<p dir="ltr">In this dissertation, we utilized the zebrafish model and the human neutrophil model to investigate the novel mechanisms that regulate neutrophil motility and chemotaxis.</p>

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Les mécanismes cellulaires et moléculaires qui sous-tendent la mémoire et l’apprentissage chez les mammifères sont incomplètement compris. Le rythme thêta de l’hippocampe constitue l’état « en ligne » de cette structure qui est cruciale pour la mémoire déclarative. Dans la région CA1 de l’hippocampe, les interneurones inhibiteurs LM/RAD démontrent des oscillations de potentiel membranaire (OPM) intrinsèques qui pourraient se révéler importantes pour la génération du rythme thêta. Des travaux préliminaires ont suggéré que le courant K+ I(A) pourrait être impliqué dans la génération de ces oscillations. Néanmoins, peu de choses sont connues au sujet de l’identité des sous-unités protéiques principales et auxiliaires qui soutiennent le courant I(A) ainsi que l’ampleur de la contribution fonctionnelle de ce courant K+ dans les interneurones. Ainsi, cette thèse de doctorat démontre que le courant I(A) soutient la génération des OPM dans les interneurones LM/RAD et que des protéines Kv4.3 forment des canaux qui contribuent à ce courant. De plus, elle approfondit les connaissances sur les mécanismes qui régissent les interactions entre les sous-unités principales de canaux Kv4.3 et les protéines accessoires KChIP1. Finalement, elle révèle que la protéine KChIP1 module le courant I(A)-Kv4.3 natif et la fréquence de décharge des potentiels d’action dans les interneurones. Nos travaux contribuent à l’avancement des connaissances dans le domaine de la modulation de l’excitabilité des interneurones inhibiteurs de l’hippocampe et permettent ainsi de mieux saisir les mécanismes qui soutiennent la fonction de l’hippocampe et possiblement la mémoire chez les mammifères. / Cellular and molecular mechanisms underlying learning and memory in mammals are incompletely understood. The theta rhythm in the hippocampus constitutes the « on-line » state of this structure which is crucial for declarative memory. In the CA1 hippocampal area, LM/RAD inhibitory interneurons exhibit intrinsic membrane potential oscillations (MPOs) that could be important for the generation of theta rhythm. Preliminary work suggested that K+ current I(A) could be involved in the generation of these oscillations. Nevertheless, little is known about the identity of the principal and auxiliary protein subunits underlying I(A) current and the extent of the functional contribution of this K+ current in hippocampal interneurons. Thus, this Ph.D. thesis shows that I(A) current underlies MPO generation in LM/RAD interneurons and that Kv4.3 proteins form channels that contribute to this current. Also, it deepens the knowledge on the mechanism controlling the interactions between Kv4.3 channel-forming principal subunits and KChIP1 auxiliary proteins. Finally, it reveals that KChIP1 modulates native I(A)-Kv4.3 current and the action potential discharge frequency in interneurons. Our work takes part in advancing the knowledge on the field of modulation of excitability in hippocampal inhibitory interneurons and allows a better understanding of the mechanisms underlying the function of the hippocampus and possibly memory in mammals.

Le rythme lent lié à la respiration est une caractéristique proéminente de l’activité du bulbe olfactif de rat. Il se définit par des oscillations de grande amplitude du potentiel de champ local, une activité de décharge des cellules mitrales/ à panache (M/P) synchronisée à la respiration et des oscillations lentes de leur potentiel de membrane (OPLM). Des relations spécifiques entre ces activités détermineraient la participation d’une cellule M/P au traitement de l’information olfactive. Jusqu’à présent, il n'existait que très peu de données sur ces relations. L’objectif de cette thèse a été de caractériser les OLPM des cellules M/P et d’étudier les relations qu’elles entretiennent avec l’activité de décharge et l’activité du réseau bulbaire. Pour répondre à cet objectif, nous avons mis au point une technique d’enregistrements simultanés de l’activité intracellulaire des cellules M/P et du potentiel de champ local du bulbe olfactif chez l’animal anesthésié et libre de respirer. Nous montrons que plusieurs types d’OLPM existent. Des relations spécifiques s’établissent entre ces OLPM et la synchronisation de l’activité de décharge à la respiration. Nous observons également l’existence de relations complexes entre les OLPM et les oscillations du réseau bulbaire. L’ensemble de ces résultats nous permet d’intégrer la dynamique lente, et plus particulièrement celle du potentiel de membrane, à un schéma général du traitement de l’information olfactive et de proposer son implication dans la formation d’assemblée de neurones. / The respiration-related slow rhythm strongly shapes the activity of the olfactory bulb. This rhythm is characterized by high amplitude oscillations in the local field potential, respiration synchronization of mitral/tufted (M/T) cell discharge and slow oscillation of M/T cell membrane potential (OLPM). Specific relationships between these activities could determine the M/T cell participation to olfactory processing. However, little is known on these relationships. The aim of my thesis has been to characterize M/T cell OLPM and to study the relationships between OLPM and both discharge and bulbar network activities. In this way, we recorded simultaneously intracellular activity of M/T cell and local field potential of olfactory bulb in anesthetized freely breathing rat. We showed that several types of OLPM can be distinguished and exhibited specific relationship with the respirations ynchronization of M/T cell discharge activity. We observed also specific and complex relationships between OLPM and local field potential oscillation. Taken together, our results allowed us to integrate slow rhythm, and more particularly OLPM, in the more general scheme of olfactory processing.

La fraîcheur est un paramètre clé de la qualité du poisson. Les méthodes actuelles appliquées en routine pour déterminer la fraîcheur du poisson ne sont pas applicables à toutes les espèces et reflètent davantage un début d'altération du produit. Ainsi, la recherche d'indicateurs précoces de fraîcheur du poisson représente encore un défi majeur et d'actualité dans l'industrie de la pêche. Le but de ces travaux de thèse était de démontrer que les fonctions et l'intégrité mitochondriales étaient susceptibles de constituer des indicateurs précoces de la fraîcheur de filets de poisson. En effet, la mitochondrie est la "centrale" énergétique de la cellule eucaryote et joue un rôle clef dans les mécanismes de mort cellulaire tels que l'apoptose et la nécrose. Les fonctions et l'intégrité mitochondriales de cellules musculaires de filets de poisson ont été étudiées à différents temps de conservation post mortem à 4°C. Le modèle d'étude était la daurade royale (Sparus aurata) (lignée cellulaire de fibroblastes (SAF-1) et muscles de poisson). Dans un premier temps, la structure des mitochondries de poisson a été étudiée par microscopie électronique à transmission. De nombreuses dégradations de la structure des mitochondries ont été observées dans les filets à partir de 72 heures (J3) de conservation à 4°C. Ces altérations se sont accentuées à J4 et J6. La fonctionnalité des mitochondries a ensuite été évaluée selon deux approches : la respiration mitochondriale (oxygraphie) et le potentiel membranaire mitochondrial (ΔΨₘ) estimé avec la sonde fluorescente Rhodamine 123. A partir de 96 heures de conservation à 4°C (J4), ces deux paramètres ont été significativement impactés témoignant d'une altération des fonctions et de l'intégrité mitochondriales.Ces résultats sont ainsi en corrélation avec l'altération structurale observée par microscopie. En parallèle, une méthode d'évaluation du potentiel membranaire a été développée avec un fluorimètre à microvolume à partir d'un modèle bactérien puis de mitochondries isolées. Ces travaux de thèse ont démontré que l'étude des fonctionnalités mitochondriales constitue un marqueur fiable et précoce de la fraîcheur des filets de poisson. Des connaissances supplémentaires sur les mécanismes cellulaires post mortem ont également été apportées. Ces résultats constituent ainsi le point de départ pour le développement d'un kit d'évaluation de la fraîcheur et ouvrent la voie pour la recherche de marqueurs de fraîcheur et de congélation/décongélation basés sur les fonctionnalités et intégrité mitochondriales. / Freshness is a key parameter of fish quality. Current routine techniques to determine fish freshness are not applicable to all species and reflect a late stage of alteration. Thus, research on early indicators of fish freshness still represents a major and topical challenge in fishing industry. This PhD research project aimed to demonstrate that mitochondrial functions and integrity constitute early indicators of fish fillet freshness. Mitochondria are the powerhouse of the cell and play a central role in cell death mecanisms such as apoptosis and necrosis. Mitochondrial function and integrity in fish filet muscle cells were studied at different times of storage post mortem at 4°C. The species studied as a model was the gilthead seabream (Sparus aurata) (gilthead seabream fibroblast cell line (SAF-1) and fish fillets). Firstly, the structure of fish mitochondria was studied by transmission electron microscopy. Numerous mitochondrial structural alterations have been observed in fish fillet from 72 hours (D3) of storage at 4°C. These alterations were more pronounced at D4 and D6. Then, mitochondrial functionality was assessed with two approaches: mitochondrial respiration (oxygraphy) and mitochondrial membrane potential (ΔΨₘ) estimated with the fluorescent probe Rh123. From 96 hours of storage at 4°C (D4), these two parameters were significantly disrupted demonstrating the alteration of mitochondrial function and integrity. The results are in correlation with the mitochondrial structural alterations described by microscopy. In parallel, a method of mitochondrial membrane potential evaluation has been developed with a micro-volume fluorimeter, first using bacteria and then isolated mitochondria. This work demonstrated that the mitochondrial functionality study constitutes a reliable and early fish filet freshness indicator. Additional knowledge on cell mechanisms in post mortem condition has been brought. These results constitute the starting point for the development of a fish freshness assay kit and pave the way to research on others freshness and freeze-thawing indicators based on mitochondrial integrity and functionality.

Apesar da melhoria no sistema de produção e cultivo dos embriões in vitro, cerca de 60% dos oócitos que entram no sistema, não atingem o estágio de blastocisto e a qualidade dos embriões obtidos é bastante variável quando comparadas com embriões produzidos in vivo. Este bloqueio pode ser afetado por íons inorgânicos, tampões, aminoácidos e composição da atmosfera gasosa. Partindo-se da premissa que há influência da mitocôndria sobre a ativação da morte celular programada levou-nos a formular a hipótese que ausência de fragmentação nuclear nos embriões antes das 72 hpi está relacionada com a ausência do potencial de membrana mitocondrial e a inibição da OXPHOS pela utilização de bloqueadores leva a manutenção de baixos níveis de potencial de membrana mitocondrial e baixas taxas de fragmentação nuclear nos embriões. Embriões foram produzidos in vitro mediante maturação durante 22 horas, fecundação e cultivo 18 horas após a inseminação (hpi). Decorridas 24hpi realizou-se o cultivo com 0% de oxigênio, a fim de bloquear o processo de OXPHOS. Após 48 hpi realizou-se o feeding do meio de cultivo (SOF) com inibidores da OXPHOS (antimicina A e/ou oligomicina, cianeto de potássio) em diferentes doses. O número de embriões 8 células foi determinado às 80 hpi, mesmo momento em que foram realizadas as técnicas de JC-1 e TUNEL. Verificou-se as 168 hpi o efeito dos tratamentos no desenvolvimento embrionário. Os resultados obtidos com a inibição da OXPHOS após 48 hpi com oligomicina e/ou antimicina A nas doses utilizadas não alterou a capacidade do embrião atingir o estádio de 8 células. Entretanto, esta inibição inviabilizou o desenvolvimento até o estádio de blastocisto. O tratamento com KCN permitiu o desenvolvimento até o estádio de 8 células e a blastocisto em taxas semelhantes ao controle. A inibição do cultivo na ausência do O2 inviabilizou o processo de cultivo. Já os resultados obtidos quanto ao &#936;mm e TUNEL evidenciam que os tratamentos dos embriões antimicina e/ou oligomicina levaram a um aumento do &#936;mm e fragmentação nuclear na maioria dos embriões testados. Portanto, não foi possível testar a hipótese de que o &#936;mm é necessário para o estabelecimento da MCP, todavia, foi observada uma correlação positiva entre &#936;mm e fragmentação nuclear. / Although in vitro embryo production has been improved in the last 2 decades, about 60% of the oocytes do not reach the blastocyst stage and embryo quality is very variable when compared with in vivo produced embryo. This developmental block can be affected by inorganic ions, buffers, aminoacids and gaseous atmosphere composition. The knowledge that there influence of mitochondria on the activation of the programmed cellular death led to formulate the hypothesis that nuclear fragmentation absence in embryos before 72 post insemination is related with absence of mitochondrial membrane potential and OXPHOS blockage by inhibiting agents, would cause the maintenance of low levels of mitochondrial membrane potential and low rates of embryo nuclear fragmentation. Embryos were cultured in vitro for 18 hours post insemination (hpi) and after 24 hpi, they were submitted to 0% oxygen culture, in order to block the OXPHOS process. At 48 hpi, feeding was performed with SOF medium containing OXPHOS inhibitors (antimycin A and/or oligomycin, potassium cyanide) in different concetrations. The numbers of 8 cell embryos were estimated at 80 hpi, the same moment that they were submitted to JC-1 probes and TUNEL for mitochondrial membrane potential and DNA damages evaluation, respectively. At 168 hpi the effect of the treatments was verified on embryonic development. The results obtained with the OXPHOS inhibition after 48 after hpi using oligomycin and/or antimycin A did not modify embryo capacity to reach 8 cell stage. However, this inhibition prevented development to the blastocyst stage. KCN treatment allowed development up to the 8 cell stage and blastocyst similar to controls. The absence of O2 prevented embryo development. The &#936;mm and TUNEL results showed that antimycin and/or oligomycin treatment increased &#936;mm and nuclear fragmentation in the majority of the embryos tested. In conclusion, it was not possible to test the hypothesis that &#936;mm is necessary to the establishment of MCP, but a positive correlation between &#936;mm and nuclear fragmentation was observed.

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

A obesidade está relacionada com uma grande variedade de disfunções, tais como as dislipidemias, resistência à insulina e diabetes tipo II. Estas patologias estão relacionadas com alterações da fisiologia mitocondrial. As mitocôndrias são o principal sítio do metabolismo oxidativo e são importantes fontes de espécies reativas de oxigênio. Alberici et al. (2006) mostraram que a atividade de canais de potássio sensíveis a ATP mitocondriais (mitoKATP), via dissipativa branda, estava aumentada em fígados de animais hipertrigliceridêmicos transgênicos. Este dado sugere um papel adaptativo importante para o mitoKATP como regulador do metabolismo e da massa corpórea. O principal objetivo deste trabalho foi investigar o papel deste canal em fígados de camundongos submetidos a uma dieta hiperlipídica, obtida por meio de suplementação de óleo de soja à única fonte de água dos animais. Foram estabelecidas correlações entre a atividade do canal e parâmetros da fisiologia mitocondrial ou parâmetros sorológicos clássicos como colesterolemia e trigliceridemia. Sendo realizados experimentos de espalhamento de luz, quantificação de peróxido de hidrogênio, potencial de membrana e consumo de oxigênio em mitocôndrias. Nossos resultados mostram aumento de massa corpórea sem dislipidemias e aumento da atividade do mitoKATP, dependente do tempo da suplementação, diretamente correlacionada com a colesterolemia. Nossos resultados apontam aumento da geração de EROs e diminuição da eficiência de conversão energética no grupo suplementado. Como conclusão: manipulações de dieta são eficazes para a indução da atividade do mitoKATP, sendo este um novo e interessante sítio de regulação do metabolismo oxidativo e da massa corpórea. / Obesity is associated with multiple dysfunctions including dyslipidemia, insulin resistance and diabetes. These malfunctions are associated with modifications in mitochondrial physiology. Furthermore, mitochondria are the most important site of energy metabolism and reactive oxygen species production. Alberici et al. (2006) demonstrated that hypertriglyceridemic transgenic mice display increased expression ad activity of mitochondrial ATP sensitive potassium channels (mitoKATP), a mild uncoupling pathway, in liver. This suggests that mitoKATP is an important adaptation to regulate body mass and metabolism. The aim of this work is to investigate the role of mitoKATP in a high fat diet induced by soy oil supplementation, correlating changes in channel activity with metabolic and mitochondrial parameters. Mitochondria were isolated from mouse livers and serological parameters were measured for each animal. Light scattering (to estimate mitoKATP activity), hydrogen peroxide generation, membrane potentials and oxygen consumption were measured in the mitochondrial suspensions. Our results indicate an increase in body mass, without dyslipidemia; and increases in mitoKATP activity, in time-dependent manner, directly correlated to cholesterol levels. In addition, we found increases in ROS generation and decreased capacity of energy conversion (ADP/O) in the high fat diet group. In conclusion, our results indicate that the activity of mitoKATP could be induced by high fat diets and that this is an novel site for metabolic and body mass control.

Due to two different mutations in the gene that encodes the a1A subunit of voltage-activated CaV 2.1 calcium ion channels, the compound heterozygous tottering/leaner (tg/tgla) mouse exhibits numerous neurological deficits. Human disorders that arise from mutations in this voltage dependent calcium channel are familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6. The tg/tgla mouse exhibits ataxia, movement disorders and memory impairment, suggesting that both the cerebellum and hippocampus are affected. To gain greater understanding of the many neurological abnormalities that are exhibited by the 90-120 day old tg/tgla mouse the following aspects were investigated: 1) the morphology of the cerebellum and hippocampus, 2) proliferation and death in cells of the hippocampal dentate gyrus and 3) changes in basic biochemical parameters in granule cells of the cerebellum and hippocampus. This study revealed no volume abnormalities within the hippocampus of the mutant mice, but a decrease in cell density with the pyramidal layer of CA3 and the hilus of the dentate gyrus. Cell size in the CA3 region was unaffected, but cell size in the hilus of the dentate gyrus did not exhibit the gender difference seen in the wild type mouse. The cerebellum showed a decrease in volume without any decrease in cerebellar cellular density. Cell proliferation and differentiation in the subgranular zone of the hippocampal dentate gyrus remained normal. This region also revealed a decrease in cell death in the tg/tgla mice. Basal intracellular calcium levels in granule cells show no difference within the hippocampus, but an increase in the tg/tgla male cerebellum compared to the wild type male cerebellum. There was no significant difference in granule cell mitochondrial membrane potential within the wild type and mutant animals in either the hippocampus or cerebellum. The rate of reactive oxygen species (ROS) production in granule cells revealed no variation within the hippocampus or cerebellum. The amount of ROS was decreased in cerebellar granule cells, but not granule cells of the hippocampus. Inducing ROS showed no alteration in production or amount of ROS produced in the hippocampus, but did show a ceiling in the amount of ROS produced, but not rate of production, in the cerebellum.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease with an annual mortality rate of 15% despite current therapies. Uncontrolled proliferation of pulmonary artery smooth muscle cells (PASMCs) results in adverse vascular remodeling contributing to PAH. Understanding the mechanisms of PASMC proliferation may identify new targets for treatment. Chloride currents/channels (ICl) are expressed in PASMCs and their roles in proliferation have been suggested based on their importance in resting membrane potential and cell volume regulation. The present study explored the role of ICl in proliferation in rat and human PASMCs. We found that either nonspecific ICl inhibitors (DIDS or NPPB) or a putative specific blocker of swelling-activated ICl (ICl,swell) reduced proliferation of PASMCs cultured in serum-containing media. Patch-clamp studies showed that proliferating PASMCs had increased baseline ICl and ICl,swell in association with depolarized membrane potentials. Quantitative real-time RT-PCR studies identified expressions of CLC-3, a candidate gene of ICl,swell, and several other CLC genes in proliferating PASMCs. While selective knockdown of CLC-3 with lentiviral shRNA reduced PASMC proliferation, it had no effect on ICl,swell. These findings are consistent with the conclusion that ICl regulate proliferation of PASMCs and suggest that selective ICl inhibition may be useful in treating pulmonary arterial hypertension.