Le Deoxynivalenol (DON) est une mycotoxine majeure retrouvée dans l’alimentation animale et celle-ci est connue pour réduire la fertilité des truies en inhibant la sécrétion de progestérone par les cellules de granulosa. Chez le bétail, DON est métabolisée en de-epoxy DON (DOM-1) dans le rumen, et DOM-1 peut atteindre des concentrations élevées dans le sang et les liquides folliculaires. Une des voies majeures de signalisation activée par DON est le ribotoxic stress response (RSR), lequel induit une auto-phosphorylation de la protéine kinase R (PKR) et réduit l’activation des MAP kinases incluant la MAPK3/1. Il n’a pas encore été démontré que ces mycotoxines affectent la reproduction chez les bovins. Les objectifs de cette thèse sont (1) de déterminer comment et à quelles doses DON affecte la fonction des cellules de granulosa et d’élucider les mécanismes d’action entrant en jeu; et (2) déterminer comment et à quelles doses la mycotoxine majeure DON et son métabolite DOM-1, affectent la fonction des cellules de la thèque chez le bétail. Les résultats sont présentés dans trois articles distincts. Dans le premier article, nous explorons les effets de DON sur les cellules de granulosa bovines; les traitements avec DON résultant en une inhibition significative de la sécrétion d’oestradiol et de progestérone (P4), et en une augmentation de la proportion de cellules apoptotiques après 4 jours de traitement. Les expériences de Western-Blot démontrent une stimulation significative de la phosphorylation de ERK1/2 et de MAPK14 entre 15 et 30 minutes après le début du traitement des cellules par DON. Par la suite, nous avons déterminé les effets de DON sur les gènes cibles de ERK1/2. En effet, les niveaux d’ARNm de EGR1 et FOS sont transitoirement augmentés avec des niveaux maximum à 1h de traitement par DON, tandis que les niveaux d’ARNm de iv COX2 et GADD45B sont augmentés mais plus de 24h après le début du traitement par DON. Dans le second article, les effets de DON et DOM-1 sur les cellules de thèque ont été étudiés. Le traitement des cellules par DOM-1 résulte en une inhibition dosedépendante de la sécrétion de P4 et de testostérone, et en une augmentation de la proportion de cellules apoptotiques, tandis que DON inhibe la sécrétion de P4 sans altérer celle de la testostérone ou bien le pourcentage de cellules mortes. Les deux mycotoxines sont effectives de manière maximale à des concentrations de 1 ng/ml (en revanche, DON affecte les cellules de granulosa à 100 ng/ml). Les résultats de Western-Blot démontrent la phosphorylation rapide de MAPK3/1, PKR et de JUN kinase après un traitement par DON ou DOM-1. En présence d’un inhibiteur spécifique de PKR, DON et DOM-1 sont incapables d’induire la phosphorylation de MAPK3/1, et l’effet inhibiteur de DON sur la phosphorylation de MAPK14 est en partie abrogé. Néanmoins, l’inhibiteur de PKR augmente davantage la phosphorylation de MAPK14 induite par DOM-1. Ensemble, ces résultats suggèrent que DON active le RSR dans les cellules de thèque et les cellules de granulosa bovines, et que les cellules de la thèque sont plus sensibles que les cellules de granulosa aux effets de DON. Ces données démontrent pour la première fois l’habilité de DOM-1 à affecter les fonctions et la survie cellulaires. / Deoxynivalenol (DON) is a major mycotoxin found in animal feed and is known to reduce fertility in pigs by inhibiting progesterone secretion from granulosa cells. In cattle, it is metabolized to de-epoxy DON (DOM-1) in the rumen, and DOM-1 can reach high concentrations in blood and follicular fluid. One of the major pathways activated by DON is the ribotoxic stress response (RSR), which involves autophosphorylation of protein kinase R (PKR) and downstream activation of MAP kinases including MAPK3/1. It is not known if these mycotoxins affect bovine reproduction. The objectives of present thesis were (1) to determine how and at what doses DON affects ovarian granulosa cell function and to elucidate its mechanism of action; and (2) to determine how and at what doses major mycotoxin DON and its metabolite DOM-1 affect theca cell function in cattle. The results are separated into three articles. In the first article the effects of DON on granulosa cells were explored; treatment with DON resulted in a significant inhibition of estradiol and progesterone (P4) secretion, and an increase in the proportion of apoptotic cells after 4 days of treatment. Western blot demonstrated significant upregulation of ERK1/2 and MAPK14 phosphorylation within 15-30 minutes of adding DON. We then determined the effect of DON on ERK1/2 target genes; EGR1 and FOS mRNA levels were transiently stimulated with maximum levels at 1 h of adding DON, whereas COX2 and GADD45B mRNA levels were upregulated but not until 24 h after DON treatment. In the second article, the effects of DON and DOM-1 on theca cells were assessed. Treatment with DOM-1 resulted in a dose-dependent inhibition of P4 and testosterone secretion, and an increase in the proportion of apoptotic cells, while DON inhibited P4 but did not alter testosterone secretion or the percentage of dead cells. Both ii DON and its metabolite were maximally effective at concentrations of 1 ng/ml (in contrast, the effects of DON on occur at 100ng/ml). Western blot demonstrated rapid phosphorylation of MAPK3/1, PKR and of JUN kinase after addition of DOM-1 or DON. Interestingly, phosphorylation of MAPK14 was significantly increased by DOM-1 but decreased by DON. The addition of a PKR inhibitor abrogated the ability of DON and DOM-1 to increase phosphorylation of MAPK3/1, and partly abrogated the inhibitory effect of DON on MAPK14 phosphorylation, however, the PKR inhibitor further increased the phosphorylation of MAPK14 caused by DOM-1. Together, these results suggest that DON activates the RSR in bovine granulosa and theca cells, and that theca cells are more sensitive than granulosa cells to the effects of DON. The data also demonstrate for the first time in any cell type the ability of DOM-1 to affect cell function and health.

The obligate anaerobe Porphyromonas gingivalis is the etiological agent responsible for periodontal disease. It must withstand high levels of reactive nitrogen species in the oral cavity generated by the host and other oral flora. The mechanisms allowing for protection against such stress remain poorly understand. HcpR is an FNR-CRP family regulator that has been implicated in regulation of the nitrosative stress response. In this study we characterize the biochemical properties of HcpR. It is a homo-dimer that is composed of 3 domains – a heme-binding domain, dimerization helix, and a DNA-binding domain. Our studies show that HcpR binds the heme cofactor. UV-Vis and Raman spectroscopy reveal that the bound heme is capable of binding the diatomic gas molecule Nitric Oxide (NO)-a source of nitrosative stress. Binding of NO causes a change in the oxidation state of the iron. SAXS reveals the protein bears a structural resemblance to homology models generated from an ortholog. Promoterr studies reveal that mechanisms P. gingivalis-HcpR uses to modulate expression are novel and different than those found in E. coli and P. aeruginosa.

Staphylococcus aureus is human pathogen and is causative factor of many diseases with different character, from light infections of the skin to life-threatening sepsis. S. aureus often colonizes cystic fibrosis patient's lungs and causes long-standing pneumonia, which can cause death in these patients. Infection caused by S. aureus are, as most of the infections, treated with antibiotics. Failure in treatment is caused because of presence of resistant strains. It was shown, that treatment failure can be caused by different type of the antibiotic tolerance - by the persistence. Persisters are phenotypic variants of isogenic bacterial population, that is unaffected by antibiotic treatment even though they don't have genetics determinants of the resistance. The phenomenon of the persistence in bacteria is still relatively poorly understood. The aim of my thesis was to contribute to the characterization of the persistence in S. aureus. We have optimized a method for rapid identification of persisters' amount in the bacterial population after treatment with antibiotics using the method of measuring the killing curves. We have found out, that exposure to osmotic stress causes increase in number of persistence in the bacterial population by 1 - 2,5 order. Using quantitative PCR method we have analyzed the...

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Experiments were conducted to evaluate the ability of prebiotic and probiotic supplementation of commercial fish feed to improve the physiological, immunological, and growth responses of stressed Nile tilapia. To investigate these objectives, tilapia were divided in two major groups: control fish (fed regular commercial feed) and stressed fish (induced by dietary cortisol supplemented to regular commercial feed). Stressed fish were further divided into three sub-groups: stressed fish fed regular feed, stressed fish fed probiotic-supplemented feed, and stressed fish fed a mixture of prebiotic and probiotic supplemented feed. Fish were maintained and tested over an eight-week long experimental period. A variety of physiological, immunological, and growth parameters were measured over the course of the experimental period. These parameters include: serum cortisol, blood glucose, plasma protein, packed cell volume, hepato-somatic index (HSI), spleen-somatic index (SSI), lysozyme activity, feed conversion ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), length gain, weight gain, length gain, and condition factor (K). The results of this study does not support the use of these specific prebiotic and probiotic as functional feed additives in Nile tilapia at the levels tested in this study. Further research is needed to determine which probiotic species are best suited for use in Nile tilapia and which prebiotic, when used in combination, will allow these probiotics to have maximum effect.

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.

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O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

Lors d'un stress, les cellules activent un mécanisme de défense appelé “la réponse au stress”. Ce mécanisme empêche notamment l'accumulation de protéines mal enroulées grâce à la synthèse des protéines du choc thermique, les HSPs (Heat Shock Proteins),via l'activation du facteur de transcription HSF1 (Heat Shock Factor 1). Les HSPs empêchent l'agrégation des protéines et aident au repliement protéique. Deux partenaires associés à HSF1 ont récemment été identifiés : le chaperon moléculaire VCP (Valosin Containing Protein) et l'histone déacétylase 6 (HDAC6). Notre projet était d'étudier les rôles d'HDAC6 et de VCP dans la réponse au stress thermique. Nous avons mis en évidence un rôle prépondérant du domaine de fixation à l'ubiquitine d'HDAC6 dans la régulation de la durée d'activation d'HSF1 suite à un choc thermique. Lorsque HDAC6 ne peut pas se fixer à l'ubiquitine, VCP favorise une inactivation rapide de HSF1 empêchant la transcription du chaperon HSP25. Ces travaux montrent également que la réponse activée suite à un stress dépend de la nature de celui-ci. En effet, nous avons montré que les mécanismes activés dans la réponse au stress suite à un choc thermique sont différents de ceux activés suite à une inhibition du protéasome. La myopathie à corps d'inclusion associée à la maladie osseuse de Paget et à une démence fronto-temporale, appelée IBMPFD, Inclusion Body Myopathy associated with Paget disease of the bone and Frontotemporal Dementia, est une maladie autosomale dominante rare. La myopathie est la caractéristique clinique la plus commune parmi celles qui sont causées par des mutations de VCP. La perturbation de la fonction de VCP entraîne l'accumulation de protéines poly-ubiquitinées et la formation de corps d'inclusion en partie responsables de la pathogenèse de l'IBMPFD. Nous avons montré que l'activation de la réponse au stress via un choc thermique dans des cellules murines déficientes en VCP mimant le phénotype de l'IBMPFD, entraîne une diminution du taux de cellules ayant des agrégats de protéines poly-ubiquitinées. Nos résultats préliminaires montrent que HSF1 ne serait pas à l'origine de cette diminution des agrégats au contraire de HSP90 et HDAC6 qui interviendraient suite à l'activation de la réponse au stress. Cette stratégie est actuellement en cours de test sur des modèles murins. / Under stress, cells activate a defense mechanism named “cellular stress response”. This mechanism prevents especially unfolded proteins accumulation thanks to Heat Shock Proteins (HSPs) synthesis through the activation of Heat Shock Factor 1 (HSF1) transcription factor. HSPs prevent aggregation and help protein refolding. Two partners associated to HSF1, have recently been identified: the molecular chaperone, VCP (Valosin Containing Protein) and the Histone DeACetylase 6 (HDAC6). Our project was to characterize the roles of HDAC6 and VCP in the Heat Shock Response (HSR). We have highlighted a preponderant role for the HDAC6 ubiquitin binding domain in the HSF1 activation time regulation after a heat shock. When HDAC6 can't bind ubiquitin, VCP promotes a rapid HSF1 inactivation preventing HSP25 chaperone transcription. This work also emphasizes a stimulus-dependent stress response. Indeed, we showed that mechanisms activated during the stress response following a heat shock differ from those activated after a proteasome inhibition. Inclusion Body Myopathy, Paget disease of the bone, and Frontotemporal Dementia (IBMPFD) is a rare autosomal dominant disorder. Myopathy is the most common clinical feature of IBMPFD. It is caused by mutations of VCP. Alteration of VCP function leads to the accumulation of poly-ubiquitinated proteins and to the formation of inclusion bodies thought to be responsible, at least in part, for the pathogenesis of IBMPFD. We have shown that activation of the heat shock stress response in mouse cells VCP deficient mimicking IBMPFD phenotype, results in the decrease of cells with ubiquitinated protein aggregates. Our preliminary results show that HSF1 is not responsible for this decrease unlike HSP90 and HDAC6 that seems to intervene following the stress response activation. This strategy is currently tested on mouse models.