La cryptococcose chez les patients atteints du VIH-1 est principalement causée par Cryptococcus neoformans var. grubii tandis que Cryptococcus gattii infecte surtout les personnes immunocompétentes. Afin d’élucider les mécanismes causant la susceptibilité différentielle à l’égard de ces deux espèces de Cryptococcus dans le contexte de l’infection au VIH-1, nous avons utilisé un modèle novateur de la cryptococcose chez la souris transgénique CD4C/HIVMutA, qui exprime les gènes nef, env et rev du VIH-1. L’expression du transgène VIH-1 a augmenté le recrutement pulmonaire des macrophages alvéolaires mais a diminué celui des lymphocytes T CD4+ et CD8+ en réponse à l’infection par le C. neoformans ou le C. gattii. La production pulmonaire des chimiokines MCP-1 (CCL2) et RANTES (CCL5) était également réduite chez les souris transgéniques infectées par l’une ou l’autre de ces espèces de Cryptococcus. La production pulmonaire de MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 et IL-13 était augmentée chez la souris infectée au C. neoformans comparativement à C. gattii. In vitro, les macrophages alvéolaires prélevés chez la souris Tg et stimulés par des agonistes ont produit davantage de MIP-1β, alors que les chimiokines MCP-1 et RANTES n’ont pas été détectées. / The most common cause of cryptococcosis in HIV-1-infected patients is Cryptococcus neoformans var. grubii, while Cryptococcus gattii usually infects immunocompetent people. We used a novel inhalation model of cryptococcosis in CD4C/HIVMutA transgenic mice expressing nef, env, and rev of HIV-1 to examine the mechanisms that cause differential susceptibility to these species of Cryptococcus in the context of HIV-1 infection. HIV-1 transgene expression increased alveolar macrophage but decreased pulmonary CD4+ and CD8+ T lymphocyte recruitment. Pulmonary production of the CC chemokines MCP-1 (CCL2) and RANTES (CCL5) was reduced in transgenic mice infected with C. neoformans or C. gattii, and concentrations were lower after infection with C. gattii compared to C. neoformans. Production of MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 and IL-13 was increased in mice infected with C. neoformans compared to C. gattii. Production of MIP-1β by alveolar macrophages harvested from Tg mice was enhanced after agonist exposure in vitro, but production of the chemokines MCP-1 and RANTES was undetectable.

Les ganglions de la base (GB) sont un ensemble de structures sous-corticales interconnectées impliquées dans l'apprentissage et le contrôle moteur mais aussi dans des processus motivationnels. Le fonctionnement des GB est fortement dépendant de l'équilibre d'activité entre les voies directe (striatonigrale) et indirecte (striatopallidale) par lesquelles le striatum, la principale structure d'entrée du réseau, contrôle les structures de sortie. L'objectif de ce travail était de développer et caractériser un modèle d'ablation sélective des neurones de la voie directe pour appréhender leur rôle dans les comportements impliquant les GB. Ce modèle repose sur l'expression, par transgénèse additive, du récepteur humain à la toxine diphtérique (DT) couplé à la GFP sous le contrôle du promoteur du gène slc35d3 exprimé dans les neurones striatonigraux et pas dans les striatopallidaux. La caractérisation cellulaire a été réalisée 15 jours après injection unilatérale de DT dans le striatum dorsal. La spécificité de l'atteinte est vérifiée par la diminution sélective (70%) de l'expression génique du précurseur de la substance P, marqueur de la voie directe, sans changement de celle du précurseur des enképhalines, marqueur de la voie indirecte. Les populations d'interneurones sont préservées à l'exception des interneurones cholinergiques dont le nombre est réduit de 50%. Un faisceau d'arguments démontre que cette baisse ne serait pas due à un effet direct de la DT sur les interneurones cholinergiques mais serait secondaire à la perte des neurones striatonigraux, mettant en évidence un lien étroit entre ces deux populations. / The basal ganglia (BG) are a set of subcortical structures implicated in motor learning and motor function as well as in motivational processes. BG functioning is thought to be highly dependent on the balanced activity between the direct (striatonigral) and indirect (striatopallidal) pathways by which the striatum, the main input station of the network, controls the output structures. This study aimed at developing and characterizing a model of selective ablation of the direct pathway to decipher its specific role in BG-related functions and disorders. The promoter of the slc35d3 gene, which is enriched in the striatonigral neurons, has been used to drive expression of the human diphtheria toxin (DT) receptor coupled to GFP selectively in these neurons by additive transgenesis. The cellular characterization has been performed 15 days after unilateral DT injection in the dorsal striatum. The ablation specificity is demonstrated by the selective decrease (70%) in substance P precursor mRNA levels, a marker of the direct pathway, with no change in enkephalin precursor gene expression, a marker of the indirect pathway. Striatal interneuron populations are spared, except the cholinergic population, which is reduced by about 50%. Evidence is provided that this loss may not be a direct effect of DT but a consequence of striatonigral neuron loss, revealing their crucial role for cholinergic interneuron viability. Then, we analyzed the functional consequences of the bilateral lesion of the striatonigral pathway (50-60% neuronal loss) either in the dorsal striatum or in the nucleus accumbens (NAc).

A faringite estreptocócica desencadeada pelo Streptococcus pyogenes pode resultar em uma série de doenças humanas e complicações como a febre reumática (FR) em indivíduos predispostos não tratados. A FR é uma doença autoimune que afeta mais de 20 milhões de crianças em países em desenvolvimento. A proteína M presente na membrana do S. pyogenes representa o maior fator de virulência da bactéria, e é objetivo de estudos para o desenvolvimento de uma vacina contra essa patologia. Atualmente mais de 200 tipos de proteínas M foram descritos na literatura e a sua porção Cterminal é conservada entre os diferentes tipos. Desenvolvemos um protótipo de vacina que compreende 55 resíduos de aminoácido da porção C-terminal, denominado StreptInCor. Neste trabalho analisamos a resposta humoral e celular específica contra o peptídeo sintético StreptInCor, usando camundongos transgênicos portadores de HLA de classe II humanos DR2, DR4, DQ6 e DQ8. O protocolo de imunização consistiu em administrar 50 g do StreptInCor adsorvido em 300 g de hidróxido de alumínio nos dias 0 e 14. Os grupos controles foram injetados com salina nas mesmas condições. O soro obtido no 28º dia foi testado por ensaio imunoenzimático (ELISA) para verificarmos a presença de anticorpos contra o StreptInCor e os esplenócitos destes animais, obtidos nessa data, foram utilizados para ensaios de proliferação celular na presença do StreptInCor. Testes de segurança foram efetuados e não observamos reação cruzada contra a miosina cardíaca e após 12 meses de acompanhamento, amostras de tecidos desses animais foram submetidas à análise histológica. Em conclusão não verificamos indícios de reações autoimunes nos animais imunizados com o StreptInCor e os resultados obtidos mostram a capacidade do StreptInCor em desencadear uma resposta imune, duradoura e segura em camundongos portadores de moléculas HLA de classe II / Streptococcal pharyngitis triggered by Streptococcus pyogenes throat infection can result in rheumatic fever (RF) and rheumatic heart disease (RHD) in untreated susceptible individuals. RF is an autoimmune disease that affects more than 20 million children in developing countries. M protein is the major factor of virulence of the bacteria, and it has been studied to develop a vaccine. Currently more than 200 M protein types have been described and its Cterminal domain is conserved in many different serotypes. We developed a vaccine epitope (StreptInCor) composed by 55 amino acid residues of the Cterminal portion of the M protein. In the present work we analyze the ability of the StreptInCor of induce immune response in HLA class II transgenic mice. The transgenic mice harboring the HLA Class II DR2, DR4, DQ6 and DQ8 were immunized subcutaneously with 50 g StreptInCor adsorbed onto 300 g of aluminum hydroxide gel on days 0 and 14. Control groups were immunized with vehicle (Saline) in same conditions. The sera were obtained on day 28 and tested by ELISA to verify the presence of antibodies. The specific cellular immune response was evaluated by proliferation assay using splenocytes. No cross reaction with cardiac myosin were observed. Tissue samples from immunized mice followed by 12 months were analyzed in order to verify if StreptInCor induces some histological damage. No autoimmune or deleterious reactions were observed. In conclusion our results indicate that StreptInCor Induces a good and prolonged and safe immune response in HLA class II transgenic mice

Myosin II, das Motorprotein des kardialen Muskels, besteht aus zwei schweren und vier leichten Ketten. Der Hebelarmbereich der schweren Myosinkette (MyHC) enthält das IQ-Konsensus-Motiv für die Bindung der essentiellen leichten Myosinkette (ELC), welche wesentlich für eine normale Kraftentwicklung des Myosinmoleküls ist. Im Rahmen dieser Arbeit wurden fünf, mit hypertropher Kardiomyopathie assoziierte, Mutationen im humanen essentiellen ventrikulären leichten Myosinketten (hVLC1)-Gen (MYL3) untersucht (E56G, A57G, E143K, M149V, R154H). Von keiner dieser Mutationen war der Pathomechanismus bekannt. Ziel der Arbeit war es, die Effekte der Mutationen im MYL3-Gen auf Proteinstruktur und Funktion zu untersuchen und daraufhin einen möglichen Pathomechanismus zu formulieren. Dazu erfolgten Strukturanalysen (CD-Spektren, Schmelzkurven, FLIM), Versuche auf Protein- und Zellebene (Protein-Protein-Interaktionsstudien, Sorting Assay) sowie Untersuchungen in vitro (Zell-Verkürzungsmessungen, isoliert perfundierte Herzen nach Langendorff) und in vivo (Echokardiographie) im transgenen Mausmodell. / Myosin II, the motor protein of cardiac muscle, is composed of two heavy chains (MyHC) and four non-covalently linked light chains (MLC). The lever arm of the MyHC contains the IQ motif that binds the essential myosin light chain (ELC), which is necessary for the normal force production of the myosin molecule. Five with HCM associated mutations in the human ventricular essential myosin light chain (hVLC1) -gen (MYL3) were investigated in this study (E56G, A57G, E143K, M149V, R154H). The pathomechanisms of the mutations were not known. Aim of the study was i) to test the hypothesis that mutations in the ventricular essential myosin light chain affect the protein structure, the binding to the IQ motif of MyHC and the force production of the myosin molecule as well as ii) to postulate an accompanying pathomechanism. Structural analyses (circular dichroism, melting curves, fluorescence lifetime imaging microscopy), functional investigations (surface plasmon resonance spectroscopy, sorting assay) and in vivo (echocardiography) and in vitro studies in a transgenic mouse model were performed.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-13T17:59:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CRISTIANO SPIAZZI.pdf: 6934724 bytes, checksum: 23cec5179331e6652ba17452be7c25eb (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-13T17:59:34Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CRISTIANO SPIAZZI.pdf: 6934724 bytes, checksum: 23cec5179331e6652ba17452be7c25eb (MD5) / Made available in DSpace on 2017-06-13T17:59:34Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) CRISTIANO SPIAZZI.pdf: 6934724 bytes, checksum: 23cec5179331e6652ba17452be7c25eb (MD5) Previous issue date: 2017-03-11 / A população mundial está envelhecendo e doenças neurodegenerativas, tais como a Doença de Alzheimer (DA), estão se tornando cada vez mais comuns. Novas formas de retardar o progresso da doença vem sendo estudadas, como a utilização de substâncias com propriedades antioxidantes e anti-inflamatórias, tais como compostos de selênio. O objetivo desse estudo foi avaliar o efeito do selenofuranosídeo, um composto orgânico de selênio sintético, em dois modelos animais de demência esporádica tipo-Alzheimer. Primero experimento: Uma única administração do peptídeo Aβ (fragmento 25-35; 3 nmol/3 μL) ou água destilada foi injetada via intracerebroventricular (i.c.v.) em camundongos machos. O selenofuranosídeo (5 mg/kg SE) ou veículo (óleo de canola, VEH) foi administrado por via oral 30 min antes da Aβ e durante os 7 dias subsequentes. A memória foi testada por meio do labirinto aquático de Morris (MWM) e esquiva passiva de descida (SDPA). O estresse oxidativo foi analisado através das atividades das enzimas SOD, CAT, GPx, GR e GST, bem como os níveis de espécies reativas (RS) e GSH. Os níveis de citocinas inflamatórias foram avaliados para medir o processo inflamatório, e a atividade da AChE para estimar o dano sináptico. No grupo Aβ houve redução significativa da atividade da SOD e aumento da atividade da AChE, níveis de RS, de IL-6 e GSH. Observou-se uma redução na latência de decida no SDPA, e aumento na latência para encontrar a plataforma no MWM. O SE foi capaz de proteger contra perda de memória em camundongos, provavelmente através da modulação da atividade da AChE. O composto foi capaz de proteger contra a redução da atividade da SOD, reduzindo os níveis de RS. E apesar do grupo SE mostrar aumento nos níveis de IL-6, o grupo Aβ+SE apresentou níveis iguais aos do controle. Segundo experimento: camundongos C57BL/6J de ambos sexos do tipo selvagem (WT) e transgênicos para os genes APP/PS1 (TG) foram utilizados. O SE (10 mg/kg) ou VEH foram administrados oralmente, duas vezes por semana, durante 5 meses. Testes comportamentais incluindo MWM, labirinto em Y, labirinto em cruz elevada (EPM), e o teste de preferência social/novidade social (SPSN), foram realizados para avaliar a memória e comportamento tipo ansiedade. Ambos genótipos tiveram performance semelhante no labirinto em Y, EPM e SPSN. Na fase de aquisição do MWM, foi visto efeito do genótipo nos dias 7, 9 e 10, onde animais TG apresentaram aumento da distância percorrida e latência para encontrar a plataforma. Animais WT passaram mais tempo no quadrante alvo do MWM durante segundo teste sem plataforma. Secções coronais cerebrais foram avaliadas para GFAP, Aβ e depósitos de placas amiloides através de imuno-histoquímica. SE foi capaz de reduzir significativamente a produção de GFAP e depósitos de Aβ em camundongos TG, mas isso não foi observado nos testes comportamentais. No entanto, o fato do selenofuranosídeo ser capaz de reduzir depósitos de Aβ em camundongos APP/PS1 e proteger contra várias alterações observadas com a administração do fragmento Aβ é promissor para estudos futuros. / World population is increasingly older and neurodegenerative diseases, such as Alzheimer’s disease (AD), are becoming more common. New ways to retard AD progress are being studied, such as substances with antioxidant and anti-inflammatory properties, like organoselenium compounds. The aim of this study was to evaluate the effect of selenofuranoside, a synthetic organoselenium compound, in two Alzheimer-like sporadic dementia animal model. First experiment – Single administration of Aβ peptide (fragment 25–35; 3 nmol/3 μL) or distilled water were administered via intracerebroventricular (i.c.v.) in male mice. Selenofuranoside (5 mg/kg, SE) or vehicle (canola oil, VEH) was administered via oral 30 min before Aβ and for 7 subsequent days. Memory was tested through Morris water maze (MWM) and step-down passive avoidance (SDPA) tests. Oxidative stress balance was assessed via SOD, CAT, GPx, GR and GST activities along with reactive species (RS) and GSH levels. Inflammatory cytokines levels were measured to assess inflammation progress, and AChE activity to estimate synaptic status. Aβ group presented a significant decrease in SOD activity and an increase in AChE activity, RS levels, IL-6 and GSH. A reduction in step-down latency in SDPA, and also latency to reach the platform former location in MWM was observed. SE was able to protect against memory loss in mice, probably due to AChE activity modulation. The compound was also able to protect against SOD activity reduction, reducing RS levels. Although an increase in IL-6 was observed in SE group, Aβ+SE showed the same levels of control group. Second experiment – male and female wild-type (WT) mice (C57BL/6J) and APP/PS1 transgenic (TG) mice were used. SE (10 mg/kg, SE) or VEH were administered via oral twice a week, for 5 month-period. Behavioural tests including Morris water maze (MWM), Y-Maze, elevated plus-maze (EPM), Social Preference/Social Novelty test (SPSN), were performed to assess memory and anxiety-like behavior. Both genotypes performed alike at Y-maze, EPM and SPSN tests. At MWM acquisition phase, a main-effect of genotype was observed at days 7, 9 and 10, where TG mice presented an increase in path length and latency to find the platform. Also, WT mice spent more time in the target quadrant during the second probe of MWM. Immunohistochemical analysis was done in coronal brain sections to evaluate Aβ deposits, GFAP production and amyloid plaques. SE treatment significantly decreased GFAP production and Aβ1-16 deposits at TG mice, but this was not reflected at the behavioral tests. Nonetheless, the fact that selenofuranoside was able to reduce Aβ1-16 deposits in APP/PS1 mice and protected against various alterations exhibited by Aβ fragment is promising for further studies.

Ce travail porte sur l’étude fonctionnelle de deux gènes préférentiellement exprimés dans deux régions du cerveau touchées par des pathologies neurodégénératives : Capucine, un marqueur du striatum, structure qui dégénère au cours de la maladie de Huntington et Agpat4, un marqueur de l’aire tegmentaire ventrale et de la substance noire compacte, dont les neurones dopaminergiques sont sélectivement atteints lors de la maladie de Parkinson. Des lignées de souris invalidées pour ces gènes ont été générées au laboratoire et au cours de ma thèse j’ai procédé à leur caractérisation. L’expression striatale du gène de la Capucine étant significativement diminuée dans des modèles murins de la maladie de Huntington, nous avons souhaité évaluer son rôle éventuel dans la pathogenèse de cette maladie. Pour ce faire, nous avons examiné, dans le cadre d’une collaboration, l’effet du knock-out et de la surexpression du gène de la Capucine sur la vulnérabilité des neurones striataux à un fragment de la Huntingtine mutée dans un modèle murin de la maladie de Huntington. Les données montrent que la Capucine n’a pas d’effet significatif sur la toxicité du fragment de la Huntingtine mutée dans le modèle étudié.La protéine Agpat4 présente des homologies de séquence avec des acyltransférases impliquées dans le métabolisme des phosphoglycérides. J’ai réalisé des études d’expression par différentes techniques de biologie moléculaire qui montrent que le gène d’Agpat4 est exprimé dans la plupart des tissus catécholaminergiques. Pour déterminer l’activité endogène d’Agpat4 et son rôle physiologique dans les tissus où elle est exprimée, j’ai comparé le métabolome de tissus de souris invalidées pour le gène d’Agpat4 et sauvages par chromatographie en phase liquide couplée à la spectrométrie de masse. Mes résultats indiquent que l’invalidation du gène d’Agpat4 perturbe le métabolisme non seulement de différentes classes de lipides, notamment les lysophosphatidyléthanolamines, mais aussi celui des catécholamines. / This work concerns the functional study of two genes preferentially expressed in two brain regions affected by neurodegenerative diseases: Capucine, a marker of the striatum, a structure that degenerates in Huntington's disease and Agpat4, a marker of the ventral tegmental area and the substantia nigra pars compacta, whose dopaminergic neurons are selectively affected in Parkinson's disease. Mouse lines deficient for Capucine and Agpat4 have been generated in the laboratory and during my PhD thesis I carried out their characterization.As the striatal gene expression of Capucine is significantly reduced in mouse models of Huntington's disease, we wished to evaluate its possible role in the pathogenesis of this disease. In a collaborative work, we examined the effect of the knockout and overexpression of the Capucine gene on the vulnerability of striatal neurons to a mutant Huntingtin fragment in a mouse model of Huntington’s disease. The data show that Capucine has no significant effect on the toxicity of the mutant Huntingtin fragment in the considered model.The Agpat4 protein has sequence homologies with acyltransferases involved in the metabolism of phosphoglycerides. I conducted expression studies using different molecular biology techniques, which showed that the Agpat4 gene is expressed in most catecholaminergic tissues. To determine the endogenous activity of Agpat4 and its physiological role in the tissues where it is expressed, I compared the metabolomes of Agpat4-deficient and wild-type mice tissues by liquid chromatography coupled with mass spectrometry. My results indicate that Agpat4 deficiency alters not only the metabolism of different lipid classes, in particular lysophosphatidylethanolamines, but also the metabolism of catecholamines.

The eye contains closely related but widely different tissues, offering a unique opportunity to investigate the phenotype and function of monocyte-derived cell populations within functionally unique microenvironments in a single complex organ. The uveal tract and retina contain rich networks of immune cells that reside and traffic through the eye, these cells having been implicated in various ocular inflammatory processes and immune-mediated diseases. One such inflammatory condition is human posterior uveitis, an autoimmune disease mainly affecting the retina. As current treatments for posterior uveitis only serve to slow down disease progression, studies using animal models, namely, experimental autoimmune uveoretinitis (EAU), have focused on determining the key cellular and molecular mediators involved in disease initiation in order to expand the potential for novel therapeutic applications. The overall purpose of experiments in this thesis was to explore monocyte-derived cell populations of the uveal tract and retina, this being achieved by utilising a novel transgenic mouse model. Cx3cr1gfp/gfp transgenic mice on both BALB/c and C57Bl/6 backgrounds contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene, disrupting its expression but facilitating GFP expression under the control of the Cx3cr1 promoter. Heterozygous (Cx3cr1+/gfp) mice were generated by crossing Cx3cr1gfp/gfp mice to wild-type (WT) mice. This transgenic model allowed for the exquisite visualisation of Cx3cr1-bearing monocyte-derived dendritic cells (DC) and macrophages in ocular tissues, whilst also enabling the investigation of a potential role for Cx3cr1 in recruiting monocyte-derived cells to the eye in steady-state and inflammatory conditions.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

For many years, it has been known that Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer’s disease (AD). It has been commonly assumed that RA patients’ usage of non-steroidal anti-inflammatory drugs (NSAIDs) have helped prevent the onset and progression of AD pathogenesis. Furthermore, experiments in animal models of Alzheimer’s disease have looked to inhibit inflammation, and have demonstrated some efficacy against AD-like pathology in these models. Thus many NSAID clinical trials have been performed over the years, but all have proven unsuccessful in AD patients. This suggests that intrinsic factors within RA pathogenesis itself may underlie RA’s protective effect. My dissertation research goal was to investigate this inverse relationship between RA and AD, in order to more precisely pinpoint critical events in AD pathogenesis toward developing therapeutic strategies against AD. It seemed improbable that any secreted factors, produced in RA pathogenesis, could maintain high enough concentrations in the circulatory system to cross the blood brain barrier and inhibit AD pathogenesis, without affecting all other organ systems. It did seem possible that the leukocyte populations induced in RA, could traverse the circulatory system, extravasate into the brain parenchyma, and impede or reverse AD pathogenesis. We thus investigated the colony-stimulating factors, which are up-regulated in RA and which induce most of RA’s leukocytosis, on the pathology and behavior of transgenic AD mice. We found that G-CSF and more significantly, GM-CSF, reduced amyloidosis throughout the treated brain hemisphere one week following bolus intrahippocampal administration into AD mice. We then found that 20 days of subcutaneous injections of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) significantly reduced brain amyloidosis and completely reversed cognitive impairment in aged cognitively-impaired AD mice, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, the recombinant human GM-CSF analogue, in elderly leukopenic patients, suggested that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD patients. It was also implied that age-related depressed hematopoiesis may contribute to AD pathogenesis.