelles elle peut être soumise. En tant que chaperons, les HSPs participent aux mouvements intracellulaires des protéines, préviennent l'agrégation des protéines altérées, éliminent les protéines anormales et contribuent à la conformation correcte des peptides nouvellement synthétisées. Mon équipe d'accueil s'intéresse aux rôles des HSPs dans des processus cellulaires tels que l'apoptose et la différenciation cellulaire. Le but de mon travail de thèse consiste à étudier le rôle des protéines de choc thermique HSP90 et HSP70 au cours de la différenciation des monocytes en macrophages. J'ai dans un premier temps étudié l'implication de HSP90 dans la différenciation macrophagique. c-IAP1 est un membre de la famille des protéines inhibitrices de l'apoptose impliqué dans la régulation de l'apoptose, dans le cycle cellulaire et dans la signalisation cellulaire. Nous avons précédemment montré que c-IAP1 migre du noyau vers le cytoplasme au cours de la différenciation cellulaire. Nous démontrons dans ce travail que c-IAP1 est une protéine cliente de la protéine de choc thermique HSP90β. Dans trois différents modèles de différenciation, ces protéines interagissent et migrent ensemble du noyau vers le cytoplasme au cours de la différenciation cellulaire. L'inhibition de HSP90 ou la déplétion spécifique de l'isoforme β par des siRNA conduisent à sa dégradation par le protéasome. La fonction de chaperon moléculaire de HSP90 envers c-IAP1 est spécifique de l'isoforme β car la déplétion de l'isoforme α n'a pas d'effets sur c-IAP1. De plus l'inhibition de HSP90 ou la déplétion de HSP90β bloquent la différenciation cellulaire tout comme la déplétion de c-IAP1 par siRNA. La deuxième partie de montre travail a consisté à étudier le rôle de HSP70 dans la différenciation macrophagique. Nous montrons que cette protéine est fortement induite après stimulation des cellules par le facteur de croissance M-CSF et que son inhibition bloque la différenciation des monocytes en macrophage. HSP70 interagit avec la protéine Spi-1/Pu.1, facteur de transcription clé de la différenciation macrophagique. L'expression de Spi-1/Pu.1 augmente également au cours de la différenciation macrophagique et ce de manière similaire à celle de HSP70. Ceci suggère l'implication des facteurs de transcription responsables de l'induction des HSPs, les Heat Shock Factor (HSF). L'étude du promoteur de Spi-1/Pu.1 a révélé la présence d'une séquence ressemblant fortement aux éléments de réponse classiques sur lesquels se fixe HSF1. HSF1 est capable de se fixer sur le promoteur de Spi-1/Pu.1 et l'inhibition de HSF1 bloque l'expression de Spi-1/Pu.1. HSF1 participe donc au contrôle de l'expression de Spi-1/Pu.1 lors de la différenciation macrophagique. HSP90 et HSP70 sont donc essentielles à la différenciation macrophagique. Comprendre les mécanismes cellulaires impliqués dans les voies de différenciation se révèle extrêmement important puisque des altérations des mécanismes de l'hématopoïèse sont retrouvées dans plusieurs types de leucémies (leucémies aiguës myéloblastiques et leucémies myélo-monocytaires chroniques). Connaître le rôle des HSPs dans la différenciation cellulaire permettrait donc de développer de nouvelles stratégies thérapeutiques pour le traitement de ces pathologies.