[pt] Criação seletiva bidirecional de uma resposta defensiva ou qualquer outra característica fenotípica é uma técnica na qual animais são criados com o objetivo de modificar a frequência dos genes que estão subjacentes a um fenótipo em particular. O acasalamento de animais de uma determinada população com base nos extremos opostos de uma característica observável vai propagar, após diversas gerações, este fenótipo particular em direções opostas, levando-se à criação de duas linhagens contrastantes. No presente trabalho empregamos o congelamento condicionado em resposta a estímulos contextuais previamente associados com choques elétricos nas patas como critério de seleção para o desenvolvimento de duas novas linhagens de ratos. O protocolo básico consistiu de acasalamento entre machos e fêmeas Wistar com as maiores e as menores taxas de congelamento condicionado em resposta a sinais contextuais da câmara experimental onde os animais foram expostos a três choques elétricos não sinalizados no dia anterior. O Estudo 1 apresenta os resultados iniciais de quatorze gerações de criação seletiva. Os resultados mostraram que diferenças significativas entre estas duas linhagens foram encontradas após 3 gerações, indicando um forte componente hereditário deste tipo de aprendizagem. As linhagens foram denominadas Cariocas com Alto Congelamento Condicionado (CAC) e Cariocas com Baixo Congelamento condicionado (CBC). Além disso, nós introduzimos um terceiro grupo de animais aleatoriamente selecionados (CTRL) em nosso programa de criação seletiva. No Estudo 2 investigamos os diferentes padrões de extinção e da reaquisição do medo condicionado nestas duas novas linhagens. Por fim, no Estudo 3, nossos resultados sugeriram uma dissociação entre o medo contextual e o medo discreto entre animais CAC e CBC. / [en] Bidirectional selective breeding of a defensive response or any other phenotypic characteristic is a technique in which animals are bred to modify the frequency of the genes that underlie a particular phenotype. Mating animals within a population based on the opposite extremes of an observable characteristic will push, over many generations, this particular phenotype in opposite directions, leading to two separately bred lines. In the present work we employed the conditioned freezing in response to contextual cues previously associated with footshock as the phenotype criterion for developing two new rat lines. The basic protocol consisted of mating male and female albino Wistar rats with the highest and lowest conditioned freezing in response to the contextual cues of the experimental chamber where animals were exposed to three unsignaled electric footshocks on the previous day. Study 1 presents the initial results of fourteen generations of selective breeding. We found that after three generations, reliable differences between these two lines were already present, indicating a strong heritable component of this type of learning. The lines were named Carioca High conditioned Freezing (CHF) and Carioca Low conditioned Freezing (CLF). Also, we introduced a third group of randomly selected animals (RND) in our selective breeding program. In Study 2, we investigated the different patterns of fear extinction and reacquisition in these two new lines. Finally, in Study 3, results showed dissociation between contextual and phasic fear between CHF and CLF rats.

La restriction alimentaire chronique correspond à un des troubles du comportement alimentaire observé en particulier dans l’anorexie mentale (AN) de type restrictif, pathologie qui touche essentiellement les adolescentes et les jeunes femmes. En plus de ce comportement restrictif, une activité physique importante est observée chez un grand nombre de patientes (40 à 80% des cas). Cette maladie se traduit par de nombreuses altérations physiologiques comme des perturbations neuroendocrines, métaboliques, osseuses (ostéopénie, ostéoporose) et ce quelle que soit la cause psychiatrique qui a conduit au développement de ce comportement. De plus, de nombreux arguments suggèrent que l’AN pourrait être considérée comme un trouble « addictif » qui se manifesterait par une addiction à la perte de poids et/ou à la restriction alimentaire ou encore à l’activité physique suggérant une altération du système dopaminergique de récompense. Ainsi, quelles que soient les origines de la maladie, l’AN entraîne des perturbations périphériques et centrales susceptibles d’être impliquées dans une première phase dite « d’adaptation » permettant aux malades de survivre à ces conditions drastiques. Les patients peuvent par la suite tomber dans une seconde phase de « chronicisation » dans laquelle ces mêmes facteurs pourraient être responsables de la dégradation de l’état des malades et conduire, dans les cas les plus graves d’épuisement, à la mort.Notre étude a comme pivot la ghréline, hormone orexigène, dont les concentrations plasmatiques sont augmentées significativement chez les patients anorexiques. Sécrétée principalement en périphérie par les cellules de l’estomac, elle va cibler plusieurs organes aussi bien périphériques que centraux. En particulier, au niveau périphérique, cette hormone agit au niveau du foie dont la principale fonction connue est le maintien de l’homéostasie glucidique. Elle agit également au niveau du tissu adipeux qui est alors stimulé, favorisant ainsi sa croissance avec un stockage des réserves et au niveau musculaire en entrainant entre autre une diminution des réserves de triglycérides. Au niveau du système nerveux central, parmi les sites d’action de la ghréline, on trouve les structures impliquées aussi bien dans le contrôle, qualifié d’homéostatique, de la prise alimentaire représenté par l’hypothalamus, que dans le contrôle, dit hédonique (motivation/récompense), de ce même comportement correspondant au circuit méso-limbique. Pour étudier son implication dans les mécanismes adaptatifs, et éventuellement, dans l’aggravation de la maladie, nous avons mis au point un modèle animal de restriction alimentaire chronique mimant les symptômes physiologiques de l’AN. Notre premier objectif a été de caractériser (« phénotypage ») ce modèle sur le plan physiologique (métabolique, endocrinien) afin de l’utiliser pour notre deuxième objectif : évaluation du rôle de la ghréline comme potentiel facteur prédictif de l’évolution de la maladie.Les données obtenues valident notre modèle comme un modèle pertinent pour étudier sur le long terme les altérations physiologiques et centrales décrites dans l’AN de type restrictif. Nous montrons que l’exercice physique modéré associé à la restriction alimentaire a des effets stabilisateurs sur de nombreux paramètres métaboliques limitant ainsi un épuisement prématuré des ressources énergétiques. En ce qui concerne la ghréline, les concentrations plasmatiques élevées observées dans notre modèle pourraient contribuer également à une régulation adaptative du métabolisme énergétique. / Chronic food restriction is one of the major features observed in anorexia nervosa (AN), especially in the restrictive type. This major eating disorder affects mainly teenager girls and young women. Additionally to the restriction behavior, important physical activity is observed in a large number of patients (40-80% of cases). This disease induces various physiological alterations that concern neuroendocrine, metabolic and bone (osteopenia, osteoporosis) pathways, which have dramatic consequences on the patient’s health. Moreover, many arguments suggest that AN could be considered like an "addictiv" disorder supported by an addiction to weight loss and/or food restriction or physical activity. It thus suggests modifications of the central dopaminergic reward system. Furthermore, whatever the origins or the causes of this disorder, AN leads to peripheral and central alterations that might be involved in an "adaptation" phase allowing patients surviving to these drastic conditions. For some patients, a phase of "chronicity" is described in which these physiological changes may worsen the patient conditions and contribute, when exhaustion is amplifying, to death. Our study points out ghrelin, an orexigenic hormone whose plasma concentrations are significantly increased in AN patients. Mainly secreted by stomach cells, it targets multiple peripheral organs as well as numerous neuronal structures in the brain. At the peripheral level, this hormone acts among others in the liver whose main function is the maintenance of glucose homeostasis. It acts also on the adipose tissue to promote its growth that is associated with lipid storage and on muscle resulting in a reduction of triglycerides stock. At the central nervous system level, ghrelin have various targets like the structures involved in the homeostatic as well as hedonic (motivation/reward) control of food intake through the hypothalamus and the meso-cortico-limbic system respectively.To study the involvement of ghrelin in the potential adaptive mechanisms, and even in the worsening of the disease, we have developed an animal model of chronic food restriction associated or not with physical activity, mimicking the physiological symptoms of AN. Our first objective was to characterize and to phenotype the mouse model by evaluating various physiological (metabolic, endocrine) factors in order to study in our second objective the role of ghrelin as a potential predictor of disease progression.We showed that our mouse model constitutes a pertinent model to study on a long term duration the physiological and central altérations described in the restrictive type AN. Moreover, we showed that moderate physical activity associated with food restriction had stabilizing effects on numerous metabolic parameters that may reduce an early exhaustion of energy stocks. Concerning the role of ghrelin in such model, its plasma concentrations were increased like in AN patients and were suggested to contribute to the adaptive regulation of energy metabolism.

La rapamycine est un inhibiteur de la voie mTOR dont la propriété immunosuppressive est reconnue pour son action préférentielle sur les lymphocytes T effecteurs et sa capacité à favoriser l’expansion de lymphocytes T régulateurs (Treg) et d’autres cellules immunosuppressives. En transplantation hépatique, la rapamycine a été retirée de l’arsenal thérapeutique dans le traitement de novo du rejet aigu, suite aux résultats obtenus et décrits au sein du « Rapamune Liver Transplant Study Group » qui a conclu à une forte incidence de thromboses de l’artère hépatique. Des travaux récents montrent que l’introduction retardée d’une immunosuppression après une greffe permet de créer, dans certains cas, une fenêtre d’opportunité pour freiner la réponse allogénique initiée. Cette nouvelle configuration pourrait remettre en de question l’utilisation en jeu des inhibiteurs de mTOR en transplantation hépatique. L’objectif de ma thèse a été d’évaluer l’efficacité d’un traitement court et retardé par la rapamycine dans un modèle de greffe hépatique immunogène chez le rat. Notre étude montre qu’un traitement de 8 jours débuté à J4 post transplantation permet de prolonger de manière significative la survie des animaux greffés en améliorant la fonction des greffons. De plus, l’analyse cellulaire des organes lymphoïdes secondaires, montre une prédominance de cellules myéloïdes suppressives (MDSC) et de lymphocytes CD8+CD45RClow à un stade précoce chez les animaux tolérants et dont le taux reste stable à un stade tardif sans modifications majeures du pourcentage de Treg CD4+CD25+Foxp3+ . Ainsi, nos résultats suggèrent un effet protecteur de la rapamycine lorsqu’elle est administrée de façon retardée et courte dans un modèle de greffe hépatique et dont le mécanisme d’induction de tolérance ne semble pas être dépendant des Treg. Cette approche permet de reconsidérer la place de la rapamycine en transplantation hépatique et suggère d’évaluer quelles sont les populations cellulaires impliquées dans le maintien de tolérance. / Rapamycin is an mTOR pathway inhibitor with immunosuppressive property recognized for its preferential action on effectors T cells and its ability to promote the expansion of regulatory T cells (Treg) and other immunosuppressive cells. In liver transplantation, rapamycin has been removed from the therapeutic arsenal for the treatment of de novo acute rejection, following the results of "Rapamune Liver Transplant Study Group" which concluded of a high incidence of thrombosis of the hepatic artery. Recent studies show that the delayed introduction of immunosuppression following transplantation creates, in some cases, a window of opportunity to curb the allogeneic response initiated. This therapeutic approach has restored hope in the future of mTOR inhibitors in liver transplantation. The aim of my thesis was to evaluate the efficacy of a delayed and short course rapamycin treatment in an immunogenic liver transplantation model in rats. Our study shows that treatment of 8 days started at day 4 post-transplantation can significantly prolong the survival of grafted animals by improving the function of the grafts. Cellular analysis of secondary lymphoid compartments shows a predominance of myeloid suppressor cells (MDSC) and CD8+CD45RClow T cells at early stage in tolerant animals and the rate remains stable at a late stage without any major changes in the frequency of CD4+CD25+Foxp3+ Treg. Thus, our preliminary results suggest a protective effect of rapamycin when administered at delayed and short way in a liver transplantation model. Early induction mechanism does not appear to be Treg-dependent. This approach allows to reconsider the place of rapamycin and the cell population involved in the maintenance of tolerance.

As distrofias musculares constituem um grupo heterogêneo de doenças genéticas, caracterizadas por uma degeneração progressiva e irreversível dos músculos. Modelos murinos distróficos, como o mdx, SJL/J, Largemyd e Lama2dy-2J/J, são ferramentas importantes para o estudo destas doenças. O objetivo deste trabalho consistiu em estabelecer parâmetros de avaliação funcional que visem a sua utilização para elucidar os benefícios clínicos de futuras terapias. Para tanto, foram avaliadas as quatro linhagens distróficas, em diferentes idades, e comparadas a controle normal. Os testes padronizados consistiram em nado forçado, avaliação de resistência/equilíbrio pelos membros anteriores e pelos quatro membros, caminhar em plataforma suspensa, suspensão pela cauda, grip strength e rota rod. Comprovou-se a existência de diferentes padrões de força, resistência, coordenação motora e aprendizagem/memória ao longo do tempo de vida de cada linhagem, o que permitiu traçar parâmetros a serem utilizados em futuras pesquisas de terapia celular e farmacológica. / Muscular dystrophies are a heterogeneous group of genetic diseases characterized by a progressive and irreversible degeneration of the muscles. Dystrophic mouse models, like the mdx, SJL/J, Largemyd and Lama2dy-2J/J, are important tools for studying these diseases. The aim of this study was to establish parameters for functional evaluation aiming its use to elucidate the clinical benefits of future therapies. Thus, we evaluated the four strains of dystrophic mice, at different ages, and compared to normal control. Standardized tests consisted of forced swimming, evaluation of resistance/balance by forelimb and four members, walking on suspended platform, suspension by the tail, grip strength and rota rod. We observed the existence of different patterns of strength, endurance, coordination and learning / memory over the lifetime of each strain, which allowed tracing parameters to be used in future studies of cell and pharmacology therapies.

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling long-term transgene regulation in future clinical developments of gene transfer.

There is now considerable evidence that both schizophrenia and affective disorders have their origin at least in part in events that occur during early pre- and post-natal development. In the case of schizophrenia, many observations, for example, increased risk for schizophrenia in the offspring of mothers who had influenza A during their second trimester of pregnancy and evidence for abnormal neuronal migration in the cerebral cortex of post mortem tissue from schizophrenic patients, suggest that a second trimester insult may have occurred and that this insult may have increased the risk for the development of schizophrenia in late adolescence or early adulthood. Animal studies have found that rats that undergo excitotxic damage to the ventral hippocampus on postnatal day 7 develop exaggerated sensitivity to dopamine-stimulating drugs or to stressful stimuli that becomes apparent after sexual maturity but not before, providing a neurodevelopmental model of schizophrenia. Similarly, post-weaning social isolation leads to nehanced responses to dopaminergic drgus and to stress that emerges after sexual maturity. These animal models are proving to be valuable tools to study the neurobiological mechanisms mediating the influence of early insults to the nervous system on later behavioural functins. In the case of affective disorders, although the evidence is not as strong, a number of the same observations have been made suggesting that an insult during early ontogeny may lead to the development of affective disorders later in life. For example, retrospective studies of people with affective disorders showed that they were more likely to have attained motor milestones at a later age and to have had poorer academic performance as children. There is a wealth of evidence suggesting hyperfunctioning of the hypothalamic-pituitary-adrenal (HPA) axis in affective disorders. Animal studies have shown that early matenal deprivation can lead to lasting changes in the reactivity of the HPA axis to stressful stimuli, providing another link from early experience to adult psychopathology. Continued studies of the effects of pre- and early post-natal events on the development of the nervous system and the relationships of these events to schizophrenia or affective disorder will provide new insights into the mechanisms underlying these common neuropsychiatric illnesses.

La survenue d’une infection péri prothétique (IPP) est la principale complication de la chirurgie prothétique articulaire, depuis son invention par Robert et Jean Judet en 1947. Comme le nombre de prothèses articulaires posées chaque année augmente de façon importante, ces infections sont de plus en plus fréquentes et l’optimisation de leur prise en charge est un enjeu important sur le plan médical et économique.Les modèles animaux d’IPP permettent de comprendre les mécanismes éthio-pathogéniques et tester de nouvelles thérapeutiques. Une analyse critique de la littérature a été effectuée en évaluant chaque modèle selon son type d’inoculation qui influence les taux et la sévérité de l’infection expérimentale obtenue.Un modèle expérimental d’IPP chez le lapin obtenu par remplacement partiel du genou et inoculation locale a été utilisé pour tester l’efficacité de nouvelles thérapeutiques au cours d’infections à deux bactéries multi résistantes qui posent des problèmes en thérapeutique humaine.Dans un 1er temps nous avons évalué l’efficacité de la ceftaroline (CPT) céphalosporine bactéricide in vivo contre le Staphylococcus aureus résistant à la méticilline (SARM) en la comparant à la vancomycine en association ou non à la rifampicine. 5.107UFC (Unités Formant Colonies) de SARM (Concentration Minimale Inhibitrice (CMI) de 0,38, 0,006, et 1 mg/l pour CPT, RIF, et VAN, respectivement) était injecté dans le genou. Les animaux infectés ont été randomisés et recevaient : aucun traitement (contrôles), CPT (60 mg/kg im), VAN (60 mg/kg im), CPT plus RIF (10 mg/kg im), ou VAN plus RIF débutant 7 jours après l'inoculation et durant 7 jours. L’efficacité des traitements a été évaluée sur la quantité de bactéries persistantes dans l’os (tibia proximal) après traitement. Ce travail a montré que la CPT et la VAN étaient efficace en monothérapie mais que seule l’association avec la rifampicine permettait de stériliser la quasi totalité des animaux. La CPT apparaît donc comme un traitement potentiellement efficace dans cette infection.Dans un 2ème temps nous avons étudié l'efficacité de la colistine (COL) dans le ciment, seule ou en combinaison avec des injections intramusculaires (im) de COL et/ou de méropénème (MRP) dans des infections à Klebsiella pneumoniae résistantes aux carbapénèmes (KPC). Un modèle proche de celui décrit pour le SARM a été utilisé. La souche KPC99YC est une souche clinique, résistante à la gentamicine (CMI 8mg/l) intermédiaire à l'imipénème (CMI 4mg/l), et sensible à la COL (CMI 0,25mg/l). L’inoculum était de 1.109UFC. Sept jours après l'infection, les prothèses étaient remplacées par espaceur sans antibiotique (contrôle), ou par espaceur imprégné de COL (3 MUI de COL/40g de ciment), ou par espaceur sans antibiotique et injections de COL (12 mg/kg im), ou l’association des deux, ou injections de COL avec espaceur en ciment imprégné de COL associé ou non à des injections de MRP (80 mg/kg im). Le traitement durait 7 jours. Tous les lapins témoins étaient infectés à J15, avec une moyenne de densité bactérienne de 6,17 [5,69, 7,04] CFU/g d'os. Contrairement à la COL locale, la COL systémique seule ou combinée avec le MRP était plus efficace que le contrôle sur le nombre de bactéries dans l'os à la fin du traitement. L’association COL locale + systémique était significativement plus efficace que le groupe témoin sur le dénombrement bactérien. D’ailleurs, c'était le seul schéma efficace sur le nombre de lapins avec un os stérile et à la limite de significativité par rapport au traitement systémique seul. Une souche résistante à la COL a été détectée dans le traitement local seul mais pas avec l’association de COL locale et systémique.Les modes d’inoculation directs sont les plus efficaces pour reproduire une IPP aigue. Les études expérimentales permettent de tester des traitements innovants en particulier pour les infections à bactéries multi résistantes. / The occurrence of prosthetic joint infection (PJI) is the main complication of joint prosthetic surgery since its invention by Robert and Jean Judet in 1947. Since the number of articular prostheses placed each year increases significantly, these infections are more and more frequent and the optimization of their management is an important medical and economic stake.The animal models of PJI make it possible to understand the ethiopathogenic mechanisms and to test new therapeutics. A critical analysis of the literature was carried out by evaluating each model according to its type of inoculation which influences the rates and the severity of the experimental infection obtained.An experimental model of PJI in rabbits obtained by partial replacement of the knee and local inoculation was used to test the efficacy of new therapeutics during infections with two multi-resistant bacteria which pose problems in human therapeutics.In a first step we evaluated the efficacy of ceftaroline (CPT) cephalosporin bactericidal in vivo against methicillin-resistant Staphylococcus aureus (MRSA) by comparing it with vancomycin (VAN) in combination with or without rifampin (RIF). 5.107UFC MRSA (Minimum Inhibitory Concentration (MIC) of 0.38, 0.006, and 1 mg/l for CPT, RIF, and VAN, respectively) was injected into the knee. Infected animals were randomized to receive no treatment (control), CPT (60 mg/kg im), VAN (60 mg/kg im), CPT plus RIF (10 mg/kg im) or VAN plus RIF, 7 days after inoculation and for 7 days. The efficacy of treatments was evaluated on the amount of persistent bacteria in the bone (proximal tibia) after treatment. This work has shown that CPT and VAN were effective as monotherapy, but only the combination with RIF allowed the sterilization of almost all animals. CPT appears to be a potentially effective treatment in this infection.In a second step we studied the efficacy of colistin (COL) in cement, alone or in combination with intramuscular (im) injections of COL and/or meropenem (MRP) in carbapenem-resistant Klebsiella pneumoniae infections (KPC). A model close to that used for MRSA was used. The strain KPC99YC is a clinical strain, resistant to gentamicin (MIC 8mg/L) intermediate to imipenem (MIC 4mg/l), and sensitive to COL (MIC 0,25mg/l). The inoculum was 1,109UFC. Seven days after the infection, the prosthesis were replaced by antibiotic-free spacer (control), or by COL-impregnated spacer (3 MIU of COL/40g of cement), or by antibiotic-free spacer and COL injections (12 mg/kg im), or the combination of the two, or COL injections with COL-impregnated cement spacer associated or not with MRP injections (80 mg/kg im). The treatment lasted 7 days. All control rabbits were infected at D15, with median and interquartile range (IQR) bone bacterial count of 6.17 [5.69, 7.04] CFU/g of bones. In contrast to local COL, systemic COL alone or combined with MRP was more effective than control on bacterial counts in bone at the end of treatment. The combination of COL local + systemic was significantly more effective than control group on bacterial counts. Interestingly it was the only effective regimen on the number of rabbits with sterile bone and at the limit of significance vs systemic treatment alone. One COL-resistant strain was detected in the COL local treatment alone but not with the combination of local and systemic COL.Direct inoculation modes are most effective in reproducing an acute PJI. The experimental studies allow testing innovative treatments in particular for the infections with multi-resistant bacteria.

In vitro models are often used both to characterize and test therapeutics for neurodevelopmental disorders (‘NDDs’). While in vitro models have extraordinary potential to develop therapies for patients, they have historically been confounded by absence of robust phenotypes and/or in vitro phenotypes that fail to translate from laboratory bench to bedside. Within this thesis work, we attempt to address three areas in which in vitro models may be improved – gene selection, model validation and identification of disease-relevant functional assays suited for therapeutic testing. Publicly available databases aggregating identified and annotated disease-causing variants for Mendelian diseases have rapidly expanded over the past two decades. Elucidating mechanisms of disease and developing therapies using in vivo model systems often is both time and cost intensive. Thus, determining which subsets of genes are more likely to generate addressable signals in a dish may lead to more effective drug development. In chapter 1, we identify a set of genes ideally suited for therapeutic inhibition. Specifically, we leverage the aforementioned large genetic databases to identify a set of genes likely to act through a gain-of-function mechanism that are both tolerant to loss-of-function mutations and in the druggable genome. In chapter 2, we aim to characterize the degree of conservation of transcriptomic dysregulation between a human in vitro cortical organoid (‘hCOs’) model, and two mouse models of a severe neurodevelopmental disorder resulting from HNRNPU deficiency. Human model systems may improve upon animal models when human pathogenesis and patient phenotypes are divergent from animal models due to species-specific etiology. However, human model systems often lack the heterogeneity and cell-type specificity and maturity seen in primary fetal samples. Importantly, some mouse models of HNRNPU deficiency have muted phenotypes compared with human patients. We hypothesized that while there are distinctions between humans and mice with HNRNPU deficiency, there will be overlap in transcriptomic dysregulation between human and mouse models. In fact, we find 45-day-old HNRNPU+/- hCOs have consistent transcriptomic dysregulation to embryonic mouse models, but not to perinatal mice. Our findings suggest hCOs are a viable model for characterizing HNRNPU deficiency; however, such models may only be appropriate for elucidating a transcriptomic disease state at a specific developmental time period. Functional assays for neurodevelopmental disorders can aid in understanding whether transcriptomic dysregulation is relevant to patient symptoms, as genomic findings may not always correlate to disease-relevant phenotypes. Further, relevant functional phenotypes can then be utilized for testing potential therapeutics. Importantly, seizures are commonly present in a significant subset of neurodevelopmental disorders and seizure phenotypes have been described as driven by aberrant synchrony in neuronal networks. Using a multielectrode array platform, investigators can use a variety of computational methods to quantify aspects of synchrony in vitro. In chapter 3a, we introduce topological approaches capable of identifying novel synchrony phenotypes in primary neuronal networks from mouse models of neurodevelopmental disorders. Certain mouse models will be confounded by species-specific pathogenesis and/or vastly different developmental timelines and fail to generalize to human patients, motivating the need for functionally active and physiologically relevant human in vitro models. In chapter 3b, we attempt to generate human networks with balanced levels of excitation and inhibition and find confounding lack of functional maturation of inhibitory neuronal subtypes in 90-day-old stem cell-derived neuronal networks. Future work generating in vitro human neuronal networks with functionally mature inhibitory neurons would complement the findings in chapters 1 and 2 and allow for more efficient therapeutic development strategies that may lead to improved patient outcomes.

Cardiovascular disease (CVD) is the leading cause of death for people of most ethnicities on a global scale, and countless research efforts on the pathology of CVD has been well-characterized over the years. However, advancement in modern technologies, such as nanotechnology, has generated environmental and occupational health concerns within the scientific community. Current investigation of nanotoxicity calls into question the negative effects nanomaterials may invoke from their environmental, commercial, and therapeutic usage. As a result, further research is needed to investigate and characterize the toxicological implications associated with nanomaterial-exposure and CVD. We investigated the toxicity of multi-walled carbon nanotubes (MWCNT) and titanium dioxide (TiO2), which are two prominently used nanomaterials that have been previously linked to upregulation of inflammatory and atherogenic factors. However, the mechanistic pathways involved in these nanomaterials mediating detrimental effects on the heart and/or coronary vasculature have not yet been fully determined. Thus, we utilized two different routes of exposure in rodent models to assess alterations in proinflammatory and proatherogenic signaling pathways, which are represented in contrast throughout the dissertation. In our MWCNT study, we used C57Bl/6 mice exposed to MWCNTs (1 mg/m3) or filtered air (FA-Controls), via inhalation, for 6 hr/d for 14d. Conversely, intravenous TiO2 was administered to F344 male fisher rats, following 24h and 28d post-exposure to a single injection of TiO2-NPs (1 mg/kg), compared to control animals. MWCNT-exposed endpoints investigated the alterations in cholesterol transport, such as lectin-like oxidized low-density lipoprotein receptor (LOX)-1 and ATP-binding cassette transporter (ABCA)-1, inflammatory markers [tumor necrosis factor (TNF)-α], interleukin (IL)-1β/IL-6, nuclear-factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signaling factors involved in activation of the pathway, as well as intracellular/vascular adhesion molecule(s) (VCAM-1, ICAM-1), and miRNAs (miR-221/-21/-1), associated with CVD, were analyzed in cardiac tissue and coronary vasculature. Cardiac fibrotic deposition, matrix-metalloproteinases (MMP)-2/9, and reactive oxygen species (ROS) were also assessed. TiO2-exposure endpoints also involved alterations on cholesterol transport proteins via LOX-1 and ABCA-1, factors of inflammation, namely intracellular macrophages and interleukin (IL)-1β, MMP-2/9 activity and protein expression, fibrotic deposition, and ROS generation were analyzed via quantitative detection or histologically in both cardiac tissue and coronary vasculature. Results from both studies found alterations in fibrotic deposition, upregulation in LOX-1 expression and MMP-2/9 activity, and ROS generation; with a concurrent decrease in ABCA-1 expression in cardiac tissue and coronary vasculature. Individually, MWCNT-exposed endpoints had shown induction of cardiac TNF-α, MMP-9, IκB Kinase (IKK)-α/β, and miR-221 mRNAs; as well as increased coronary expression of TNF-α and VCAM-1. TiO2 studies found increases in IL-1β and MMP-9 protein expression, as well as intracellular macrophage induction. Both studies also found, through pre-treatment of NADPH oxidase inhibitor, apocynin, resulted in attenuation of nanomaterial-exposure mediated ROS production; with nitric oxide synthase inhibitor, L-NNA, also showing attenuation, but only in our MWCNT-exposed inhalation study. The results from both studies have demonstrated, through different routes of administration, exposures, and rodent models; that exposure to nanomaterials can mediate signaling pathways involved in initiation and/or progression of CVD.

Fifty twelve-month female Sprague-Dawley rats were used to study a lysine requirement for tissue maintenance. Animals were randomly assigned into five groups with dietary lysine levels ranging from 0.097 to 0.317 percent and fed for 60 days. Liver composition and carcass composition were determined, and a lysine requirement was also predicted. Results showed that the group fed 0.317 percent lysine had significantly increased liver fat content and decreased protein content. Neither liver moisture content nor total liver nitrogen content was related to dietary lysine levels. There was no significant finding on the analysis of carcass composition. The data indicated that the mature rat had a requirement for lysine lower than 0.097 percent in the diet. It was suggested that either adequate lysine was provided by wheat gluten in the diet, or the mature rat did not require lysine in order to maintain tissue level of protein. In future studies, it was necessary to use a diet with lysine levels lower than 0.097 percent to determine the minimum lysine requirement, and a concurrent baseline group for comparison with the treatment animals. / M.S.