Les Granule de Stress (GS) sont des inclusions cytoplasmiques contenant des protéines et des ARNm qui s’assemblent en réponse à l’exposition à un stress. Leur formation fait partie intégrante de la réponse cellulaire au stress et est considérée comme une étape déterminante pour la résistance au stress et la survie cellulaire. Actuellement, les GS sont reliés à divers pathologies allant des infections virales aux maladies neurovégétatives. L’une d’entre elle, la Sclérose Latérale Amyotrophique (SLA) est particulièrement agressive, caractérisée par une perte des neurones moteurs aboutissant à la paralysie et à la mort du patient en cinq ans en moyenne. Les mécanismes de déclenchement de la pathologie restent encore à déterminer. TDP-43 (TAR DNA binding protein 43) et FUS (Fused in liposarcoma) sont deux protéines reliées à la pathologie qui présentent des similarités de structure et de fonction, suggérant un mécanisme commun de toxicité. TDP-43 et FUS sont toutes les deux recrutées au niveau des GS en condition de stress. Nous avons démontré pour la première fois que la fonction des GS est de protéger les ARNm de la dégradation induite par l’exposition au stress. Cette fonction n’était que suspectée jusqu’alors. De plus nous avons mis en évidence que G3BP1 (Ras GTPase-activating protein-binding protein 1) est l’effectrice de cette fonction via son implication dans la dynamique de formation des GS. TDP-43 étant un régulateur de G3BP1, nous prouvons ainsi que la perte de fonction de TDP-43/G3BP1 aboutit à un défaut de réponse au stress aboutissant à une vulnérabilisation cellulaire. Le mécanisme de toxicité emprunter par FUS diffère de celui de TDP-43 et ne semble pas passer par une perte de fonction dans le cadre de la réponse au stress. / Stress Granule (SGs) are cytoplasmic inclusions sequestering proteins and mRNAs following a stress exposure. Their assembly is part of the cell stress response and is considered an important step for stress resistance and cell survival. SG are currently linked to different pathogenesis from viral infection to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).ALS is an aggressive disease, characterized by neuronal death leading to paralysis and death within five years. Pathogenesis mechanisms are still not fully understood. TDP-43 (TAR DNA binding protein 43) and FUS (Fused in liposarcoma) are two proteins linked to the disease that share many structural features and functions suggesting a common toxicity mechanism. TDP-43 and FUS are both recruited to SGs in stress conditions. We demonstrate for the first time that SGs function to protect mRNA from degradation induced after stress exposure, a function that was only suspected until now. We also prove that G3BP1 (Ras GTPase-activating protein-binding protein 1) is the effector of this function via it’s implication in the dynamics of SG formation. As TDP-43 is a regulator of G3BP1, we prove that loss of TDP-43/G3BP1 function leads to a stress response defect yielding increased cellular vulnerability. Furthermore, we have discovered that the mechanism of toxicity for FUS is different from TDP-43, and does not implicate a loss of function mechanism during the cell stress response.

Stress is a widespread phenomenon in the western society of these days. It is a risky factor for health and well-being of the majority of people. Based on these facts, it is the main subject for the field of "stress physiology" research, which aims to study processes occurring during stress response and tries to elucidate mechanisms leading to stress-induced health impairment. The first aim of this thesis was to describe effects of psycho-social stress on organism. The second aim was to find out if can stress applied in juvenile age affect the stress response in adulthood. If so, how is the role of glucocorticoid-metabolism enzyme 11β-HSD1 in this influence? To answer these questions, two different animal models inducing stress response in the laboratory rat were used. The first one is the model of mild social stress based on the resident-intruder paradigm. Our results show efficancy of this model. Fisher 344 male rats treated under this model for seven consecutive days show highly elevated plasma corticosterone concentrations and elevated expression of the glucocorticoid receptor gene in the pituitary. Behavioral analysis demonstrates a decreased social behavioral profile of the intruders, suggesting submisive social position of these animals in the resident-intruder paradigm. The second model used is...

De manière générale, l'inhalation de particules entraîne des réactions inflammatoires et des réactions d'oxydo-réduction responsables de la dégradation des matrices biologiques qui exercent, de plus, un fort impact cardio-vasculaire. La combustion du bois est une source majeure de composés organiques semi-volatils, parmi lesquels les méthoxyphénols (MPs), tels que le coniféryl aldéhyde (CA), le syringaldéhyde (SR), ou l'acétosyringone (AS). Les MPs sont néanmoins peu étudiés dans la littérature alors que la toxicité d'autres composés également issus de la combustion de la biomasse, tels que le monoxyde de carbone, les suies et les hydrocarbures polyaromatiques est intensivement étudiée. Ce travail a montré par GC/MS que le vieillissement en atmosphère simulée (ozone, rayonnements lumineux) dégrade le CA en produits secondaires moins cytotoxiques, évalués sur des fibroblastes en culture, mais préserve le taux atmosphérique de SR et AS, de toxicité avérée. Un dispositif original de production de MPs particulaires (Ø~50nm, N~7E4particules/cm3, m~5µg/m3) en atmosphère contrôlée a été validé et permet la 1ièreétude in vivo des MPs. L'exposition chez le rat (1-3mois) montre une modification des défenses antioxydantes et des changements cardiaques principalement avec AS, puis CA et un peu moins pour SR. Des processus adaptatifs sont démontrés après 5mois d'exposition.Par ailleurs, il a été montré in vitro sur des adénocacinomes pulmonaires A549 en culture, que le CA induit une destructuration du tapis cellulaire et l'apoptose (caspase 3) mais pas d'effet pro-inflammatoire (IL8, Cox-2). En conclusion, ce travail contribue à étudier l'impact des MPs in vitro et in vivo. / In general, inhalation of particles is at the origin of inflammatory and oxidative reactions who are responsible of the degradation of biological cellular constituents, and could have a strong cardiovascular impact. The wood combustion is a major source of semivolatile organic compounds such as the methoxyphenols (MPs) including coniferyl aldehyde (CA), syringaldehyde (SR), or acetosyringone (AS). The MPs are however few studies into literature while toxicity of other compounds also from biomass combustion, as carbon monoxide, soot and polycyclic aromatic hydrocarbon are intensively studies.This work has shown by GC/MS that aging in simulated atmosphere (ozone + light rays) degraded CA in secondary products less cytotoxic, studies on fribroblastes culture but keep the atmospherical level of SR and AS which have a toxicity proved.A device of MPs particle production original by atomization, with a check system (height, composition, weight) and exposition flow continuous (Ø~50 nm, N~7E4 particles/cm3, m~5 µg/m3) adapted to little animals, was developed and validated, allowed the first study in vivo with these molecules. Between 1 and 3 month of exposition to rat Wistar, show modified antioxidant defences and cardiac modification (ischaemia/reperfusion) principally with AS, then CA and less SR. The adaptatives processes (remodeling) are demonstrated after 5 month of exposition.Furthermore, it is showed in vitro on lung adenocacinum cell lines (A549), CA induced a monolayer destructuration and apoptosis (caspase 3) but no effect proinflammatory (IL8, Cox-2 and iNOS).To conclude, this work contributes to study the impact of MPs in vitro and in vivo.

Com a construção da linhagem RlhE fusionada a um epitopo, fomos capazes de investigar o acúmulo da proteína após choque frio, em fase estacionária, durante o ciclo celular de Caulobacter crescentus, sob diferentes condições de estresses e, finalmente, após a adição de cloranfenicol, para o estudo a estabilidade protéica. Foi observado um aumento no acúmulo da proteína após choque frio. Além disso, quando em diferentes condições de estresses, RlhE obteve uma leve indução na presença de NaCl e Sacarose; mas permanaceu constante em fase estacionária e durante o ciclo celular. Finalmente, vimos que a estabilidade de RlhE varia de acordo com a temperatura, tendo um aumento da estabilidade a 10°C. As análises de expressão do gene rhlE foram realizadas por ensaios de atividade de betagalactosidase. Demonstramos que a presença da região 5UTR é importante para a indução, e que rlhE possui, pelo menos em parte, uma regulação pós-transcricional. Uma análise transcriptômica global da linhagem selvagem e mutante para rhlE, após o choque frio, foi realizada por RNAseq, o qual nos auxiliou na identificação de genes envolvidos em diversos processos biológicos. Finalmente, a co-imunoprecipitação e identificação dos RNAs por sequenciamento em larga escala revelou que RlhE interage com 51 mRNAs. / Here, we constructed a strain in which the RhIE protein was fusioned to an epitope that allowed the investigation of the protein profile after cold-shock, at stationary phase, during cell cycle of Caulobacter crescentus, under different environmental stresses, and, finally, after chloramphenicol addition to study protein stability. The results showed an increase in protein concentration after cold shock stress. When exposed to different stresses RhlE was slightly induced in the presence of NaCl and Sucrose; but its abundance remained constant at stationary phase and during C. crescentus cell cycle. Lastly, the protein stability varies depending on temperature - increasing at low temperature. Gene expression analysis was performed using beta-galactosidase assays. We showed that the presence of the 5UTR is important for the induction of rhlE, and that RhlE is posttranscriptionally regulated. A global transcriptome analysis was performed using RNAseq after cold shock stress of the wild type strain and null mutant for rhlE, and several genes involved in a wide range of biological process were identified. Finally, High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation revealed interactions of RhlE with 51 mRNAs.

A fosforilação reversível de proteínas em resíduos de serina e treonina, catalisada por quinases e fosfatases desempenha papel chave na regulação do crescimento e na diferenciação celular em eucariotos. As serina/treonina proteínas fosfatases (PSTPs) são atualmente divididas em três famílias denominadas PPP (PhosphoProtein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) e FCP/SCP (RNA polymerase II CTD phosphatase), sendo que os membros da família PPP são, frequentemente, holoenzimas compostas de uma subunidade catalítica associada a uma ou mais subunidades reguladoras, as quais definem a função, localização e especificidade ao substrato da fosfatase. Neste trabalho, analisamos, através de RT-qPCR, o perfil de expressão dos genes codificadores de subunidades catalíticas de PPPs de Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c e PP5c) e de 16 potenciais parceiros moleculares de algumas destas subunidades catalíticas, tais como DdI-2 e DdI-3, sabidamente inibidores da PP1c. Em resposta ao estresse térmico de células da fase de crescimento, detectamos o aumento dos níveis de transcritos de PP4c e PP6c e também de DdI-2, DdI-3 e DDB_G0292194, esta última, uma proteína de função desconhecida que interage com a PP1c em ensaios de duplo-híbrido em leveduras. Por outro lado, durante o estresse hiper-osmótico observamos a diminuição dos níveis de transcritos de quase todos os genes analisados com exceção de DdI-2 e DDB_G0292194. O nível de expressão de DdPP1c, DdI-2, DdI-3 e DDB_G0292194 também foi analisado em resposta ao estresse oxidativo e apenas o DDB_G0292194 foi induzido nesta condição. Os genes de PP1c, PP4, PP5c e PP6c são expressos durante todo o ciclo de vida de D. discoideum, mas a expressão de alguns dos genes analisados aumenta em uma fase definida do ciclo de desenvolvimento como é o caso de DDB_G0292194 que tem níveis de transcritos aumentados na fase de agregação. Este gene codifica uma proteína hipotética de 559 aminoácidos, que apresenta um domínio FHA (ForkHead-Associated) em sua região aminoterminal, além de uma sequência similar ao motivo consenso de ligação à PP1c. Ensaios no sistema de duplo-híbrido em leveduras confirmaram que a interação entre DDB_G0292194 e DdPP1c independe do domínio FHA. Verificamos, também, que o mutante nocaute de DDB_G0292194 apresenta uma morfologia alterada em condições padrões de cultivo, tanto na fase de crescimento como durante o desenvolvimento, além de uma maior sensibilidade ao estresse oxidativo causado pelo peróxido de hidrogênio quando comparado à linhagem selvagem. Em conjunto, nossos resultados evidenciam a importância das PPPs na resposta a diferentes tipos de estresse e para o crescimento e desenvolvimento de D. discoideum. / Reversible phosphorylation of proteins on serine and threonine residues, catalyzed by kinases and phosphatases plays a key role in growth and cell differentiation regulation in eukaryotes. Protein serine/threonine phosphatases (PSTPs) are currently divided into three families named PPP (Phosphoprotein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) and FCP/SCP (RNA polymerase II CTD phosphatase). The PPP family members are often holoenzymes composed of a catalytic subunit associated with one or more regulatory subunits, which define function, localization and substrate specificity of the phosphatase. In this work, we have examined, by RT-qPCR, the expression profile of genes encoding PPP catalytic subunits of Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c and PP5c) and 16 potential molecular partners for some of these catalytic subunits, such as DdI-2 and DdI-3, both known as PP1c inhibitors. In response to heat stress of growth phase cells, we detected increased levels of transcripts of PP4c and PP6c as well as of DdI-2, DdI-3, and DDB_G0292194, the latter a protein of unknown function that interacts with PP1c in yeast two-hybrid assays. Moreover, during the hyperosmotic stress we observed decreased transcript levels of nearly all genes examined except DdI-2 and DDB_G0292194. The expression level of DdPP1c, DdI-2, DdI-3 and DDB_G0292194 was also analyzed in response to oxidative stress and only DDB_G0292194 was induced in this condition. PP1c, PP4c, PP5c and PP6c genes are expressed throughout growth and development of D. discoideum while transcript levels of some the analysed genes were increased at a defined stage of the developmental cycle as in the case of DDB_G0292194, which increased during aggregation. This gene encodes a hypothetical protein of 559 amino acids bearing a FHA (ForkHead-Associated) domain in its aminoterminal region and a sequence matching the PP1c binding consensus motif. Yeast two-hybrid assays confirmed that DDB_G0292194 and DdPP1c interaction does not depend on FHA domain. We also found that DDB_G0292194 knockout mutant exibits an altered morphology on standard growth and developmental conditions and shows an increased sensitivity to oxidative stress induced by hydrogen peroxide in comparison to the wild type strain. Taken together, our results highlight the importance of PPPs in the response to different types of stress and for growth and development of D. discoideum.

No description available.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

<p>Stress responsive genes play a key role in shaping the manner in which plants process and respond to environmental stress. Their gene products are linked to DNA transcription and its consequent translation into a response product. However, whilst these genes play a significant role in manufacturing responses to stressful stimuli, transcription factors coordinate access to these genes, specifically by accessing a gene&rsquo / s promoter region which houses transcription factor binding sites. Here transcriptional elements play a key role in mediating responses to environmental stress where each transcription factor binding site may constitute a potential response to a stress signal. Arabidopsis thaliana, a model organism, can be used to identify the mechanism of how transcription factors shape a plant&rsquo / s survival in a stressful environment. Whilst there are numerous plant stress research groups, globally there is a shortage of publicly available stress responsive gene databases. In addition a number of previous databases such as the Generation Challenge Programme&rsquo / s comparative plant stressresponsive gene catalogue, Stresslink and DRASTIC have become defunct whilst others have stagnated. There is currently a single Arabidopsis thaliana stress response database called STIFDB which was launched in 2008 and only covers abiotic stresses as handled by major abiotic stress responsive transcription factor families. Its data was sourced from microarray expression databases, contains numerous omissions as well as numerous erroneous entries and has not been updated since its inception.The Dragon Arabidopsis Stress Transcription Factor database (DASTF) was developed in response to the current lack of stress response gene resources. A total of 2333 entries were downloaded from SWISSPROT, manually curated and imported into DASTF. The entries represent 424 transcription factor families. Each entry has a corresponding SWISSPROT, ENTREZ GENBANK and TAIR accession number. The 5&rsquo / untranslated regions (UTR) of 417 families were scanned against TRANSFAC&rsquo / s binding site catalogue to identify binding sites. The relational database consists of two tables, namely a transcription factor table and a transcription factor family table called DASTF_TF and TF_Family respectively. Using a two-tier client-server architecture, a webserver was built with PHP, APACHE and MYSQL and the data was loaded into these tables with a PYTHON script. The DASTF database contains 60 entries which correspond to biotic stress and 167 correspond to abiotic stress while 2106 respond to biotic and/or abiotic stress. Users can search the database using text, family, chromosome and stress type search options. Online tools have been integrated into the DASTF&nbsp / database, such as HMMER, CLUSTALW, BLAST and HYDROCALCULATOR. User&rsquo / s can upload sequences to identify which transcription factor family their sequences belong to by using HMMER. The website can be accessed at http://apps.sanbi.ac.za/dastf/ and two updates per year are envisaged.</p>

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.