Salmonella enterica serotypes have been linked to outbreaks associated with low water activity foods. The ability of biofilm forming pathogens, such as Salmonella, to survive thermal and chemical processes is improved; it is unclear if biofilms will also improve survival to desiccation and gastric stresses. The purpose of this study was to quantify the effect of physiological state (planktonic versus biofilm) and prior exposure to desiccation on Salmonella survival and gene expression after passage through an in-vitro digestion model. Cells of Salmonella enterica serotype Tennessee were deposited onto membranes for planktonic cells or on glass beads to create biofilms. The cells were subsequently dried at room temperature and stored in dried milk powder (aw = 0.3) for up to 30 days. Salmonella survival was quantified by serial dilution onto brilliant green agar before desiccation, after desiccation, after 1-day storage and after 30-day storage. At each sampling both physiological states were tested for survival through a simulated gastrointestinal system. RNA was extracted at the identical time points and relative gene expression determined for genes associated with stress response (rpoS, otsB), virulence (hilA, hilD, invA, sipC) and a housekeeping gene 16S rRNA using quantitative real-time PCR. The physiological state and length of storage effected the survival and gene expression of Salmonella within the desiccated milk powder environment and after passage through an in-vitro digestion system (p<0.05). Larger numbers of S. Tennessee were recovered by plate counts for biofilm cells, compared to planktonic cells. However, the numbers of 16S rRNA gene copies were not significantly different suggesting entry of S. Tennessee into a viable but non-culturable state. Prolonged storage in dry milk powder was not associated with increased cross-protection to gastric stress. Increased expression of stress response genes rpoS and otsB correlated with survival, indicating cross protection of low water activity and acid stress. Increased expression of virulence-associated genes was seen in cells exposed to short periods of dry storage, suggesting an increased virulence potential. / Master of Science in Life Sciences

Die myotone Dystrophie ist eine autosomal dominant vererbte, multisystemische Erkrankung mit skelettmuskulärem Fokus und wird in zwei Untergruppen eingeteilt, die myotone Dystrophie Typ I (MD1), verursacht durch eine CTG-Trinukleotidexpansion im DMPK-Gen auf Chromosom 19, und die myotone Dystrophie Typ II (MD2), verursacht durch eine Tetranukleotidexpansion im CNBP-Gen auf Chromosom 3. Die Nukleotidexpansionen akkumulieren als mRNA intranukleär und tragen über die Beeinflussung des alternativen Splicings von über 30 Genen zum Erkrankungsmechanismus bei. Daneben akkumulieren die mRNA-Repeats im Zytoplasma und induzieren über die Repeat-assoziierte-non-AUG-Translation (RAN-Translation) die Akkumulation aberranter Proteine. Wenngleich die Patienten der MD2 einen milderen Krankheitsverlauf erleben, zeigen sie ein erhöhtes Auftreten von Autoimmunerkrankungen. In der Pathogenese der Autoimmunerkrankungen spielt die durch Interferon-Typ-I vermittelte Immunreaktion eine wichtige Rolle. In der AG Günther konnte eine erhöhte Expression interferonstimulierter Gene und die erhöhte Expression von Markern des Stresszustandes des endoplasmatischen Retikulums in Fibroblasten von MD2-Patienten nachgewiesen sowie die erhöhte Prävalenz von Autoimmunerkrankungen in MD2 gezeigt werden. Die Auswertung serologischer Parameter im Rahmen dieser Arbeit erlaubte eine klinische Charakterisierung der Patienten und ergab einen positiven Zusammenhang zwischen Höhe der Myoglobin-Konzentration im Serum und Länge der CCTG Repeats. Es wurde die Hypothese aufgestellt, dass die zytoplasmatischen RNA-Repeats über die RAN-Translation und Akkumulation aberranter Proteine zur Induktion von ER-Stress führen, welcher über die gemeinsame Kontaktflächen der Mitochondrien-assoziierten Membranen (MAMs) in die Mitochondrien übertragen wird. Im Rahmen des mitochondrialen Stresszustandes könnte es zu einer Freisetzung mitochondrialer DNA und zur Aktivierung des zytosolischen DNA-Rezeptors cGAS und einer nachfolgenden Expression von Interferon und Interferon-stimulierten Genen kommen, die für das vermehrte Auftreten der Autoimmunerkrankungen verantwortlich sind. Zur Überprüfung der Hypothese sollte nach Analyse der Proteinexpression der ER-Stress-Marker eIF2α und peIF2α eine Hemmung der ER-Stressreaktion vorgenommen werden. Die anschließende Untersuchung des mitochondrialen Stresszustandes und die Unterbrechung der MAMs sollte Aufschluss über die Rolle der Mitochondrien in der Pathogenese der Autoimmunität liefern. In dieser Arbeit konnte die erhöhte Expression des ER-Stressmarkers peIF2α nach Stressinduktion in Fibroblasten von MD2-Patienten gezeigt werden. Dies könnte auf eine Sensibilisierung des Signalweges im Rahmen eines chronischen Stresszustandes hinweisen. Der Versuch einer Behandlung der Fibroblasten mit Ursodesoxycholsäure (UDCA), einem chemischen Chaperon, mit Ziel der Suppression der ER-Stressreaktion erzielte keine Reduktion der Expression der ER-Stressmarker unter Nativniveau. Nach Stimulation des ER-Stresses konnte mit UDCA-Vorbehandlung dennoch die Reduktion auf Nativniveau erzielt werden. Zur Analyse des mitochondrialen Stresszustandes wurde eine Färbung mit Mitotracker-Farbstoffen und anschließender Durchflusszytometrie und eine Messung der reaktiven Sauerstoffspezies (ROS) vorgenommen, welche ein verringertes mitochondriales Membranpotential und eine erhöhte ROS-Expression in den MD2-Fibroblasten ergaben und mit einem mitochondrialen Stresszustand einhergehen könnten. Nach Unterbrechung der räumlichen Verbindung zwischen ER und Mitochondrium durch die Herunterregulation des gemeinsamen Strukturproteins PERK konnten Hinweise auf eine verringerte ROS-Expression in den MD2-Fibroblasten gefunden werden. Die Ergebnisse dieser Arbeit unterstützen die Hypothese der Funktion der mitochondrialen Stressreaktion in den Fibroblasten der MD2-Patienten, wenngleich die Autoimmunität-vermittelnden Mechanismen noch Gegenstand weiterführender Untersuchungen sein sollten.

Résumé : Introduction. Les collisions routières représentent un grave problème à travers le monde. Elles causent des dizaines de millions de blessés chaque année et figurent comme l’une des principales causes de décès chez les jeunes adultes. La conduite avec les capacités affaiblies, la vitesse et d’autres comportements routiers à risque (CRR) sont souvent associés à ces accidents. Notre équipe de recherche s’est intéressée à l’étude de marqueurs de risque de l’implication dans les CRR. Les résultats indiquent qu’une faible réponse au stress, mesurable par l’hormone cortisol, est liée à un nombre plus élevé de condamnations passées pour conduite avec les capacités affaiblies chez les contrevenants et à l’implication future dans les accidents et quasi-accidents chez les jeunes conducteurs. Aucune étude n’avait encore étudié le lien direct entre la réponse au stress et la présence de CRR suite à la consommation d’alcool. But et hypothèse. L’étude a pour but principal de mesurer l’association entre la réponse au stress des jeunes conducteurs et la présence de CRR suite à la consommation d’alcool. L’hypothèse principale soutient qu’une faible réponse au stress est liée à la présence de CRR suite à la consommation d’alcool. L’objectif secondaire de cette étude est d’explorer si d’autres variables (c.-à-d., la recherche de sensations, la consommation de cigarettes et la consommation problématique d’alcool), peuvent expliquer la variance de réponse au stress chez les jeunes conducteurs. L’hypothèse secondaire propose que la recherche de sensations, la sévérité de la consommation d’alcool et de cigarettes sont des variables qui expliquent la variance de réponse au stress. Méthode. Quarante conducteurs masculins âgés de 20 à 24 ans ont pris part à l’étude. Les comportements routiers suite à la consommation d’alcool (c.-à-d., vitesse et non-respect de la signalisation lors du premier trajet, décision de conduire un deuxième trajet sous l’effet de l’alcool) ont été observés à l’aide d’un simulateur de conduite. La réponse au stress a été mesurée à l’aide d’une tâche d’induction du stress standardisée et des échantillons de cortisol salivaire ont été recueillis avant et après la tâche. Les variables de l’objectif secondaire ont été mesurées à l’aide de questionnaires. Résultats. Les résultats démontrent qu’une faible réponse au stress est négativement et significativement associée à la présence de CRR lors de la conduite du simulateur sous l’influence de l’alcool (r (38) = -0,30, p = 0,032). La recherche de sensations explique significativement la variance de la réponse au stress (β = -0,33; p = 0,041); la sévérité de la consommation d’alcool et la consommation de cigarettes n’expliquent pas significativement la variance de réponse au stress entre individus. Implications. Les résultats de cette étude ont permis de mettre en lumière le lien existant entre la réponse au stress et l'implication des jeunes conducteurs dans les CRR suite à la consommation d’alcool. L’efficacité d’outils de prévention et d’intervention adaptés aux caractéristiques des individus qui réagissent moins au stress devrait être étudiée dans d’autres études. // Abstract : Introduction. Motor vehicle crashes are a serious problem worldwide. They cause tens of millions of injuries each year and represent one of the leading causes of death in young adults. Impaired driving, speeding and other risky driving behaviours (RDBs) are often associated with these crashes. Examination of risk markers associated with involvement in RDBs by our research team has shown that a low response to a stressful task, measurable by cortisol, is linked to a higher number of past convictions for impaired driving in offenders and to future involvement in crashes and near-crashes in young drivers. No study had yet examined the direct link between stress response and the presence of RDBs following alcohol consumption. Goals and Hypotheses. The primary goal of the study is to measure the association between young drivers’ stress response and the presence of RDBs following alcohol consumption. The main hypothesis is that a low stress response is linked to RDBs following alcohol consumption. The study’s secondary objective is to explore whether other variables (i.e., sensation seeking, cigarette use, and problem drinking) may explain the variance in stress response exhibited by young drivers. Hence, the secondary hypothesis proposes that sensation seeking and the severity of alcohol and cigarette use are variables that explain the variance in stress response. Method. Forty male drivers aged 20 to 24 years old participated in the study. Driving behaviours following alcohol consumption (i.e., speeding and failure to obey traffic signs during the first trip, deciding to drive a second trip under the influence of alcohol) were observed by means of a driving simulator. Stress response was measured using a standardized stress-inducing task, and saliva cortisol samples were collected before and after the task. Questionnaires gathered data used to test the secondary hypothesis. Findings. The findings show that a low stress response is negatively and significantly associated with the RDBs when driving the simulator under the influence of alcohol (r (38) = -0.30, p = 0.032). Sensation seeking significantly explains the variance in stress response (β = -0.33; p = 0.041); the severity of alcohol consumption and cigarette use do not significantly explain the variance in stress response across individuals. Implications. The study findings clarify the link between stress response and the involvement of young drivers in RDBs following alcohol consumption. The efficacy of prevention and intervention tools adapted to the characteristics of individuals who react less to stress should be explored in future studies.

Animal domestication, the process where animals become adapted to living in proximity to humans, is associated with the alteration of multiple traits, including decreased fearfulness and stress response. With an estimated population of 50 billion, the domesticated chicken is the most populous avian species in the world. Hundreds of chicken breeds have been developed for meat and egg production, hobby or research purposes. Multidirectional selection and the relaxation of natural selection in captivity have created immense phenotypic diversity amongst domesticates in a relatively short evolutionary time. The extensive phenotypic diversity, existence of the wild ancestor, and feasibility of intercrossing various breeds makes the chicken a suitable model animal for deciphering genetic determinants of complex traits such as stress response. We used chicken domestication as a model to gain insights about the mechanisms that regulate stress response in an avian species. We studied behavioural and physiological stress response in the ancestral Red Junglefowl and one of its domesticated progenies, White Leghorn. An advanced intercross between the aforementioned breeds was later used to map genetic loci underlying modification of stress response. The general pattern of the stress response in chickens was comparable with that reported in mammals, however we identified distinctive differences in the stress modulatory pathways in chickens. We showed that changes in the expression levels of several stress modulatory genes in the brain, the pituitary and the adrenal glands underlie the observed modified stress response in domesticated chickens. Using quantitative trait loci (QTL) mapping, several QTL underlying stress induced corticosterone, aldosterone and baseline dehydroepiandrosterone (DHEA) levels were detected. As a next step, we combined QTL mapping with gene expression (eQTL) mapping and narrowed two QTL down to the putative causal genes, SERPINA10 and PDE1C. Both of these genes were differentially expressed in the adrenal glands of White Leghorn and the Red Junglefowl, had overlapping eQTL with hormonal QTL, and their expression levels in the adrenal glands were correlated with plasma levels of corticosterone and al-dosterone. These two genes thus serve as strong candidates for further functional investigation concerning modification of the stress response during domestication. This dissertation increase the knowledge about genetics and physiology of the stress response in an avian species and its modification during domestication. Our findings expand the basic knowledge about the stress response in chicken, which can potentially be used to improve welfare through appropriate genetic selection.

In a bacterium's environment, life conditions are subject to constant changes. One of the proposed mechanisms of adaptation to these changes is the increase in mutation rate. Bacterial mutability is generally kept very low by action of various mechanisms of control and repair, one of the most important ones being the Mismatch Repair, which is the master regulator of genetic stability of organisms. When its function is impaired, larger amounts of mutations occur in cells. In adverse conditions, these might be beneficial for cells' adaptation. The role of these repair mechanisms in adaptive processes in Bacillus subtilis has not yet been definitely resolved. The previous work in our lab focused on establishing an experimental system to measure the extent of mutagenesis in B. subtilis, and the influence of several stresses on mutation rate was assessed. No significant increase in mutability was found to be triggered by nutrient limitation in stationary growth phase, hyperosmotic stress or increased cultivation temperature. Furthermore, a system to monitor the expression of mismatch repair proteins was constructed, which has not revealed significant differences between stressed and nonstressed growth conditions. This thesis follows the results of previous experiments, expanding the range of stresses...

Linhagens mutantes foram obtidas para os fatores sigma <font face=\"symbol\">sE (RpoE) e <font face=\"symbol\">sN (RpoN) da bactéria Xylella fastidiosa. O mutante rpoE mostrou-se sensível a etanol e a choque térmico. Análises de microarranjo de DNA, de RT-PCR quantitativo e mapeamento de sítios de início de transcrição permitiram definir o regulon <font face=\"symbol\">sE em resposta ao choque térmico. Verificou-se co-transcrição entre os genes que codificam para <font face=\"symbol\">sE, seu anti-sigma e uma protease, e <font face=\"symbol\">sE não se mostrou auto-regulado, mas regulou o gene do anti-sigma. Análises similares às acima indicaram que o gene pilA, codificando a pilina da fímbria tipo IV, é positivamente regulado por <font face=\"symbol\">sN, enquanto o operon codificando proteínas da fímbria tipo I é regulado negativamente, explicando a maior formação de biofilme e auto-agregação no mutante rpoN. O perfil temporal de expressão da linhagem selvagem J1a12 em carência de nitrogênio foi determinado, além de genes induzidos por carência de nitrogênio via <font face=\"symbol\">sN. Assim, <font face=\"symbol\">sN regula genes de fímbrias e de resposta à carência de nitrogênio em Xylella fastidiosa. / Mutant strains were obtained for the sigma factors <font face=\"symbol\">sE (RpoE) and <font face=\"symbol\">sN (RpoN) in Xylella fastidiosa. The rpoE mutant showed to be sensitive to ethanol and heat shock. Microarray and quantitative RT-PCR analyses and determination of transcription start sites permitted to define the <font face=\"symbol\">sE regulon under heat shock. Co-transcription of the genes encoding <font face=\"symbol\">sE , its anti-sigma factor and a protease was observed, and <font face=\"symbol\">sE did not present auto-regulation, but it regulated the gene encoding the anti-sigma. Similar analyses indicated that the pilA gene, encoding the pilin of the type IV fimbriae, is positively regulated by <font face=\"symbol\">sN, while the operon encoding proteins of the type I fimbriae is negatively regulated, what explains the increased biofilm formation and auto-aggregation in the rpoN strain. Temporal expression profile of wild type strain J1a12 under nitrogen starvation was determined, as well as genes induced by nitrogen starvation via <font face=\"symbol\">sN. Thus, <font face=\"symbol\">sN regulates genes encoding fimbriae and genes for nitrogen starvation response in Xylella fastidiosa.

As mutações pontuais nos dois domínios de CspC proporcionam fenótipos mais severos que a falta de cspC. Nenhuma CSP de C. crescentus é capaz de complementar o fenótipo de sensibilidade ao frio de E. coli BX04. Entretanto, os domínios de choque frio de CspC de C. crescentus individualmente são capazes de complementar este fenótipo. Uma análise transcricional global mostrou que a ausência de cspC afeta a transcrição de 11 genes na fase exponencial e 60 genes na fase estacionária. A meia vida dos genes sciP, aceA e CC0682 se mostrou menor no mutante cspC, sugerindo que é possível que CspC desempenhe uma regulação pós-transcricional. / Point mutations in the CspC CSDs caused a more severe phenotype than that of the null strain. None of the C. crescentus CSPs complemented the cold sensitivity of E. coli BX04 mutant. However, the individual CSDs of C. crescentus CspC complemented this phenotype. A microarray global transcriptional profiling showed the absence of cspC affected the transcription of 11 genes at exponential phase and 60 genes at stationary phase. mRNA decay experiments showed that the sciP, aceA e CC0682 mRNAs were less stable in the cspC mutant, indicating that its effect could be at least partially due to posttranscriptional regulation.

Em estudos de proteção ao genoma contendo lesões de C. crescentus, foi feita uma varredura em uma biblioteca clones mutados pelo transposon Tn5 e sensíveis à luz UV-B e MMS. Dos 249 clones selecionados conseguimos identificar mutações em 102 genes, distribuídos em dez categorias funcionais, incluindo metabolismo de DNA. Além de genes já descritos como atuantes na defesa de genoma a lesões, os dados ainda adicionam funções potenciais para outros genes. Investigação mais detalhada indica que vários dos genes identificados podem afetar o estresse e ciclo celular, podendo estar relacionados a respostas do tipo pontos de checagem. Além disso, verificamos que mutantes para genes envolvidos em reparo excisão de nucleotídeos são mais sensíveis à H2O2, indicando que nessa bactéria, este sistema de reparo atua também em lesões oxidativas no DNA. Os dados obtidos são pioneiros no estabelecimento de funções para vários genes de C. crescentus e de outras alfa-proteobactérias. / This work aimed to identify genes related to DNA damage protection in C. crescentus, by means of screening a Tn5 -mutated library for clones sensitive to UV-B light and MMS. Out of 249 selected clones, we were able to identify mutations in 102 genes, classified in ten different functional categories, including DNA metabolism. In addition to genes already described as playing a role in genome defense to lesions, the results provide new potential functions to several other genes. More detailed investigation indicates also that the mutations in some of these genes may also affect cell stress and cell cycle, being potentially involved in check-point responses. Moreover, mutants for nucleotide excision repair genes were also found to be sensitive to H2O2, indicating that this DNA repair system also acts in DNA oxidative damage. These data are the first establishing a role in genome protection for several genes in C. crescentus, as well as other alpha-proteobacteria.

La métamorphose des amphibiens est le processus rapide et irréversible par lequel un têtard aquatique se transforme en une grenouille respirant à la surface. Cette transition écologique, réminiscente de la période périnatale chez les mammifères, s'accompagne de changements spectaculaires (régime alimentaire, organes locomoteurs, système respiratoire...). Ces modifications morphologiques et physiologiques nécessitent la réponse concertée à un signal hormonal, les hormones thyroïdiennes (HT), de différents tissus vers des destin parfois opposés : apoptose (dans la queue), prolifération (dans les pattes), et remodelage (dans les intestins et le système nerveux central). Toutefois, la synchronisation de la réponse des différents tissus fait appel à d'autres signaux hormonaux, et notamment les glucocorticoïdes (GC). Ces derniers sont également les médiateurs principaux de la réponse au stress. Les processus endocriniens de la métamorphose et la réponse au stress sont fortement couplés. Les GC peuvent ainsi jouer le rôle d'interface permettant l'intégration de signaux environnementaux au niveau de réseaux de régulation. Dans le cadre de mon doctorat, j'ai analysé les transcriptomes des bourgeons de membres postérieurs et de l'épiderme caudal de têtards de Xenopus tropicalis traités ponctuellement avec des HT et / ou des GC. La comparaison de ces deux tissus a permis de caractériser la diversité des profils d'expression des gènes cibles des HT et des GC.Il en ressort plusieurs résultats majeurs. Tout d'abord, la diversité des profils d'interaction entre ces deux voies est limitée, et la majorité des types de profils sont communs aux deux tissus. Indépendamment du tissu, certains profils sont caractéristiques de fonctions biologiques spécifiques comme le remodelage de la matrice extracellulaire et le système immunitaire. Les gènes impliqués dans ces fonctions communes aux deux tissus sont cependant différents. Enfin, plusieurs facteurs impliqués dans la méthylation de l'ADN sont régulés par les deux hormones. / Amphibian metamorphosis is the rapid and irreversible process during which an aquatic tadpole transforms into an air breathing adult frog. This ecological transition, reminiscent of the mammalian perinatal period, comes with spectacular changes (diet, locmotor organs, respiratory system...). These morphological and physiological modifications necessitate the properly timed response to a single hormonal signal, the thyroid hormones (TH), in various tissues to lead them to sometimes opposite fates : apoptosis (in the tail), cell prolifération and differenciation (in the limbs) and remodeling (in the intestine and the central nervous system).However, TH do not act alone. In particular, glucocorticoids (GC) play important roles during this process. They also are the main mediator of the stress response. Endocrine processes of the metamorphosis and the stress response are deeply intertwined. GC can thus act as an interface to integrate environmental inputs into regulatory networks.During my doctorate, I analyzed the possible transcriptional crosstalks between TH and GC in two larval tissues : the tailfin (TF) and the hindlimb buds (HLB). Comparing these two tissues allowed me to caracterize the diversity of TH and GC target gene expression profiles. This resulted in several major results. First, the diversity of the profiles of crosstalk between these two pathways is limited, and the majority of the types of profiles is common to both tissues. Next, independently ofthe tissues, some profiles are caracteristic of spécific biological functions such as extracellular matrix remodeling and the immune system. Yet, the genes involved in these shared functions are different between the TF and the HLB. Finally, several factors involved in DNA methylation are subject to a crosstalk between the two hormones.

O choque frio em bactérias causa a indução de proteínas de choque frio de baixo peso molecular (CSPs), que desestabilizam estruturas secundárias do mRNA, permitindo sua tradução. Caulobacter crescentus possui quatro genes codificando CSPs: cspA e cspB são induzidos sob choque frio, e cspC e cspD, na fase estacionária. Neste trabalho, foi determinada uma nova seqüência para o gene cspC, revelando que a proteína CspC possui dois domínios CSD, como CspD. O mutante nulo para cspC apresentou sensibilidade em baixa temperatura e menor viabilidade em fase estacionária, com alterações na morfologia. A região regulatória foi mapeada por fusões de transcrição, e uma região ativadora da expressão foi identificada, mostrando uma regulação transcricional. Algumas condições nutricionais que disparam a indução do gene foram determinadas, indicando que sua expressão é influenciada pela ausência de glicose no meio, mas não pela ausência de nitrogênio. Este perfil de indução não depende da região ativadora, que, por sua vez, é necessária para os máximos níveis de expressão. / The cold shock response in bacteria involves the expression of cold shock proteins (CSPs), which destabilize secondary structures on mRNAs, allowing their translation. Caulobacter crescentus possesses four genes encoding CSPs: cspA and cspB are induced upon cold shock, while cspC and cspD are induced at stationary phase. In this work, a new sequence for the coding region of the cspC gene was determined, revealing that CspC contains two cold shock domains, like CspD. A null cspC mutant was sensitive to low temperature, presented reduced viability at stationary phase, and altered morphology. The regulatory region of cspC was mapped by transcriptional fusions, identifying a region responsible for activation of cspC expression, suggesting a transcriptional regulation. Some nutritional conditions triggering cspC induction were determined, indicating that its expression is influenced by glucose starvation, but not by nitrogen starvation. This expression profile was not dependent on the activation region, which, in turn, was required for maximum levels of expression.