Dissertations / Theses on the topic 'Stress modulation'

This thesis investigates the role of central 5-hydroxytryptamine (5-HT) in the process of adaptation to chronic stress. Central 5-HT of male rats was depleted by bilateral intraventricular (i.c.v.) infusion of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) with the use of specific re-uptake blockers to protect the dopamine (DA) and noradrenaline (NA) system. They were then exposed to different chronic stress paradigms. The effect of 5-HT depletion on social defeat was studied. Lesioned or sham-operated rats were either exposed daily for 10 days to a second larger aggressive male in the latter's home cage, or simply transferred to an empty cage (control procedure). Lesioned rats failed to show the increased freezing behaviour during the pre-defeat phase of the social interaction test characteristics of sham-operated animals. Core temperature increased during aggressive interaction in sham-operated rats, and this did not adapt with repeated stress. By contrast, stress-induced hyperthermia was accentuated in 5-HT-reduced rats as the number of defeat sessions increased. Basal core temperature was unaffected by 5-HT depletion. Heart rate increased during social defeat, but this did not adapt with repeated stress: 5-HT depletion had no effect on this cardiovascular response. Basal corticosterone was increased in lesioned rats, but the progressive reduction in stress response over days was not altered. C-fos expression patterns in lateral preoptic area (LPOA) and medial amygdala (Me-AMY) in rats with repeated defeat were altered by 5-HT depletion. These results suggest that 5-HT is important in the modulation of overall adaptation process in exposure to chronic stress.

Selenium (Se) is an essential micronutrient necessary for human health. In humans, Se de ciency has been associated with in ammatory bowel disease (IBD) and increased risk of certain cancers, including colorectal cancer. Se has well established antioxidant and anti-in ammatory properties which are medi- ated, in part, though the actions of the selenoproteins, in which Se is present in the form of the amino acid selenocysteine (Sec). The cells of the gastrointestinal tract are exposed to stresses from pro-oxidative and hypoxic conditions, which have been suggested to be involved in the pathogenesis and pathology of IBD. Further characteristics of IBD are inappropriate immune responses of the gut epithelial cells to the gut microbiota. Thus, to help explain the roles of Se in IBD, it is important to understand the modulatory e ects of Se on the cell innate immune responses following challenge of intestinal epithelial cells with pathogen-associated molecular patterns (PAMPs), as well as oxidative and hypoxic stresses. The present work aimed to assess the roles of Se and the selenoproteins, SelH and TR1, in the responses of Caco-2 cell, modelling the gut epithelium, to hypoxia and infection, the latter replicated by challenge with S. typhimurium agellin. To investigate the responses of gut cells to low Se and PAMPs, undi erentiated Caco-2 cells with either supplemented with Se (40 nM selenite) or depleted of Se for 72 h before challenging with agellin (F) (500 ng/mL). The gene expression of the pro-in ammatory cytokines IL-8 and TNF- were measured in addition to the genes encoding the antimicrobial peptides (AMPs) hBD1 and hBD2. Data showed that Se depletion signi cantly a ected hBD1 expression (0.88-fold increase, P < 0.05), but that Se depletion plus F signi cantly increased the induced expression of all genes (IL-8: 1.68-fold, P < 0.001; TNF- : 0.71-fold, P < 0.001; hBD2: 1.74-fold, P < 0.001) compared with the Se supplemented cells. F and Se depletion were also associated with a signi cant increase in expression of TR1 (F: 1.68-fold, P < 0.001; Se depletion: 0.33-fold, P < 0.01) and GPX2 (F: 3-fold, P < 0.001; Se depletion: 11-fold, P < 0.001), but a signi cant decrease due to Se depletion in SelH (62 %, P < 0.001) and GPX1 (47 %, P < 0.001). The selenoprotein TR1 is an antioxidant enzyme and the primary regulator of the thioredoxin system (TXN), which has previously been shown to regulate immune responses. Knockdown of TR1 expression resulted in the reduced agellin-induced expression of IL-8 (40 %, P < 0.001), TNF-a (45 %, P < 0.01), hBD1 (40 %, P < 0.01) and hBD2 (45 %, P < 0.001). These data suggested that Se, through TR1, is involved in regulating the expression of agellin-induced immune e ectors. The selenoprotein SelH has also been suggested to have antioxidant functions. Knockdown of SelH was associated with the increased expression of the oxidative stress-associated genes NQO1 (0.41-fold, P < 0.001), and HMOX1 (1.78-fold, P < 0.001), supporting a role for SelH in the expression of oxidative stress-associated genes. The role of Se, through SelH and oxidative stress, in regulating the gut responses to agellin, has been discussed. The Caco-2 cell model is more representative of intestinal epithelial cells in vivo, when the cells are di erentiated and placed in a gaseous environment re ecting the oxygen gradient of the gut. Thus the F challenge experiments using di erentiated Caco-2 cells were repeated using a dualoxic environment. Interestingly, no potentiation of gene expression relating to the pro-in ammatory agents IL-8 and TNF- , and the defensins hBD1 and hBD2 was observed. These data suggested that the dualoxic environment completely diminished the e ects of Se depletion on the expression of immune e ectors IL-8, TNF- , hBD2 and hBD1, following agellin challenge. These data suggested the e ects of Se in more physiologically relevant intestinal epithelial cell models, more representative of the in vivo state, are required.

Mestrado em Biologia Aplicada
As mitocôndrias são organelos altamente dinâmicos com um papel crucial na homeostase celular. Uma rede de mitocôndrias funcionais é mantida por processos de biogénese e mitofagia, regulando desta forma o conteúdo e o metabolismo mitocondriais. Espécies reactivas de oxigénio (ROS) são formadas como consequência do processo normal de fosforilação oxidativa mitocondrial, desempenhando um papel importante na sinalização redox e regulação da função celular. Um aumento ligeiro na formação de ROS mitocondrial desencadeia o fenómeno de hormese mitocondrial, uma resposta adaptativa ao estado metabólico celular, ao stress e outros sinais intracelulares ou ambientais. Este mecanismo induz maior resistência a um stress posterior, tendo por isso efeitos benéficos para a saúde. Compostos que são tóxicos em doses maiores são conhecidos por induzir adaptações mitocondriais em doses mais baixas. O excesso de equivalentes redutores fornecidos à cadeia transportadora de electrões (ETC) em condições de sobrenutrição/inactividade física ou danos acumulados/defesas antioxidantes mais baixas associadas com o envelhecimento, causam um aumento nas taxas de produção de ROS, provocando stresse oxidativo e danos irreversíveis em proteínas, lípidios e DNA. A disfunção mitocondrial resultante é permanente e compromete o estado energético de todo o organismo, aumentando a susceptibilidade a lesões, provocando a aceleração do envelhecimento e desenvolvimento de doenças metabólicas, tais como a resistência à insulina e fígado gordo. Um dos principais reguladores do sistema de defesa antioxidante celular é a Sestrina 2 (SESN2), induzida em condições de stress. A diminuição da actividade da SESN2 está associada a um aumento de danos oxidativos, disfunção mitocondrial, degeneração muscular e acumulação de gordura, resultando num envelhecimento mais rápido dos tecido. No entanto, os mecanismos pelos quais a SESN2 afecta as funções mitocondriais não estão definidos. A compreensão dos mecanismos moleculares e de como a SESN2 afecta a mitocôndria, pode fornecer novas pistas para alvos terapêuticos, a fim de atenuar e prevenir o envelhecimento e as patologias relacionadas com a obesidade. Tendo em conta isto, este trabalho teve como objectivo avaliar se a SESN2 medeia uma resposta mitocondrial adaptativa protectora, desencadeada pela exposição de células C2C12 a menadiona, um estimulador da formação de aniões superóxido. Adicionalmente, e tendo em conta que a Sirtuina 1 (SIRT1) é um conhecido sensor metabólico e regulador da função mitocondrial, este estudo avaliou de que forma a modulação de SIRT1 afecta a SESN2 no contexto de fígado gordo induzido por uma dieta rica em gordura. Os resultados obtidos mostram um efeito da menadiona, dependente da dose, na viabilidade celular e a função mitocondrial. O tratamento com 10 μM de menadiona durante 1 h não alterou a formação de ROS, redução do MTT e o potencial de membrana mitocondrial, como avaliado 24 e 48 horas após a remoção de menadiona. No entanto, a exposição de células C2C12 a 30 μM menadiona durante 1 h, resultou no aumento da formação de ROS, diminuiu a redução do MTT e o potencial de membrana mitocondrial. Um aumento no conteúdo de SESN2 foi observado após 10 h de exposição a 10 μM de menadiona durante 1 h, enquanto 30 μM de menadiona resultou na diminuição do conteúdo em SESN2. Estes resultados sugerem que a indução de SESN2 por stress moderado, induzido pela menadiona, pode activar uma resposta mitocondrial protetora que preserva a viabilidade celular. O silenciamento da SESN2 com siRNA resultou num aumento da morte celular, bem como numa diminuição no potencial de membrana mitocondrial induzida por ambas as concentrações de menadiona, sendo mais drásticas as alterações induzidas por 30 μM. Na presença de SESN2, a exposição a menadiona causou um aumento no padrão pontuado de distribuição de LC3, indicando a indução de autofagia. Contrariamente, a depleção de SESN2 com siRNA resultou numa diminuição da pontuação de LC3, quer em condições controlo quer após a exposição a menadiona. Colectivamente estes resultados sugerem que o stress moderado provocado pela menadiona induz a SESN2 e activa autofagia/mitofagia como uma estratégia de sobrevivência celular. A ausência de SESN2 resultou na acumulação de dano mitocondrial induzido por ROS e consequente diminuição da viabilidade celular. Em relação ao impacto da modulação da SIRT1 na SESN2, os resultados obtidos mostram que a expressão hepática do factor de transcrição c/EBPα (proteína alfa potenciadora de ligação CCAAT) foi estimulada pela dieta rica em gordura (HFD) e reduzida pelo tratamento com resveratrol, um activador da SIRT1. Em ratinhos sem SIRT1 (SIRT1 - KO) a expressão c/EBPα estava diminuída comparativamente ao controlo. A expressão hepática de SESN2 apresentou-se reduzida em animais HFD e SIRT1 - KO. O tratamento com resveratrol, em animais controlo, preveniu a diminuição da SESN2 induzida por HFD. A expressão de KEAP1 (proteína kelch 1 associada a ECH) também se verificou dependente de SIRT1, sendo que, em ratinhos SIRT1-KO, o tratamento com resveratrol não induziu nenhuma alteração em KEAP1. A degradação de KEAP1 é promovida pela SESN2, permitindo a translocação de Nrf2 (factor nuclear derivado de eritróide 2) para o núcleo e, consequentemente, a indução de genes antioxidantes. A expressão hepática de Nrf2 não foi afetada pela modulação de SIRT1. O envelhecimento diminuiu a expressão de todos os genes estudados. Em conclusão, este trabalho demonstrou que a indução de SESN2 por stress ou compostos promotores de homeostase mitocondrial, como o resveratrol, aumenta a tolerância mitocondrial ao dano, através da modulação da autofagia/mitofagia. A estimulação da eliminação de mitocôndrias lesadas pela SESN2, pode ser uma via para evitar a acumulação de danos e, portanto, resultar num aumento da tolerância à sobrenutrição e ao envelhecimento.
Mitochondria are highly dynamic organelles with a crucial role in cellular homeostasis, with processes of biogenesis and mitophagy regulating mitochondrial content and metabolism and maintaining functional mitochondrial networks. Reactive oxygen species (ROS) are formed as a consequence of normal mitochondrial oxidative phosphorylation and are involved in redox signalling and regulation of cellular function. A mild increase in mitochondrial ROS triggers mitochondrial hormesis, an adaptive retrograde response to cellular metabolic state, stress and other intracellular or environmental signals that culminate in subsequently increased stress resistance with health promoting effects. Compounds that are toxic at higher doses are known to induce mitochondrial adaptations at lower doses. Overflow of reducing equivalents to the electron transport chain (ETC) under conditions of overnutrition/physical inactivity or accumulated damage/lower antioxidant defenses associated with aging, causes higher rates of ROS formation, resulting in oxidative stress and irreversible damage to proteins, lipids, and DNA. As a result, permanent mitochondrial dysfunction compromises whole-body energetic status and increases susceptibility to injuries, resulting in accelerated aging and development of metabolic diseases such as insulin resistance and fatty liver. One of the main regulators of the cellular antioxidant defense system is Sestrin 2 (SESN2), which is induced by several stress conditions. Decreased SESN2 activity is associated with increased oxidative damage, mitochondrial dysfunction, muscle degeneration and fat accumulation. However, the mechanisms by which SESN2 affects mitochondrial functions are not defined. Understanding the molecular mechanisms and how SESN2 affects mitochondria may provide new insights for novel therapeutic targets for attenuation and prevention of aging and obesity-related pathologies. In view of this, this work aimed to evaluate if SESN2 mediates an adaptive protective mitochondrial response in C2C12 cells triggered by menadione, a stimulator of superoxide anion formation. Additionally, and since Sirtuin 1 (SIRT1) is a known metabolic sensor and regulator of mitochondrial function, this work evaluated how modulation of SIRT1 affects SESN2 in the context of fatty liver induced by a high-fat diet. Results showed a dose-dependent effect of menadione on cellular viability and mitochondrial function. Treatment with 10 μM menadione for 1 h did not alter ROS formation, MTT reduction and mitochondrial membrane potential, as evaluated 24 and 48 h after menadione removal. However, exposure of C2C12 cells to 30 μM menadione for 1 h resulted in increased ROS generation, reduced MTT reduction and mitochondrial membrane potential. An increase in SESN2 content was observed 10 h after exposure to 10 μM menadione for 1 h, while 30 μM menadione resulted in SESN2 depletion. These results suggest that induction of SESN2 by mild stress induced by menadione may be involved in a mitochondrial protective response that preserves cell viability. SESN2 silencing with siRNA resulted in increased cellular death as well as a decrease in mitochondrial membrane potential induced by both concentrations of menadione, being more potent the alterations induced by 30 μM menadione. In presence of SESN2, exposure to menadione caused an increase in the punctuated pattern of LC3 (microtubule-associated protein 1A/1B-light chain 3 - PE phosphatidylethanolamine) distribution, showing induction of autophagy. However, depletion of SESN2 with siRNA resulted in a decrease in LC3 punctuation, both in control and menadione conditions. Altogether these results suggest that mild stress induced by menadione induces SESN2 and activates autophagy/mitophagy as a cell survival strategy. Absence of SESN2 results in accumulation of mitochondrial damage induced by ROS and consequent decrease in cell viability. Regarding the impact of SIRT1 modulation on SESN2, results showed that hepatic expression of transcription factor c/EBPα (CCAAT-enhancer-binding protein - α) was up-regulated by high-fat diet (HFD) and down-regulated by resveratrol treatment, a SIRT1 activator. In SIRT1-knock-out (SIRT1 - KO) mice c/EBPα expression was decreased when compared to control. SESN2 expression was reduced by HFD and SIRT1-KO. Resveratrol treatment in wild-type animals prevented the decrease in SESN2 induced by HFD. KEAP1 (Kelch-like ECH-associated protein 1) expression was also dependent on SIRT1 and resveratrol treatment showed no effect on KEAP1 in SiRT1-KO mice. KEAP1 degradation is promoted by SESN2 and when degraded, induces Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) translocation to the nucleus and consequently induction of antioxidant genes. Hepatic Nrf2 expression was not affected by SIRT1 modulation. Aging decreased the expression of all of the evaluated genes. The current work shows that SESN2 induction by mild stress or promotors of mitochondrial homeostasis, such as resveratrol acting on SIRT1, increase mitochondrial tolerance to damage, through modulation of autophagy/mitophagy. Elimination of damaged mitochondria is stimulated by SESN2 and may be the pathway to prevent accumulation of damage and thus result in increased tolerance to overnutrition and extend lifespan.

RNA dysregulation is a recently recognized disease mechanism in amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease characterized by muscle atrophy and death of both upper and lower motor neurons. A key feature of the disease is the mislocalization of the RNA binding protein TDP-43 and formation of TDP-43 containing cytoplasmic aggregates in motor neurons and surrounding glia. TDP-43 is known to associate with stress granules, and recent studies in mammalian cell culture have indicated that pathological TDP-43 aggregates may arise from RNA stress granules following prolonged stress. We set out to test this hypothesis by investigating the interaction between PolyA Binding Protein (PABP), a known core RNA stress granule component, and TDP-43. Here we show that PABP colocalizes with TDP-43 in a variant dependent manner. Given that the highest risk factor for ALS is aging, an attractive model is that age-related oxidative stress triggers formation of toxic cytoplasmic aggregates from TDP-43 containing stress granules. We have therefore begun investigations using a time course, and live imaging of RNA stress granules under oxidative stress to determine if this leads to an altered RNA stress granule dynamics in cultured motor neurons. These studies will yield a better understanding of the mechanisms that lead to the toxic cytoplasmic aggregates in cases of ALS.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

Le stress oxydant est la conséquence d'un déséquilibre au sein d'un organisme entre la production d'oxydants et les mécanismes de défenses antioxydants. La NADPH oxydase est l'enzyme clé d'une production excessive d'oxydants renforçant l'intérêt de sa modulation. Elle participe à l'initiation et à la progression des maladies associées au vieillissement comme les maladies cardiovasculaires et les démences. Au moyen d'une étude épidémiologique composée de 517 sujets (79,5 ± 7,1 ans), nous avons déterminé l'activation de la NADPH oxydase et de ses principaux déterminants en fonction de l'âge. Parmi les facteurs testés, l'homocystéine et l'inflammation sont significativement associés à l'activité de la NADPH oxydase en analyse multivariée. Ces données sont confirmées in vitro puisque l'homocystéine thiolactone et le lipopolysaccharide induisent de façon dose-dépendante l'activation et l'expression de la NADPH oxydase sur le modèle cellulaire THP-1. Dans une étape in vitro sur les THP-1, nous avons étudié la modulation négative de la NADPH oxydase par des antioxydants naturels (polyphénols) et enzymatiques (extrait de melon riche en SOD et catalase). Ces antioxydants nutritionnels modulent négativement l'activité mais aussi l'expression de la NADPH oxydase monocytaire. En conclusion, l'activité de la NADPH oxydase est associée à l'âge et est positivement régulée par l'inflammation et l'homocystéine. La production d'anion superoxyde est associée aux pathologies neurodégénératives et cardiovasculaires. La NADPH oxydase peut être modulée in vitro par des antioxydants naturels. Les liens avec la démence, les maladies cardiovasculaires ou la mortalité doivent faire l'objet d'études ultérieures. L'efficacité de manœuvres de supplémentation en antioxydants doit être précisée sur des modèles animaux puis sur une étude clinique
Oxidative stress is the result of an imbalance between production of oxidants and antioxidant defense mechanisms. The NADPH oxidase is a key enzyme for excessive production of oxidants, strengthening the interest of its modulation. NADPH oxidase is involved in the initiation and progression of age-related diseases such as cardiovascular diseases and dementia. Through an epidemiological study consisted of 517 subjects (79. 5 ± 7. 1 years), we determined NADPH oxidase activation and its main determinants with age. Among the factors tested, homocysteine and inflammation were significantly associated with NADPH oxidase activity in a multivariate analysis. These data are confirmed in vitro because homocysteine thiolactone and lipopolysaccharide induce a dose-dependent activation and expression of NADPH oxidase in cell line THP-1. In an in vitro on THP-1 and animal studies, we studied the negative modulation of the NADPH oxidase by natural antioxidants (polyphenols) and enzymatic antioxidants (melon extract rich in SOD and catalase). These nutritional antioxidants negatively modulate the activity but also the expression of NADPH oxidase. In conclusion, NADPH oxidase activity is associated with age and is positively regulated by inflammation and homocysteine. The superoxide anion production is associated with cardiovascular and neurodegenerative diseases. NADPH oxidase may be modulated in vitro by natural antioxidants. Links with dementia, cardiovascular disease or mortality must be further study. The antioxidant supplements efficiency must be clarified in animal models and then on a clinical study

Viral myocarditis is an inflammatory heart disease caused by viral infection, which is a major cause of sudden death in children and young adults. Among the various viruses, coxsackievirus B3 (CVB3) is a predominant pathogen of viral myocarditis. As CVB3 replication is tightly tangled with signaling pathways in host cells, an in-depth study of CVB3-host interactions would promote the understanding of the pathogenesis of viral myocarditis and provide critical drug targets for the development of therapeutics. CVB3 infection induces different types of stress in host cells, and in turn, the cells respond to the stress via expressing certain stress-responsive proteins (SRPs) to counteract the stress for cell survival. During the co-evolution of virus and host, CVB3 has developed sophisticated strategies to modulate and utilize SRPs to benefit its own replication. The main objective of this dissertation is to investigate the modulation and functional roles of SRPs in CVB3 infection and CVB3-induced myocardium damage. I hypothesize that 1) CVB3 infection differentially regulates the expression and activity of SRPs at transcription, translation or post-translation level; 2) the dysregulation of SRPs benefits CVB3 replication and promotes CVB3-induced cell damage. This dissertation mainly focuses on two SRPs, the inducible heat shock 70 kDa protein (Hsp70) and nuclear factor of activated T-cell 5 (NFAT5), during CVB3 infection. Using in vitro (cell culture) and in vivo (mouse) models, I demonstrated an increase of Hsp70 but a decrease of NFAT5 during CVB3 infection. Further studies elucidated the mechanism underlying such changes as well as the feedback effects on CVB3 replication. Hsp70 was upregulated via CaMKII-HSF1 signaling cascade activated in CVB3 infection and in turn promoted CVB3 infectivity via stabilizing viral genome and benefiting viral translation. NFAT5 was cleaved by CVB3 proteases 2A and 3C, generating a 70 kDa dominant negative truncate, which inhibited the iNOS-mediated anti-viral activity of NFAT5. Together, my findings have uncovered the new roles of SRPs in CVB3 infection and potential novel drug targets for CVB3-induced myocarditis.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Heat shock or stress proteins (HSPs), are a large family of phylogenetically conserved molecules that can be constitutively expressed at high levels under normal physiological conditions. These proteins are selectively synthesized following metabolic and environmental insult. This study was designed to determine whether cognate and/or inducible HSP production are altered in CD4-expressing lymphocytic cell lines concomitant with acute and chronic human immunodeficiency virus type 1 (HIV-l) infection. Our findings indicate that in CEM.NKR cells, HSP27 production is selectively altered at early stages of acute HIV-infection (6-24h post-infection), subsequent to virus internalization but prior to synthesis of viral progeny. Levels of HSP27 induction were viral dose-dependent and were not accompanied by any alterations in cellular proliferation. In contrast, acute HIV-infection was not associated with significant quantitative changes in the constitutive expression of HSP60, HSP70, or HSP90. Nevertheless, a transient, marked induction of select HSP70 subspecies was evident at early stages of infection, finally disappearing by 48-72h. Acute-infection of Jurkat cells resulted in similar patterns of de novo induction of HSP27 and HSP70 isoforms. Uninfected and chronically-infected CEM extracts showed little detectable constitutive HSP27. However, synthesis of select HSP27 and HSP70 homologues in chronically-infected cells was observed following exposure to a mild heat shock and doses of TNF$ alpha$. Similar HSP70 homologues arose in chronically-infected cells treated with heat-shock and TNF$ alpha$. These findings indicate that HSP pathways are uniquely modulated in CD4+ cells as a consequence of acute and chronic HIV-1 infection.

In this thesis study we examined glycogen synthase kinase-3β (GSK-3β) and its effects over Nrf2 and Pin 1 as it relates to Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized by a prolonged high oxidative environment. Transcription factor Nrf2 is vital in the brain’s defense against oxidative insults through its up-regulation of over 100 antioxidants. Depletion of the brain’s antioxidant defense system results in intolerance to an oxidative environment, contributing to the progression of AD. The regulatory Pin 1 protein promotes cellular homeostasis, and when down-regulated results in increased deposits of neurofibrillary tangles (NFTs) and amyloid-β (Aβ) plaques, the two pathological hallmarks of AD. Using aged SAMP8 mice treated with antisense oligonucleotide (AO) directed at GSK-3β and random AO, the data presented here demonstrate decreased oxidative stress and increased Nrf2 transcriptional activity and Pin 1 levels as a result of the down-regulation of GSK-3β. Collectively, these results implicate GSK-3β activity in the increased oxidative stress of AD and support its inhibition as a possible therapeutic treatment for the disease. Further, we elucidate a possible mechanism connecting GSK-3β to the loss of tolerance to an oxidative environment and increased deposits of NFTs and Aβ plaques observed in AD.

Chez les espèces sociales, les vocalisations transmettent des informations qui participent au maintien et à la survie du groupe. Alors que de nombreuses études se sont intéressées aux informations stables portées par les vocalisations telles que l’identité, peu d’études se sont interrogées sur le rôle des signaux vocaux dans la transmission des informations plus labiles, telles que l’état émotionnel de l’émetteur. Le stress est un bon candidat pour l’étude de l’expression des émotions chez les animaux, puisqu’il est directement mesurable via un dosage de la concentration plasmatique en glucocorticoïdes. Le stress est connu pour modifier les paramètres acoustiques des vocalisations chez les mammifères, mais peu d’études ont traité ce processus chez les oiseaux, qui présentent pourtant des réseaux sociaux complexes. Le but de cette thèse est de déterminer de quelle manière les oiseaux expriment vocalement leur stress, et d’évaluer dans quelle mesure la corticostérone, hormone de stress principale chez l’oiseau, est impliquée dans le phénomène. Je me suis intéressée au Diamant mandarin (Taenopygia guttata), un oiseau chanteur australien au comportement grégaire qui forme des liens d’appariements à vie et prodigue des soins biparentaux à sa progéniture. En administrant de la corticostérone exogène à des oiseaux et en utilisant également des évènements sociaux stressants, nous montrons que les mâles adultes et les poussins expriment leur stress à travers l’émission de cris modifiés dans leur structure, ce qui suggère que la flexibilité des cris chez les oiseaux est plus importante que l’avaient montré des études précédentes. Par une analyse complète des paramètres temporels et spectraux des cris, nous montrons pour la première fois que le stress, par un effet direct de la corticostérone, déclenche l’émission de vocalisations présentant un spectre de fréquence déplacé vers les hautes fréquences. De plus, les receveurs du signal (respectivement les partenaires femelles et les parents) semblent capables de décoder l’information portée par ces cris de stress car ils modifient leur comportement en conséquence. Les processus physiques impliqués dans l’émission de cris modulés par le stress sont également discutés en appliquant la théorie « source-filtre » généralement utilisée chez les mammifères. Enfin, les valeurs adaptatives de ces cris sont également envisagées, en rapport avec le réseau social du Diamant mandarin et les risques de prédation encourus par l’émetteur du signal. Ce travail apporte de nouvelles preuves sur l’expression du stress chez les oiseaux, et propose une étude complète, des signaux physiologiques impliqués dans le stress aux modifications de comportement de l’émetteur, qui déclenchent une réponse adaptative des receveurs du signal
In social species, vocalisations convey information that participates in the maintenance and the survival of the group. While many studies were interested in stable information carried by vocal signals, like identity, fewer studies dealt with their potential role in informing about labile information such as the senders’ emotional state. Stress is a good candidate for the study of the expression of emotions in animals, as it is directly measurable by the plasma levels of glucocorticoïds. Stress is known to modify acoustic parameters of vocalisations in mammals, but few studies studied the process in birds, that also show complex social networks. The aim of this thesis is thus to determine how birds can vocally express their stress and to what extent corticosterone, the main stress hormone in birds, is implicated in this expression. I focused my research on the zebra finch (Taenopygia guttata), a gregarious Australian songbird that form lifelong pairbond and provides biparental care to its young. Using oral administration of exogenous corticosterone but also social stressful events, we show that both adult males and nestlings can express their stress through modulations of their calls’ structure, suggesting that flexibility in birds’ vocalisations is higher than previously expected. With a complete analysis of temporal and spectral parameters of calls, we show for the first time that stress evokes the emission of vocalisations with up-shifted frequency spectrum via a direct effect of corticosterone. Moreover, we show that females and parents are able to decode the information carried by stressed-induced calls of respectively their male partner and their young, as they exhibit modifications of behaviour in accordance with the context. The physical processes leading to the emission of stressed-induced vocalisations are discussed by applying the source-filter theory usually used in mammals. Adaptive values are also proposed, in regards with the zebra finch social network and predation risks for the caller. This work gives new evidences about the expression of stress in birds, and proposes a comprehensive study, from the physiological signals involved in stress to the resulting modifications of communication behaviour for the sender, that leads to an adaptive response from the receivers

Les protéines APOBEC3 (A3A-A3H) catalysent la désamination des cytidines (C) présentes sur l'ADN simple brin en thymidine (T). Cette activité cytidines désaminase a initialement été décrite comme impliquée dans la restriction des rétrovirus et de certains virus à ADN par leur capacité à induire de nombreuses mutations C->T, ou hypermutations, sur les génomes viraux. Il apparait néanmoins que leur activité n'est pas restreinte aux génomes viraux et que certaines A3 peuvent induire des mutations sur l'ADN mitochondrial (A3A, C, F, G et H) et nucléaire (A3A et A3B). Ainsi, l'impact somatique des A3 est désormais établi dans la formation de certains cancers, dont la majorité des mutations, portent signatures des APOBEC3. Aux vues de ces observations, nous nous sommes intéressés à la façon dont sont régulées ces enzymes dans le contexte du stress cellulaire viro-induit ou endogène. La première partie de nos travaux a porté sur la protéine A3DE, seul membre de la famille APOBEC3 ne possédant pas d'activité cytidine désaminase. De façon intéressante, il apparait qu'A3DE est surexprimée dans les cirrhoses infectées par le VHB, VHC ou co-infectées par le VHC et le VHB. Nous avons pu mettre en évidence qu'A3DE interagit et module l'activité d'A3F et d'A3G, deux cytidines désaminases exprimées dans le foie et impliquées dans la restriction du VHB. Dans un second temps, nous nous sommes intéressés à la caractérisation du potentiel génotoxique de la protéine A3B. Cette protéine, de par sa localisation strictement nucléaire, constitue la seule A3 à double domaine n'interagissant pas avec A3DE. Contrairement à A3A, A3B est faiblement active sur l’ADN nucléaire et n’induit pas de cassures de l’ADN double brin. Nous avons pu mettre en évidence par mutagénèse les régions de la protéine impliquées dans l’atténuation de la génotoxicité d’A3B par rapport à A3A et que cette atténuation est conservée chez les primates. Enfin, nous avons étudié le rôle et la régulation d’A3A dans le catabolisme. Nous avons mis en évidence que l’ADN mitochondrial cytoplasmique (ADNcymt) active la voie RIG-I/ARN polymérase III ce qui a pour effet d’induire la production d’IFN qui va activer l’expression d’A3A. A3A va ainsi jouer un rôle dans le catabolisme de l’ADNcymt et contribue à l'élimination de cette source de stress cellulaire, mais occasionnant par la même des dommages sur l’ADN nucléaire. Les A3 sont des enzymes fondamentales de la défense immunitaire innée et du catabolisme de l’ADN. Nous montrons qu’A3DE a pour fonction de moduler l’activité d’A3F et d’A3G tandis qu’A3B, possède un phénotype atténué chez tous les primates et s’avère moins génotoxique que’A3A. Cette dernière participe à la dégradation de l’ADN cytoplasmique, limitant ainsi l’inflammation. Néanmoins, A3A peut s’avérer dangereuse pour l’intégrité génomique et contribuer à l’émergence de cancers, notamment en cas d’inflammation chronique
APOBEC3 proteins (A3A-A3H) catalyse the deamination of cytosine (C) to thymidine (T) on single stranded DNA. This activity, called cytidine deaminase, has initially been described as a mechanism involved in restriction against retroviruses and DNA viruses by massively inducing C->T mutations on viral genome : this phenomenon is called "hypermutations". Nevertheless, this activity is not virus-specific and some A3 can induce mutations on mitochondrial DNA (A3A, C, F, G, H) and nuclear DNA (A3A and A3B). Thus, the impact of those proteins on cancer formation is now established in cancers where mutations mostly show an APOBEC3 signature. In view of those considerations, we decided to study how those enzymes are regulated in the context of a viral cellular stress or an endogenous cellular stress. The first part of our work is focused on A3DE, the only APOBEC3 lacking a cytidine deaminase activity. Interestingly, A3DE is upregulated in cirrhotic livers infected by HBV, HCV or coinfected with HBV & HCV. We show that A3DE inhibits A3F & A3G activity by interacting with those HBV restriction involved A3. Then, we studied the attributes of the genotoxicity potential of A3B. This protein, by his strictly nuclear localization, constitutes the only double domain A3 which is not regulated by A3DE. Unlike A3A, A3B is weakly active on nuclear DNA and does not induce double strand breaks. We determine by directed mutagenesis the clusters of A3B involved in genotoxicity attenuation compared with A3A. We also show that this attenuation is conserved among primates. Finally, we investigated the role and regulation of A3A in the context of DNA catabolism. We proved that mitochondrial cytoplasmic DNA (mtcyDNA) triggers the RIG-I/DNA polymerase III pathway, which induces IFN production leading to A3A expression. So A3A will be involved in mtcyDNA catabolism and contribute to the clearance of this stress signal, but will also induce double strand breaks on nuclear DNA. A3 are major enzymes of the innate immune response and DNA catabolism. We show that A3DE modulates A3F and A3G activity while A3B is attenuated among primates and is less genotoxic than A3A. A3A participates to cytoplasmic DNA catabolism and limits inflammation. Nevertheless, A3A could be dangerous for the genomic integrity and contributes to cancer, especially in cases of chronic inflammation

Cumulative evidence suggests that constitutively elevated levels of proteotoxic stress represent a specific vulnerability of malignant cells that can be targeted by pharmacological modulation of the intracellular proteotoxic stress response. According to this emerging mechanism, small molecule stress modulators may induce deviations from protein homeostasis causing cytotoxicity confined to malignant cells already at a high set point of constitutive proteotoxic stress leading to functional impairment and even cell death. In contrast, normal cells with sufficient protein degradation capacity can tolerate the extra dysfunctional protein overload. My graduate research has focused on testing the feasibility of repurposing clinically used non-oncological drugs for experimental chemotherapy targeting metastatic melanoma cells. The following specific aims were pursued: (1) To identify clinically used non-oncological drugs that preferentially induce cytotoxicity in melanoma cells but not primary melanocytes through upregulation of proteotoxic and/or oxidative stress; (2) To explore the specific molecular mechanisms underlying induction of melanoma cell apoptosis by lead compounds focusing on oxidative and proteotoxic stress modulation; (3) To explore efficacy of selected lead compounds for antimelanoma intervention in a murine xenograft model. First, we demonstrate feasibility of using the FDA-approved redox-active D-cysteine-derivative D- penicillamine for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo through induction of the unfolded protein response (UPR). Second, we demonstrate that the antimicrobial oligopeptide thiostrepton displays dual activity as a selective prooxidant and proteasome inhibitor causing proteotoxic stress that preferentially targets malignant melanoma and multiple myeloma cells. Third, we demonstrate for the first time that the clinically used 4-aminoquinoline antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced melanoma cell death. Taken together, our data indicate the chemotherapeutic potential of small molecule proteotoxic stress inducers and strongly suggest feasibility of repurposing specific non-oncological drugs for proteotoxic stress-directed antimelanoma intervention.

Twenty-four crossbred steer calves (235 + 6 kg initial BW) were weaned, adapted to a growing diet, trained to use Calan feeders and assigned to one of four treatments in a 2 x 2 factorial arrangement as follows: (1) no transit stress and saline injection, (2) no transit stress and trace mineral and vitamin E (TM/VitE) injections, (3) transit stress and saline injection and (4) transit stress and TM/VitE injection. TM/VitE injections consisted of MultiminTM to provide 0.3, 0.6, 0.15 and 0.6 mg of Cu, Mn, Se, and Zn/kg BW, respectively, and Vita ETM to provide 9 IU of VitE/kg BW on d 0 and 21 of the study. On d 24, transit-stressed steers were subjected to a three-d, 2000-km, transportation period. During the 56-d experiment, liver and serum samples were assayed to assess trace mineral status, antibody titers to keyhole limpet hemocyanin (KLH) measured to assess humoral immunity and phytohemagglutinin(PHA)- and ovalbumin(OVA)-induced skin swelling and lymphocyte proliferation measured to assess cellular immunity. TM/VitE-injected steers grew slower (P < 0.05) over the first 24 d of the experiment and tended (P = 0.10) to grow slower after transit. TM/VitE injections increased (P < 0.05) liver Cu and Zn concentrations 135 and 21 % by d 30, respectively. TM/VitE-injected steers had increased (P < 0.05) serum Cu on d 24 and 26, but decreased (P < 0.05) serum Zn and whole blood Se compared to saline-injected steers on d 24 and 56. TM/VitE injections increased serum α-tocopherol concentrations. TM/VitE injections did not affect humoral immunity, but decreased (P < 0.05) skin-swelling responses to PHA on d 21, and decreased (P < 0.05) lymphocyte proliferation to OVA and KLH. Transit-stressed steers had higher (P < 0.05) serum Cu on d 26, and higher (P < 0.05) liver Cu concentrations on d 56 than non-transit-stressed steers. Transit stress did not affect serum Zn or whole blood Se concentrations. Injections of TM/VitE increased Cu, Zn and vitamin E, but not Se status. The TM/VitE treatment suppressed growth and cellular immune function suggesting that levels of trace minerals used in this study were too high.

Les Formes réactives de l’oxygène (FRO) ont un rôle bien établi dans l’oncogénèse en augmentant les capacités de prolifération et d’invasion des cellules tumorales. Mais les FRO exercent aussi un effet important sur le microenvironnement tumoral, en particulier en participant à l’échappement des tumeurs au système immunitaire. Une modulation pharmacologique de l’équilibre oxydo-réductif tumoral est donc susceptible d’influencer la progression tumorale. Il a été montré que l’induction pharmacologique d’un stress oxydant dans les cellules tumorales peut induire un effet cytotoxique mais ses effets sur le microenvironnement sont moins bien connus. L’objectif de nos travaux était d’étudier l’effet d’une modulation du stress oxydant sur les cellules immunitaires du microenvironnement tumoral et in fine de préciser les conséquences potentielles sur la progression tumorale. L’arsenic trioxyde (As2O3), inducteur de FRO, présentait à faible dose, dénuée d’effet cytotoxique direct sur les cellules malignes, un effet antitumoral dans un modèle de cancer colique murin. Cet effet était lié à une déplétion sélective en lymphocytes T régulateurs (Tregs) et était médié par la génération d’anions superoxyde (O2°-) et de monoxyde d’azote (NO), eux même responsables de la formation de peroxynitrite. Les Tregs présentaient un niveau basal de FRO plus élevé que les autres populations lymphocytaires, qui pourrait expliquer leur plus grande sensibilité à un surcroît de stress oxydant induit par l’As2O3. Un cytotoxique antitumoral, la vinorelbine, s’est également montré capable d’exercer un effet sur le microenvironnement tumoral. Par co-cultures, nous avons montré que la vinorelbine induisait un effet bystander toxique sur les cellules immunitaires effectrices voisines des cellules tumorales. In vivo, le prétraitement par vinorelbine de cellules malignes implantées à la souris était responsable d’une perte de la réactivité anti-tumorale des cellules mono-nuclées. Cet effet était dépendant de la production d’O2°- et de NO par les cellules malignes. Un modulateur du stress oxydant, le mangafodipir, inhibait cet effet, permettant ainsi de restaurer la réponse immunitaire antitumorale locale. Notre travail a donc permis de mettre en évidence que des modulateurs du stress oxydant peuvent agir sur le microenvironnement, et spécialement sur les cellules immunitaires. Ils pourraient être utilisés en clinique pour restaurer la réponse immunitaire antitumorale. Une meilleure compréhension du rôle du stress oxydant dans la défaillance de l’immunité antitumorale est nécessaire
Several reports have demonstrated the involvement of reactive oxygen species (ROS) in carcinogenesis, through promotion of cancer cell proliferation and invasion. But ROS could also have consequences on non cancerous cells which are part of the tumor microenvironment, such as immune cells. Therefore, a pharmacological modulation of oxidative stress can induce a cytotoxic effect on tumor cells but its consequences on microenvironment are unknown. The aim of our studies was to evaluate the effects of a pharmacological modulation of oxidative stress on immune cells from the tumor microenvironment. At low dose, Arsenic trioxide (As203), an oxidative stress modulator, was shown to exert antitumor effects in colon tumor-bearing mice. We observed that this effect was related to As203-induced regulatory T cells (Tregs) -selective depletion in vitro and in vivo and was mediated by oxidative and nitrosative bursts. The differential effect of As203 on Tregs versus other CD4 cells was related to difference in the cells’redox status. We also observed that vinorelbine, an anticancer agent, could interfere with the antitumor immune response. We showed that vinorelbine could alter the function of immune cells surrounding tumor cells by a bystander toxic effect against tumor effector cells. In vivo experiment in A549 tumor bearing nude mice showed that adoptive transfer of A549 immune splenocytes was not able to delay tumor growth when vinorelbine-pretreated A549 cells were used for immunization. This effect was mediated by ROS and was inhibited by an oxidative stress modulator, mangafodipir, which restored antitumor immune function. Therefore, our work showed that oxidative stress modulators can influence tumor microenvironment and more specifically, immune cells. They could be used to restore antitumor immune response

Oxidative stress is characterized by the cellular accumulation of reactive oxygen species (ROS). Increased production of ROS, such as the superoxide anion (O2.-), or a deficiency in their clearance by antioxidant defenses, mediates the cellular pathology. Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder commonly manifesting in milieu of the metabolic syndrome. Oxidative stress is an important pathogenic mediator in NAFLD, and in its associated morbidities like atherosclerosis. The objective of my research was to investigate the regulation of oxidative stress and the antioxidant actions of natural health products (NHPs) in the context of NAFLD and its associated disorders. The O2.- generating NADPH oxidase contributes to atherogenesis by facilitating macrophage induced vascular injury. In manuscript I, the plant alkaloid berberine effectively abolished NADPH oxidase mediated O2.- production in lipopolysaccharide stimulated macrophages. Real-time PCR analysis and siRNA transfection studies revealed that berberine mediated its effects through down-regulation of the oxidase’s catalytic subunit gp91phox. Berberine also restored the activity of the O2.- clearing enzyme superoxide dismutase (SOD). High fat diet (HFD) fed rodents are a popular model for investigating NAFLD pathogenesis. In manuscript II, folic acid supplementation significantly reduced HFD-induced hepatic oxidative stress and liver injury in mice. Folic acid decreased NF-kB/DNA binding, down-regulated NADPH oxidase gene expression, and inhibited the oxidase. The antioxidant activities of SOD and catalase were restored and the reduced to oxidized glutathione ratio (GSH:GSSG) was re-established with folic acid supplementation. Folic acid’s hepatoprotective antioxidant effects were associated with a marked improvement in liver histology. Homocysteine (Hcy) levels are perturbed in NAFLD, but the etiology is unclear. In manuscript III, HFD fed mice exhibited decreased Hcy levels. Real-time PCR and Western Immunoblotting analysis revealed that Hcy catabolising enzymes cystathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE) were increased in the liver of these animals. The transsulfuration activities of these enzymes were elevated and coincided with enhanced hepatic hydrogen sulfide biosynthesis. Glutathione was maintained despite increased hepatic oxidative stress. Taken together, NHPs such as berberine and folate, and Hcy catabolising enzymes CBS and CSE, might have therapeutic potential for managing oxidative stress in NAFLD and its associated co-morbidities.
October 2015

It is well known that environmental contaminants such as arsenic can induce oxidative stress in fish. It is also known that thyroid hormone status affects susceptibility to oxidative stress. However, the effect of thyroid hormone status on pollutant- induced oxidative stress is unknown. This is significant, because such knowledge would help to assess the risk of xenobiotics such as arsenate, a prevalent contaminant in the environment. The interaction between arsenate- induced oxidative stress and altered thyroid hormones has not been elucidated thoroughly in previous studies. In order to address this, we used zebrafish (Danio rerio). There are many advantages of using this as a research model over rodents such as rats. For example, it is often used to study the effects of xenobiotic compounds such as arsenic. This xenobiotic is widespread in the environment due to human activities such as agricultural, industrial and military activities. For this purpose, it is more appropriate to use this fish as a research model. The reason for using the chemicals perchlorate and arsenic is that they occur together in the environment. Oxidative stress can be caused by environmental pollutants such as arsenic. One of the key defenses against oxidative stress is glutathione (GSH). GSH concentrations, GSSG/GSH ratios, and lipid peroxidation (TBARS) were used to assess the affect of arsenic, perchlorate and thyroxine (T4) on zebrafish (Danio rerio) liver, gills, and muscle tissue. Our results support the hypothesis that thyroid hormones modulates the toxicity of arsenic. Moreover, arsenite was found to cause oxidative stress as reflected by GSH levels, GSSG/GSH levels, and lipid peroxidation (TBARS). Although hypothyroidism caused by perchlorate did not cause any major difference on oxidative stress, but hyperthyroidism caused by treating the fish with T4 enhanced GSH levels. This shows that thyroxine is involved in response to oxidative stress. In addition, perchlorate abrogated or reversed the affects of arsenite on oxidative stress parameters. These results support the hypotheses that thyroid hormones modulate oxidative stress in general, and arsenite-mediated oxidative stress in particular.

Le premier objectif de cette thèse était d’améliorer l’utilisation et la compréhension des outils de mesure de la déformabilité du globule rouge (GR) dans la drépanocytose (Etudes 1 et 2). L’étude 1 a montré l’importance de la standardisation des mesures de déformabilité par ektacytométrie chez les enfants drépanocytaires. Au cours de l’étude 2, les propriétés des GR ont été modifiées et la variation des courbes de déformabilité érythrocytaire « classique » (index d’élongation en fonction de la contrainte de cisaillement en milieu isotonique) a été comparée aux résultats d’osmoscan (mesure de la déformabilité érythrocytaire en gradient osmolaire à contrainte de cisaillement fixe), méthode de référence pour étudier les anomalies de la membrane du GR. Ainsi, les variations de déformabilité érythrocytaire au-delà de 3 Pa sont affectées à la fois par la viscosité interne du GR et par des modifications de la surface cellulaire (rapport surface/volume) alors que les modifications de l’élasticité membranaire affectent la déformabilité érythrocytaire quelles que soient les forces de cisaillements utilisées (faibles, modérées ou hautes). Le deuxième objectif de cette thèse était d’apporter des éléments supplémentaires sur l’implication des facteurs génétiques, des paramètres hémorhéologiques et du niveau de stress oxydant sur la survenue des complications vaso-occlusives chez les patients atteints de syndrome drépanocytaire majeur (Etudes 3 à 6). La mise en commun des résultats d’hémorhéologie obtenus sur 165 patients de notre cohorte lyonnaise et 240 patients de la cohorte guadeloupéenne a permis de montrer que la rhéologie du GR chez les patients drépanocytaires était dépendante de l’âge. Ainsi, la viscosité sanguine augmente avec l’âge pour atteindre un plateau vers 30 ans alors que la déformabilité érythrocytaire diminue avec l’âge (Etude 3). Ces modifications participent vraisemblablement à l’apparition de complications chroniques chez l'adulte drépanocytaire. Les études 4 et 5 ont été réalisées sur la cohorte pédiatrique lyonnaise. Au cours de ces 2 études, nous avons étudié l’influence sur la rhéologie du sang et la survenue de crises vaso-occlusives (CVO) des facteurs génétiques (alpha-thalassémie, déficit en Glucose-6-Phosphate Déshydrogénase (G6PD) et haplotypes S) d’une part (Etude 4) et du niveau de stress oxydant et nitrosatif d’autre part (Etude 5). L’alpha-thalassémie augmente la déformabilité des GR et l’agrégation érythrocytaire. Ces 2 phénomènes pourraient participer à augmenter le risque de CVO. De plus, l’alpha-thalassémie, en diminuant l’hémolyse, diminuerait le niveau de stress oxydant, élément majeur impliqué dans la physiopathologie de la drépanocytose. Enfin, l’étude 6 a montré que la rhéologie sanguine des patients Sbêta+ était quasi-identique à celle des sujets sains AA mais que les patients les plus sévères pourraient avoir un déficit en monoxyde d’azote circulant. En conclusion, mon travail de thèse contribue à une meilleure compréhension de la physiopathologie de la drépanocytose
The first goal of this thesis (Study 1 and 2) was to improve the use and the comprehension of tools for red blood cell (RBC) deformability measurements in sickle cell disease (SCD). The first study showed the importance of standardization of RBC deformability measurements by ektacytometry in SCD children. In the study 2, the RBC proprieties was modified and the variation of « classic » RBC deformability curve (elongation index as a function of the shear stress in isotonic medium) was compared to osmoscan results (elongation index in hyperosmolar gradient and constant shear stress), the gold standard for RBC membrane defect studies. Thus, the modifications of RBC deformability curve above 3 Pa were affected by RBC internal viscosity and cellular surface modification (and thus surface/volume ratio) while membran elasticity modifications affected RBC deformability whatever the shear stress (low, moderate or high). The second goal of this thesis was to study the effects of genetic modifiers, hemorheological parameters and oxidative stress level on vaso-occlusive complications (VOC) in SCD (Study 3 to 6). Hemorheological parameters were measured on 165 patients from Lyon and 240 patients from Gwada and the results showed that blood viscosity increased until the age of 30 and RBC deformability decreased with age (Study 3). This modifications probably play role in the chronic complications of SCD adult patients. The studies 4 and 5 were conducted on SCD children. We studied the effects of genetic modifiers (alpha-thalassemia, glucose-6-phospho-deshydrogenase deficiency and S haplotypes ; study 3) and nitro-oxidative stress level (study 5). Alpha-thalassemia increase RBC deformability and RBC aggregation. This phenomenon could contribute to increase VOC. Moreover, alpha-thalassemia decreased hemolysis and thus oxidative stress, a major component of SCD physiopathology. Then the study 6 showed that Sbeta+ patient hemorheology was quite the same of AA ubjects but the more severe patients could have a defect in circulating nitric oxide. To conclude, my thesis contribute to a better understanding of SCD physiopathology

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.
Includes bibliographical references (leaf 18).
Cyclosporin A induced biosynthesis of colored compounds in three species of Aspergillus. Diode array HPLC MS analysis of culture extracts revealed Aspergillus terreus demonstrated the most profound response, with upregulation of more then twelve compounds from three distinct chemical families; butyrolactones, aspulvinones, and asterriquinones. Compounds from these three families are prenylated, and biosynthetically derived from homodimers of amino acids. The majority of the upregulated compounds were the aspulvinones, a class of butenolides. Structural elucidation of four isolated aspulvinones revealed both known and novel structures. Inducer concentration had a significant impact on aspulvinone profile. Bioassay revealed previously unreported antibacterial activity for the aspulvinones. A high-throughput colorimetric screen was designed to probe the response. The screen of 2480 known bioactives revealed multiple compounds capable of inducing aspulvinone production, and one compound, rapamycin, capable of inhibiting the response. Additionally, the increase in aspulvinone production was correlated with a decrease in culture density, indicating aspulvinone production is a general stress response.
by Amy Hanlon.
S.M.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2010
Consumption of sucrose with a meal containing oxidised and oxidisable lipids cause an increase in oxidative stress which is referred to as postprandial oxidative stress. The modulating effect on postprandial oxidative stress by an antioxidant-rich beverage, fermented rooibos (Aspalathus linearis) was compared to that of a commercial soft drink (soda). Both study beverages contained sucrose and were consumed with a standardised fat meal. The study consisted of two parts, a pilot study (Phase One) where participants consumed either a standardised fat meal with water (control group n = 5) or a standardised fat meal with a sucrose-containing commercial soda (treatment group n = 8) using a parallel design, and the experimental study (Phase Two) where participants (n = 14) consumed the standardised fat meal with the commercial soda (control group) or the rooibos beverage (treatment group) using a crossover design. Specific analytical techniques and methods for determination of plasma glucose, serum insulin, lipid profile, an inflammatory indicator (high sensitive C-reactive protein), plasma antioxidant capacity, whole blood redox status and plasma lipid oxidation biomarkers were used. Results from the pilot study indicated significantly (P<0.05) higher postprandial levels of glucose in the control group at 4 hr and 6hr postprandially. The inflammatory biomarker and triglyceride levels were significantly (P<0.05) elevated in both groups when compared to their respective baselines. Results also showed the total antioxidant capacity and total glutathione levels in the plasma of both groups to be significantly (P<0.05) lowered when compared to the baseline values. The level of lipid oxidation biomarkers in the plasma was significantly (P<0.05) higher at 2 hr, 4 hr and 6 hr post time intervals for thiobarbituric acid reactive substances and 4 hr post time interval for conjugated dienes in the participants consuming the standardised fat meal with soda when compared to the baseline value, while this was reflected only at 2 hr post time interval for thiobarbituric acid reactive substances, with the conjugated dienes levels being significantly (P<0.05) lowered at 6 hr post time interval in the control group. No differences were shown on inter group level for the pilot study. On inter group level, results from Phase Two showed significant (P<0.05) lower levels of plasma glucose at 6 hr post time interval in the treatment group when compared to the control group, with insulin levels being significantly (P<0.05) higher in the control group at 4 hr post time interval.

Stress has been shown to exacerbate cell death and cognitive deficits after ischemic injury in rodents, however, little is known of the effects of stress on motor recovery. The objective of this present thesis is to examine the effects of chronic stress on skilled motor recovery after devascularization lesion in rats. It was found that pre-lesion stress induced the most behavioural impairments, while post-lesion stress exacerbated infarct volume. The effects of chronic multiple stress on skilled motor recovery after lesion was also examined. Chronic multiple stress did not modulate skilled motor recovery nor did it have any influence on infarct volume. Additionally, stress had effect on edema after devascularization lesion. The present thesis suggests that the time of exposure to chronic stress in respect to the ischemic lesion, in addition to the type of stress, will differentially affect recovery and compensation in rats.
xii, 122 leaves : ill. ; 29 cm.

L'environnement social contribue au développement et à l'expression de maladies. En effet, dans les espèces sociales, la susceptibilité aux infections dépend de la position sociale mais les bases endocriniennes et immunitaires de ces différences au sein du groupe sont ignorées. De plus, une modification de la structure sociale engendre un stress important dont les conséquences immunitaires sont encore sujettes à controverse. Ce travail de thèse a pour objectifs (1) de décrire l'influence du statut social sur le fonctionnement des systèmes endocrinien et immunitaire, (2) de préciser les effets du stress social sur la production de cytokines et la susceptibilité aux infections et (3) de rechercher des facteurs à l'origine de la variabilité des conséquences immunitaires du stress social. Une étude introductive sur le porcelet mise à part, un modèle murin a été utilisé. Les résultats montrent une influence du statut social. En absence de stress, les dominants présentent des niveaux de base de corticostérone et une réponse à la tuberculine supérieurs aux dominés. Suite à une procédure de défaite sociale chronique, les dominants sont plus affectés que les dominés. La défaite sociale accroît la réactivité inflammatoire mais ne modifie pas l'équilibre de la production de cytokines de type1 et 2 et n'affecte pas la réponse à une infection mycobactérienne. Les conséquences immunitaires de la défaite sociale ne sont observées que lorsque le stress est associé à des combats et à des blessures. Ces travaux montrent enfin que la réponse au stress dépend de l'histoire sociale de l'individu, en particulier de son statut social, et son histoire immunitaire récente
In social species, social environment contributes to the development and expression of diseases. Indeed, the social position in a group modulates susceptibility to infections. However, the endocrine and immune mechanisms involved in these differences are not known. Furthermore, instability in the social organisation generates a severe stress of which immune consequences are still controversial. The purposes of this thesis are (1) to describe the influence of social status on the endocrine and immune systems, (2) to specify the effects of social stress on cytokine production and susceptibility to infections and (3) to look for possible sources of variability in the immune consequences of social stress. Excepted a single study on piglets, experiments were performed in mice. Results show an influence of social status. In the absence of stress, dominants show higher basal corticosterone levels and specific response to tuberculin than subordinates. After social defeat, dominants are more affected. Social defeat increases inflammatory reactivity but does not clearly modify the balance between Th1 and Th2 cytokines production and does not affect the development of specific immunity against a mycobacterial infection. Immune effects of social defeat can be observed only when the stress involves significant fights and injuries. Our work also shows that the response to stress depends on individual social experience, in particular on social status, and on the recent immune experience of the individual

Thesis (Ph.D.)--Tufts University, 2003.
Adviser: L. Michael Romero. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 158-180). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

>Magister Scientiae - MSc
With a decrease in water availability and arable land, and the ever-increasing reports of toxic chemical pollutants, it is crucial to elucidate plants’ mechanisms of adaptability to these abiotic stressors. South Africa alone accounts for approximately 30% of global Zirconium (Zr) production. However, reports on Zr-induced stress in plants are sparse. Increased mining activity leads to soil contamination which subsequently has harmful effects on crop plants. Under normal conditions B. rapa crop plants flourish, they are rapid in their cycling and circumvent the seed dormancy stage which enables them to have high yields over relatively short periods. However, when unfavourable conditions arise, such as exposure to toxic chemicals and metal ions like Zirconium, the development and growth of B. rapa L., much like other crop plants is affected. More specifically, the damaging effects of Zr is not only attributed directly; as with substitutions of biometals [like Iron (Fe)] in various biomolecules rendering them inactive, but more as a consequence of the production of toxic molecules such as reactive oxygen species (ROS) and methylglyoxal (MG). ROS such as superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂ ) are known to have signalling roles in plants with reports on their involvement in alleviating seed dormancy and seedling development. However, the signalling roles of MG are not known with regards to plant cells and have been reported more so in animal cells; playing vital roles in fat signalling in diseases such as diabetes. Furthermore MG, in plant and animal cells, directly converts oxygen (O₂) to O₂⁻ and thus increases the cell’s oxidative imbalance, leading to cell damage if O₂⁻ is not rapidly dismutated to H₂O₂ and H₂O by superoxide dismutase (SOD). In turn, H₂O₂ is more stable than O₂⁻ and consequently is more toxic to cells over time. Therefore, H O must be removed as well by a collection of enzymes, such as ascorbate peroxidase (APX) and catalases (CAT). In this study, possible stress-signalling of MG in seedlings under normal conditions and Zr-stress were investigated to establish whether MG at a low dose (6 μM) would benefit seedling growth and development, via a proposed preinduction of the B. rapa L. antioxidant system. Therefore, it was proposed that ROS accumulation due to the exogenous application of MG, would incite the activation of antioxidants and thus mitigate the effects of Zr stress. Physiological tests to determine dry weights (figure 3.2.3) and germination percentage (figure 3.2.2) revealed that MG-treated seedlings yielded an improved biomass and early development compared to Zr-treated seedlings and the control. Membrane damage as assessed by lipid peroxidation viz. Malondialdehyde [MDA] (figure 3.2.4) and conjugated dienes [CD] (figure 3.2.5) also indicated less damage in MG-treated seedlings compared to the Zr-treated set. The chlorophyll content observed was prominent (table 3.1). MG-treated seedlings exhibited a 40% and 15.5% increase compared to Zr-treated seedlings and the control respectively. Moreover cell viability had improved in MG-treated seedlings compared to the control, and in MG+Zr-treated seedlings only a slight increase in cell death occurred despite Zr being present. O₂⁻, H₂O₂ and •OH (figure 4.2.1 – 4.2.3) were investigated in B. rapa L. seedlings in response to Zr and MG by spectrophotometric biochemical assays, as well as their scavenging enzymes, MG accumulation and Gly-I activity. Furthermore, BrGLY1 gene expression and Zr-uptake by ICP-OES were performed. Seedlings treated with MG and Zr respectively showed an increase in ROS. However, all of the ROS observed in MG+Zr-treated seedlings were markedly lower compared to Zr-treated seedlings. SOD and CAT activity observed in MG+Zr-treated seedlings had decreased compared to Zr-treated seedlings, whereas APX activity had increased. Gly-I activity and BrGlyI gene expression had increased across all treatments, showing an elicited response to oxidative stress, due to the observed upregulation, as a result of the accumulated MG. The observed Zr-uptake in MG+Zr-treated seedlings was inhibited by 5-fold compared to Zr-treated seedlings. Clear signs of stress were evident in seedlings treated with Zr compared to the control and MG- treated seedlings, the MG-supplemented (MG and MG+Zr) seedlings displayed a vast improvement comparatively. Modulation of antioxidant activity observed in this study is indicative of an incited response to oxidative stress (figure 4.2.4 – 4.2.6). MG revealed distinct involvement in stress-signalling, ROS levels had increased, although not as severely as with Zr-treated seedlings, but seemingly enough to activate antioxidants without eliciting damage. Furthermore, the proposed early-onset activation of antioxidants has been observed in B. rapa L. seedlings of this study, and as such has resulted in improved growth, development and seed germination. The results of this study has therefore negated the previous reports on MG-toxicity (at high concentrations), and has shed light on further properties of this ubiquitous and inevitably-occurring metabolite at low levels.
National Research Foundation (NRF)

Le peroxyde d'hydrogène est nécessaire à la prolifération cellulaire mais toxique lorsqu'il est présent à des concentrations plus importantes. Les cellules tumorales sont soumises à un stress oxydatif intense, proche du seuil toxique, ce qui augmente leur sensibilité à des composés capables d'augmenter la concentration intracellulaire de peroxyde d'hydrogène. Le paclitaxel induit une accumulation précoce de peroxyde d'hydrogène qui joue un rôle significatif dans son effet antitumoral, aussi bien in vivo qu'in vitro. Le mangafodipir, mimétique de la superoxyde dismutase (SOD), accroît la production de peroxyde d'hydrogène dans les cellules tumorales et augmente l'effet antitumoral du paclitaxel. Il est de plus capable de diminuer la toxicité hématologique du paclitaxel in vivo. Cet effet différentiel du mangafodipir semble être en rapport avec sa double activité SOD et glutathion réductase lui conférant plutôt un effet antioxydant dans les cellules non tumorales
Hydrogen peroxyde is necessary to cellular proliferation but is toxic at high concentrations. Tumor cells are submitted to severe oxidative, close to toxic threshold, increasing their sensitivity to pharmacologic compounds able to increase intracellular concentration of hydrogen peroxide. Placlitaxel induces an early hydrogen peroxide accumulation which is significantly involved in its antitumoral effect, in vitro and in vivo. Mangafodipir, a superoxide dismutase (SOD) mimic, increases the hydrogen peroxide production in tumor cells and enhances the antitumoral effect of paclitaxel. Moreover, it is able the hematologic toxicity of paclitaxel in vivo. This differential effect of mangafodipir seems to be related to its dual activity, SOD and glutathione reductase, responsible for an antioxidant effect in non-tumoral cells

Les conséquences immunitaires d'un stress d'origine environnementale sont complexes et encore difficilement prévisibles. Le stress affecte le système immunitaire soit en agissant sur l'immunité innée, en altérant la réactivité inflammatoire, soit en agissant sur l'immunité acquise, en modulant la production de cytokines dites Th1 et Th2. L'environnement social
contribue largement au développement et à l'expression de maladies. Dans les espèces sociales, la position sociale occupée dans le groupe module la susceptibilité aux infections mais les supports endocriniens et immunitaires de ces différences de susceptibilité sont ignorés. La remise en cause de l'organisation sociale engendre un stress important dont les conséquences immunitaires sont encore sujettes à controverse.
Ce travail de thèse a pour objectifs (1) de décrire l'influence du statut social sur le fonctionnement des systèmes endocrinien et immunitaire, (2) de préciser les effets du stress
social sur la production de cytokines et la susceptibilité aux infections et (3) de rechercher des facteurs à l'origine de la variabilité des conséquences immunitaires du stress social.
Chez le porcelet, un regroupement après le sevrage élève transitoirement le cortisol salivaire et altère le comportement mais n'affecte pas la réactivité des lymphocytes sanguins.
La suite des travaux a utilisé une procédure de défaite sociale chronique chez la souris. Les résultats obtenus mettent en évidence une influence du statut social. En absence de stress, les
dominants présentent des niveaux de base de corticostérone et une réponse spécifique à la tuberculine supérieurs aux dominés. Suite à une défaite sociale, les dominants sont plus affectés que les dominés. La défaite sociale augmente la réactivité inflammatoire mais ne modifie pas de façon nette l'équilibre de la production de cytokines de type Th1 et Th2 et n'affecte pas l'immunité spécifique développée contre une infection mycobactérienne. Les conséquences immunitaires de la défaite sociale ne sont observées que lorsque le stress est associé à des combats et à des blessures. Ces travaux montrent que la réponse au stress dépend de l'histoire sociale de l'individu, en particulier de son statut social. De plus, les
répercussions immunitaires du stress dépendent aussi de l'histoire immunitaire récente. En effet, une réaction inflammatoire systémique inhibe la libération plasmatique de cytokines
inflammatoires en réponse à un stress psychologique ultérieur.

Kinesiology
Ph.D.
It is known that African American ethnicity is an independent risk factor for exaggerated oxidative stress which is intricately intertwined with inflammation, hypertension (HT), and cardiovascular disease (CVD). The purpose of this dissertation study was to examine the racial differences that exist between African Americans and Caucasians in oxidative stress levels at the molecular level using an in vitro model of Human Umbilical Vein Endothelial Cells (HUVECs). African American HUVECs were found to have significantly higher baseline levels of oxidative stress in vitro compared to Caucasian HUVECs. In order to establish proof of concept, three preliminary studies were conducted. The first preliminary study, an acute exercise protocol was conducted in young healthy adults in order to measure plasma oxidative stress markers in response to a single moderate intensity treadmill exercise bout. In this study, it was found that the treadmill exercise did not elicit a race-dependent responses, but that African American adults had higher level of oxidative stress at all sample times when compared to the Caucasians. A second preliminary study was conducted using a parallel cell culture design to measure basal oxidative stress levels in African American and Caucasian HUVECs without stimulation. These data were shown in relation to the plasma levels of oxidative stress in resting African American and Caucasian adults. This was done in order to show that the common oxidative stress markers measured in human plasma can also be measured in cell culture supernatant and lysate. It was found that both African American adults and HUVECs had heightened oxidative stress and inflammatory markers when compared to their Caucasian counterparts. The third preliminary study was conducted using tumor Necrosis Factor-#945; (TNF-#945;) as an inflammatory stimulant and measuring the oxidative stress response in both African American and Caucasian HUVECs. This was done in order to show that cells of different race respond differently to stimuli. It was found that the response to TNF-α was blunted in African American HUVECs. The final step was to use laminar shear stress (LSS) as an exercise mimetic in order to examine whether HUVECs from different race respond differently. HUVECs from both race were harvested under static condition (no LSS), with low LSS at 5 dyne/cm2, and with a moderate level of LSS at 20 dyne/cm2. It was found that despite the fact that African American HUVECs had higher levels of oxidative stress under static conditions, when LSS was applied, protein expressions and oxidative stress biomarkers adjusted to levels that were similar to the Caucasian HUVEC adaptations to LSS. From this, it appears that African American HUVECs have a larger response to LSS stimulus indicating that aerobic exercise prescriptions may be valuable for this population since the potential exists for large improvements in oxidative stress levels for this population.
Temple University--Theses

Background: Sex and stress steroids are metabolized to 3a-hydroxy-pregnane-steroid metabolites such as allopregnanolone (Allo) and tetrahydrodeoxycorticosterone (THDOC). Allo and THDOC are neuroactive steroids that are metabolized in the brain and act in brain as potent positive GABAA receptor function modulators. Allo as well as THDOC levels increase during stress. Allo has been associated with a number of symptoms and malfunctions such as impaired memory function and negative mood symptoms in a subgroup of individuals both for animals and humans. Pregnane steroids with 3b-hydroxy-configuration (3b-steroids) have been shown to reduce the Allo enhanced GABA effect. Aims: The aims for the present thesis were to investigate the effect of 3b-steroids on the GABA mediated GABAA receptor function in presence of positive GABAA receptor modulators. Further, the regional variances between the 3b-steroids as well as the mechanism of the effect were studied. Finally, the effect of stress steroid metabolites on the GABAA receptor function was investigated. Results: 3b-OH-5a-pregnane-20-one reduced the Allo enhanced GABA mediated chloride ion uptake into cortical microsacs. The 3b-isomer reduced the efficacy of Allo without shift the concentration response curve. It is therefore suggested that the 3b-isomer has a non-competitive effect. Further, it was shown that the 3b-isomer reduced the Allo effect in a selective way since the 3b-isomer did not interact with other positive modulators or with GABA itself. Five tested 3b-steroids reduced the Allo enhanced GABA mediated chloride ion uptake in cerebral cortex and hippocampus as well as the Allo prolongation on spontaneous inhibitory postsynaptic currents (sIPSCs) in preoptic nucleus. In cerebellum on the other hand the 3b-steroids showed to have weaker or no effect compared to the other tested regions. Interestingly, in absence of Allo, two of the 3b-steroids positively modulated the GABA stimulated GABAA receptor function. In absence of Allo, 5b-pregnane-3b,20(R)-diol increased the desensitization rate of current response. In contrast to the reducing effect on the Allo induced prolongation on sIPSCs, the effect of the 3b-steroid on GABA application, was not altered in presence of Allo. The mechanism of the 3b-steroid is therefore suggested being desensitization dependent in contrast to Allo, which has been suggested to decrease the GABA unbinding rate. In contrast to the enhanced effect of Allo, glucocorticoid metabolites reduced the GABA mediated chloride ion uptake in a concentration dependent way. The results in present thesis indicate that both sex and stress steroid metabolites interact with the GABAA receptor function. The knowledge that diversity of endogenous steroids interact with the GABAA receptor function is of importance for further understanding of different sex and stress steroid related symptoms and syndromes.

The human monocyte-derived THP-1 cell line was incubated with 10mM AAPH in Earle’s Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide formation occurred after an 8 hour lag phase which corresponded to glutathione loss observed in the cells. SDS-Page analysis confirmed protein degradation occurred after 6 hours. Cell viability measured by the MTT reduction assay also dropped after 8 hours. Reduction of intracellular glutathione levels using BSO caused reduction of the lag phase seen in protein hydroperoxide formation. Cell viability of BSO-treated cells was lower than control cells, indicating the initiation of apoptotic events. Flow cytometry analysis showed no difference between BSO-treated and control cells, indicating that GSH levels do not have an effect on the type of cell death observed in AAPH-induced oxidative damage on THP-1 cells. These results confirmed previous data in the lab suggesting THP-1 cells undergo AAPH-induced necrosis as a result of cellular damage, including the loss of GSH and the formation of protein hydroperoxides.

PTSD is a devastating disorder that affects 7-12% of individuals who experience a traumatic event in their lives. Individuals suffering from the disorder show a dysfunctional HPA axis with decreased basal cortisol release and increased glucocorticoid receptor sensitivity. Recently, epigenetic studies have shown associations between trauma and the subsequent effects on the HPA axis in abused participants who committed suicide. Importantly, findings also showed child trauma as being a significant factor in DNA methylation levels in promoter region 1F in the hippocampus. This thesis shows the possible role of epigenetic programming of glucocorticoid receptors in PTSD while examining the effects of different timing of trauma. Methods included the measurements of salivary basal cortisol levels, the expression of glucocorticoid receptors in whole blood and the DNA methylation levels in two promoter regions of the glucocorticoid receptors by sequenome epityper. Results showed that individuals suffering from PTSD have decreased salivary cortisol release in the morning, increased glucocorticoid receptors and increased total methylation levels in promoter region 1C in whole blood. Additionally, adulthood trauma seemed to be of greater importance with regards to HPA axis activity while childhood trauma showed to be more significant with regards to epigenetics. These results indicate that 1) individuals suffering from a lifetime PTSD have a dampened HPA axis activity, 2) epigenetics play a different role in PTSD compared to abused individuals who have committed suicide and 3) timing of trauma has a significant effect on both the HPA axis activity and the epigenetic modifications.
Le TSPT est un syndrome dévastateur qui touche entre 7 et 12% de gens qui vivent un événement traumatique. Les personnes qui souffrent du syndrome ont un axe HPA dysfonctionnel avec moins de cortisol salivaire et une augmentation de sensitivité des récepteurs glucocorticoïdes. Il y a récemment eu des études épigénétiques qui ont montré une association entre les traumas et les effets sur l'axe HPA chez des patients abusés qui ont commis un suicide. De plus, les résultats indiquent qu'un trauma durant l'enfance est un facteur significatif pour la méthylation d'ADN dans la région promoteur 1F de l'hippocampe. Cette thèse présente le possible rôle modulateur de l'épigénétique des récepteurs glucocorticoïdes chez les personnes touchées du TSPT tout en examinant les effets du temps du trauma. Les résultats montrent que les individus qui souffrent du TSPT ont moins de cortisol salivaire le matin, une augmentation des récepteurs glucocorticoïdes et une augmentation de niveaux de méthylation totale dans la région promoteur 1C du sang. Il semblerait aussi qu'un trauma vécu durant l'âge adulte soit plus significatif pour l'axe HPA, alors qu'un trauma durant l'enfance semblerait prendre le dessus pour les modifications épigénétiques. Ces résultats montrent que 1) les personnes souffrant du TSPT ont une activité HPA atténuée, 2) l'épigénétique joue un rôle différent chez les TSPT comparés aux patients abusés qui ont commis un suicide et 3) le temps du trauma a des effets significatifs pour l'axe HPA et les modifications épigénétiques.

The present study has established that physiological concentrations of estradiol can modulate stress-induced increases in plasma epinephrine (EPI). In anesthetized female rats, insulin-induced hypoglycemia (0.25 U/kg) increased plasma EPI concentration to a significantly greater extent in 14-day ovariectomized (OVEX) rats compared to sham-operated controls. In 17-estradiol (E2)-replaced OVEX rats, the hypoglycemia-induced rise in plasma EPI was significantly reduced compared to OVEX rats. This suppression was due to both decreased adrenal medullary output and increased clearance of EPI. Adrenal venous EPI concentration was significantly reduced in OVEX+E2 rats, suggesting that EPI secretion from the adrenalmedulla was decreased by E2 replacement. The underlying mechanism(s) of this apparent E2-mediated reduction in secretion could not be established since 1) the expression levels of the biosynthetic enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase were not affected in OVEX+E2 rats, suggesting that EPI biosynthesis is similar in these and OVEX rats; and 2) agonist-induced increases in intracellular CaP2+P were identical in isolated adrenal medullary chromaffin cells exposed to E2 (10 nM) or vehicle for 48 hr, suggesting that stimulus secretion coupling is unaffected by E2 treatment. In contrast, plasma clearance of EPI was significantly increased in OVEX+E2 rats. Although 48 hr exposure to E2 had no effect on intracellular signaling in chromaffin cells, acute (3 min) exposure to micromolar concentrations of E2 dose-dependently and reversibly inhibited agonist-induced CaP 2+Ptransients. Consistent with this observation, acute (30 min) infusions of E2 also significantly reduced the insulin-induced increase in plasma EPI in OVEX rats. These data demonstrate that physiological levels of circulating E2 can modulate hypoglycemia-induced increases in plasma EPI. This effect appears to be mediated by the steroids influence on adrenal medullary EPI output and plasma EPI clearance; however the mechanism(s) underlying these E2-mediated modulations remain undetermined. This study has also established that acute exposure to supra-physiological levels of E2 can suppress hypoglycemia-induced increases in plasma EPI, due at least in part to inhibition of stimulus-secretion coupling.

Three trials were conducted to investigate the effects of dietary immuno-stimulant supplementation on various physiological, haematological and immunological parameters of ornamental fish following stress and vaccination. The first series of experiments investigated the mucosal stress response of mirror carp, (Cyprinus carpio), using urinalysis strips and established stress indicators within peripheral blood. A netting stressor, which combined aerial exposure and confinement, of 6 minutes duration was applied and the response of fish was measured 1.5, 3, 5, 24 and 168 hours post-stress. Mucosal haemoglobin and protein were established as consistent indicators of stress and strong relationships could be drawn between them and plasma glucose concentration and peripheral monocytes and lymphocytes counts, firmly establishing them as secondary stress indicators. Visual assessment of the strips was found to overestimate mucosal parameters compared to results obtained using instrument based analysis, but the underlying changes were similar. Further experiments conducted on goldfish, (Carassius auratus), and tilapia, (Oreochromis niloticus x. 0. mossambicus), showed that the mucosal response to stress was conserved between fish species. The second trial investigated the potential of dietary immuno-stimulants to modify the immune status of fish and their response to stress. Fish were fed either a control, beta-glucan (0.2 % w/w), nucleotide (0.2 % w/w) or combined supplement (0.2 % beta-glucan + 0.2% nucleotide) diet for 3 weeks at 1 % BW.dˉ¹ and then to the same stressors as used in Trial 1. Significant stress-induced changes in mucosal protein (p = 0.006), plasma glucose (p = <0.004) and protein concentration (p = <0.003), peripheral lymphocytes (p = 0.001) and monocytes (p = <0.001) and blood NBT reduction (p = <0.001) were observed. Fish fed diets containing immuno-stimulants shower a quicker recovery from the stress-induced hyperglycaemic response than those fed the control diet. Fish that received the beta-glucan diets showed lower blood NBT reduction immediately post-stress than all other diets. The final trial investigated the effects of the control, beta-glucan and combined-supplement diet on the response of fish to vaccination. Fish were fed the diets for 2 weeks prior to and 1 week following IP vaccination against Aeromonas salmonicida at 1.0 % BW.dˉ¹. The immune response was measured every 2 weeks for 12 weeks following vaccination. Vaccination induced a significant stress response as seen by a large hyperglycaemic response (p = <0.05). Significant reductions in peripheral monocyte count were seen following vaccination (p = <0.04). All groups showed elevated NBT reduction at 4 weeks post-vaccination. Serum lysozyme activity showed evidence of long term immuno-suppression following vaccination. No clear effect of the immuno-stimulating diets could be found on any of the physiological or immune parameters tested. The trials conducted found that ornamental fish have a well developed mucosal stress response and this can be used to make inferences about the stress state of fish. The effects of beta-glucans and nucleotides on either the response to stress and immune status were subtle. The nucleotide supplement appeared most effective at reducing the effects of stress, whilst beta-glucans may exert more influence on the immune parameters tested. However, no evidence of synergy between the two types of immuno-stimulant was found in fish fed both immuno-stimulants together.

L’arsenic (PM = 74,92 g/mol) est un contaminant environnemental majeur dans le monde présent au niveau de la croûte terrestre. Sa concentration dans le sol est d’environ 2 ppm. L’arsenic peut être redistribué vers les compartiments aquatiques et atmosphériques. Par ailleurs, les diverses utilisations industrielles et agricoles de l’arsenic sont également responsables de son accumulation dans l’environnement. La consommation d’eau au niveau de nappes phréatiques contaminées conduit à un empoisonnement chronique à l’arsenic dans de nombreuses régions du monde, et les troubles développés par ces individus sont multiples. Toutefois, les bases moléculaires restent encore mal connues. Des études récentes suggèrent que l’immunotoxicité de l’arsenic pourrait expliquer certains effets chez l’homme. Au laboratoire il a été montré que l’arsenic est capable d’induire des modifications phénotypiques importantes au niveau du macrophage pouvant être assimilées à une dé-différenciation macrophagique. La différenciation macrophagique est un ensemble de mécanismes moléculaires complexes, modulant l’expression de nombreux gènes, et permettant l’induction de caractéristiques phénotypiques spécifiques du macrophage. Elle peut être induite par des facteurs de croissance de type GM-CSF et G-CSF et implique l’intervention de facteurs de transcription, tel que PU. 1. Dans un premier projet, nous avons précisé l’impact de l’arsenic sur les processus de différenciation macrophagique induits par le GM-CSF, et notamment ses effets sur l’expression de gènes associés au processus de différenciation, à l’aide d’une étude transcriptomique. Nous avons montré que l’arsenic est capable de moduler l’expression d’un grand nombre de gènes dans le macrophage exposé à ce toxique. Ce sont, d’une part, des gènes de stress mais aussi des gènes associés au processus de différenciation. Par ailleurs, ces régulations géniques impliquent des voies de signalisation sensibles au statut rédox indépendantes de la synthèse d’Espèces Activées de l’Oxygène (EAO). L’arsenic est également capable de sensibiliser le macrophage à une exposition au LPS. De plus, une exposition chronique d’individus à l’arsenic puis au LPS/IFN? ex-vivo sensibilise les PBMC à la réponse cytokinique au LPS. C’est donc qu’il existe un mécanisme opérant in vivo. Le second projet a eu pour but d’identifier les voies de signalisation impliquées dans la potentialisation par l’arsenic de la réponse cytokinique induite par le LPS dans le monocyte humain; ceci met en jeu la voie de signalisation SRC-pMKK3/6-p38-kinase et la synthèse d’EAO. Au total, ces travaux ont permis de préciser les mécanismes de toxicité de l’arsenic vis-à-vis des cellules immunitaires humaines.

Les vols spatiaux affaiblissent le système immunitaire. Les objectifs de cette thèse étaient de déterminer, à l’aide de modèles terrestres, les conséquences d’une exposition à la micropesanteur simulée, l’hypergravité ou des stress chroniques ultra légers sur l’immunité humorale murine. Nous avons ainsi montré que la suspension anti-Orthostatique, qui mime certains effets de la micropesanteur, induit une diminution de 59% du nombre de lymphocytes B spléniques et une inversion du rapport entre lymphocytes B et T. L’affaiblissement de la lymphopoïèse B mis en évidence par la diminution du nombre de progéniteurs lymphoïdes et des cellules pré-B en est probablement la cause. De plus, la position anti-Orthostatique entraîne, en l’absence de stress, une diminution de la réponse in vitro des lymphocytes B au LPS, plus importante que celle des lymphocytes T à la ConA, comme en hypergravité. Afin de déterminer les mécanismes responsables de la diminution de la réponse des lymphocytes B chez les souris hypergravitaires, les transcrits de gènes impliqués dans la voie TLR-4, le récepteur du LPS, ont été quantifiés. L’expression de plusieurs gènes de la voie MyD88-Dépendante est augmentée après 21 jours d’hypergravité, mais aucune modification n’a été observée dans les lymphocytes B stimulés avec du LPS. Enfin, la contribution de stress chroniques ultra légers a été évaluée. Les proportions de lymphocytes spléniques ne sont pas affectées par ces stress. Par contre, le taux d’IgA sériques s’est révélé augmenté et une baisse des cytokines pro-Inflammatoires de type Th1 a été observée comme chez les astronautes. Ainsi, ces stress n’expliquent pas complètement l’impact négatif des vols spatiaux sur l’immunité humorale. Ces recherches sont importantes pour identifier la/les cause(s) de l’altération de l’immunité humorale en vol, afin de tester/développer des contremesures efficaces pour renforcer le système immunitaire des astronautes, mais également de personnes stressées ou âgées
Spaceflight weaken the immune system. The aims of this thesis were to determine the effects of exposure to simulated microgravity, hypergravity or chronic ultra-Mild stress on murine humoral immunity, using ground-Based models. We were able to show that the anti-Orthostatic suspension, that mimics some of the effects of microgravity, caused a 59% decrease of the number of splenic B cells and an inversion of the ratio between B and T lymphocytes. A decrease in B lymphopoiesis, as evidenced by the decrease of lymphoid progenitors and pre-B cells is likely the cause. Furthermore, we showed that the anti-Orthostatic position, in the absence of stress, leads to a decreased in vitro response of B cells to LPS, more important than the one of T cells to ConA, as did hypergravity exposure. To determine the mechanisms responsible of the decreased response of B cells from hypergravity mice, transcripts encoding genes involved in the TLR-4 signaling pathway, the LPS receptor on B cells, have been quantified. The expression of several genes of the MyD88-Dependent pathway was increased after 21 days of hypergravity, but no change was observed in B cells stimulated with LPS. Finally, the contribution of chronic ultra-Mild stresses was evaluated. The proportions of splenic lymphocytes were not affected by these stresses. However, the levels of serum IgA were increased and those of Th1 pro-Inflammatory cytokines were decreased, as in astronauts. Thus, these stresses do not fully explain the negative impact of spaceflight conditions on humoral immunity. These researches are important to identify the cause(s) of spaceflight-Associated impaired immunity in order to test/develop effective countermeasures to strengthen the immune system of astronauts, but also of stressed or elderly people

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators. Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.