O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica. / This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results.

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke. / by Rebecca A. Crouch. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Serotonin syndrome (SS) is a drug-induced toxicity caused by an excess of serotonin (5-HT) in the central nervous system (CNS). The symptoms of the disorder range from mild to severe, with the severe state evoking life-threatening hyperthermia. Autonomic dysfunction is controlled in part by serotonin receptors, with the 5-HT2A receptor responsible for increasing core body temperature (Tcor). Our results show that the 5-HT2A receptors on the preoptic/anterior hypothalamus (PO/AH) and prefrontal cortex (PFC), in particular, are sensitive to changes in ambient temperature (Tamb). The toxic increase of 5-HT is postulated to occur due to the temperature-dependent activation of these receptors that promotes a positive feedback mechanism. Our results suggest that changes in Tamb can either exacerbate or alleviate the symptom and that this is mediated by the 5-HT2A receptors. Understanding the mechanism involved in elevating Tcor is imperative in treating and preventing the disorder. / by Swapna Krishnamoorthy. / Vita. / Thesis (M.S.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

Taurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study the neuromodulatory role of taurine in the CNS and to gain insights into its potential sites of action. A combination of techniques was used, including whole-cell patch clamp recording to study taurine's regulation of voltage-gated potassium (K+) and Ca2+ channels and Fluo-4AM Ca2+-imaging to study taurine's regulation of glutamate-induced [Ca2+] I,. Taurine was shown to suppress of glutamate-induced [Ca2+] l, in a dose dependent manner. This suppression was mostly sensitive to the glycine rece ptor antagonist Strychnine but insensitive to any GABA receptor antagonist. The remaining strychnine-insensitive effect was inhibited with the protein kinase A (PKA) inhibitor, PKI, suggesting that there was an additional metabotropic pathway. Moreover, using the protein kinase C (PKC) inhibitor, GF109203X, there was an enhancement in strychnine-insensitive taurine's regulation. Taurine inhibits voltage-gated Ca2+ channels in the retinal neurons and has a dual effect on voltage-gated K+ channels. Taurine causes an increase in K+ current amplitude which is further enhanced with PKI and blocked with GF109203X, suggesting that it is through a PKC-dependent pathway negatively controlled by PKA-dependent pathway. / There is a suppression of K+ current by taurine with intracellular application of GF109203X, suggesting that the reduction is through a PKA-dependent pathway. With both PKC and PKA inhibitors there is no longer an enhancement in maximum amplitude but a shift of volt dependence on a hyperpolarizing direction. Taurine's enhancement of K+ current is blocked by the Kv1.3 subtype antagonist Margatoxin, with Kv1.3 accounting for the majority of delayed-rectifier sustained current in bipolar and amacrine cells, as well as 50% of ganglion cells. Interestingly, the enhancement of K+ current by taurine is blocked by 5HT2A antagonist MDL11939, suggesting that activation of PKC is through this metabotropic serotonin receptor subtype. The suppression of voltage-gated Ca2+ channels is reversed with a combination of MDL11939 and the 5HT1A antagonist NAN-190. These results provide the evidence that the natural effect of taurine in the retinal neurons might be dependent on the activation of both 5HT1A and 5HT2A receptors. The high apparent activity of taurine on 5HT receptors could have important implication for the actions of taurine in central brain in which taurine has been known to be beneficial for improving mental health, as well as learning and memory processes. / by Simon Bulley. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Performance during prolonged exercise capacity diminishes with increasing temperatures. The onset of fatigue under these conditions is not adequately explained by peripheral mechanisms. Recently, drugs which inhibit the reuptake of dopamine and noradrenaline in the brain have been found to improve exercise performance in warm conditions. The aim of this thesis was to further explore and characterise the role of these neurotransmitters during prolonged exercise in warm conditions by manipulating their reuptake or synthesis. The first series of experiments were designed to further investigate the effects of bupropion, a dopamine and noradrenaline reuptake inhibitor, which has been found to improve performance in warm conditions. To explore gender differences in response to acute bupropion administration, the effects of bupropion on prolonged exercise performance in warm conditions in women was investigated in Chapter 3. The results of this study suggest that during the follicular phase of the menstrual cycle, acute administration of bupropion improves exercise performance. To determine whether there are any dose-dependent effects of bupropion, the experiment in Chapter 4 was designed to test three different doses of bupropion. Exercise performance was only improved for the maximal dose, suggesting a threshold for the performance effects of bupropion. Catecholamine precursors do not appear to improve exercise performance as consistently as reuptake inhibitors. In agreement with previous studies, the dopamine precursor L-DOPA did not affect exercise performance in warm conditions in Chapter 5. In Chapter 6 the effect of the atypical antidepressant nutritional supplement S-adenosylmethionine was investigated for its role in the synthesis of dopamine and noradrenaline. S-adenosylmethionine appeared to negatively influence cognitive function, increased skin temperature and circulating prolactin concentrations, but no effects on exercise performance were observed.

A raiva é uma doença do sistema nervoso central que é quase invariavelmente fatal. Apesar de causar cerca de 60.000 mortes/ano, a raiva ainda permanece uma doença negligenciada na maioria dos países, principalmente naqueles em desenvolvimento. O objetivo do nosso estudo foi verificar nos microambientes meningeal, perivascular e intraparenquimatoso do sistema nervoso central, o processo inflamatório, a resposta imune do hospedeiro e a morte dos neurônios frente à infecção rábica transmitida por morcegos. Verificamos que a raiva humana transmitida por morcegos é uma meningoencefalomielite. Através de reação imuno-histoquímica caracterizamos e quantificamos in situ a distribuição do antígeno viral, o fenótipo de células inflamatórias, as células expressando citocinas pró inflamatórias, citocinas representativas de padrão Th1 e Th2 e células em apoptose. O antígeno viral foi encontrado difusamente no parênquima cerebral, em maior abundância em neurônios, não diferindo sua distribuição em relação as regiões cerebrais. As células da glia, em especial os astrócitos, estavam imunomarcadas com o antígeno da raiva, assim como as células endoteliais e células mononucleadas da luz vascular. Esses achados contribuíram para a hipótese da ocorrência de uma via hematogênica alternativa de disseminação viral, através da infecção de células endoteliais pelo vírus, posterior infecção de astrócitos e finalmente infecção de neurônios. A expressão significativa de IL-12 nos pacientes rábicos provavelmente traduz imunidade de base preservada. Identificamos, outrossim, falta de resposta efetiva das células NK e comprometimento da resposta imune adaptativa seqüencial, demonstrada pela depleção de linfócitos TCD4+ no parênquima cerebral, prejuízo da atividade citotóxica dos linfócitos TCD8+ com proporcionalmente baixa expressão de granzima e expressão rarefeita de IFN-g e IL-2r. Essa situação deve ser reflexo da manipulação do vírus sobre a resposta imune inata e adaptativa do hospedeiro tendo como conseqüência uma reposta ineficaz para clareamento do vírus. A expressão aumentada de citocinas pró-inflamatórias (IL-1b, IL-6 e TNF-a) contribuiu para o dano neuronal. O padrão citocínico predominante no sistema nervoso central na raiva foi o Th2, com alta expressão de IL-4 e IL-10. O TGF-b aumentado nos casos de raiva favorece um ambiente imunossupressor para manter intacta a rede neuronal, mas também contribui para permanência local do vírus. A verificação de grau pouco acentuado de neurônios apoptóticos em contraposição a apoptose expressiva de linfócitos TCD4+ e TCD8+, indicaria a utilização pelo vírus de mecanismos ora anti-apoptóticos para preservar neurônios e favorecer a disseminação viral, ora pró-apoptóticos, destruindo linfócitos e atenuando o processo inflamatório. Em síntese, o envolvimento do sistema nervoso central em decorrência da raiva se faz às custas de processo inflamatório com predomínio local de linfócitos TCD8+, um padrão Th2 de expressão de citocinas, acometendo os microambientes meningeal, perivascular e intraparenquimatoso, sendo o bulbo a região mais afetada pelo processo. / Viral disease of central nervous system almost invariable fatal, rabies causes about 60.000 deaths yearly, and still remain a neglected disease in most countries, specially in developing ones. We study the meningeal, perivascular and parenquimal environment in central nervous system from patients who dies after bat transmitted rabies, looking for the inflammatory process, host immune response and neuronal death. We show that human rabies transmitted by bats is a meningoencephalomyelitis. Immunohistochemistry allows the in situ quantification of viral antigen distribution, inflammatory cellular phenotype, expression of cytokines representing both Th1 and Th2 profile and pro-inflammatory and cell apoptosis. Viral antigen was found disseminatted in cerebral parenquima, more abundant in neurons, without cerebral area preference. Glial cells, specially astrocytes, were immunostained with rabies antigen, as well as endothelial and vascular mononuclear cells. These findings contributed for an alternative hypothesis of the occurrence of hematogenic viral dissemination, through viral infection or passage by endothelial cells, followed by astrocyte infection, and finally reaching neurons. IL-12 significant expression in central nervous system from rabies patients probably reflect preservation of immunity. The lack of effective NK cells could compromise adaptive immune response, demonstred by TCD4+ lymphocytes depletion in cerebral parenquima, ineffective TCD8+ cytotoxic activity with low granzyme expression and low expression of IFN-g and IL-2r. This situation reflects viral effect on host innate and adaptive immune response leading to an ineffective response for viral clearance. High pro-inflammatory cytokine expression, IL- 1b, IL-6 and TNF-a, contribute for neuronal damage, with predominant Th2 cytokine profile in central nervous system in rabies patients, with high expression of IL-4 and IL-10. Increased TGF-b expression also shows an immune suppressor environment, which maintains the neuronal network intact, but also contributes for viral permanence. The low degree of apoptotic neurons opposed with expressive apoptosis of TCD4+ and TCD8+ lymphocytes, could indicate viral mechanisms anti-apoptotic for neurons preservation and favour viral dissemination, or pro-apoptotic, destroying lymphocytes and attenuating the inflammatory process. In synthesis, the involvement of central nervous system in rabies is consequence of inflammatory process with TCD8+ lymphocytes predominance, Th2 profile cytokines expression, affecting all brain compartments and areas, especially the medulla oblongata.

O presente trabalho compreende o estudo da resposta imune e inflamatória no sistema nervoso central (SNC) de camundongos ou ratos-veadeiros (Peromyscus maniculatus) frente a infecção por dois diferentes tipos de vírus neurotrópicos, o vírus da estomatite vesicular (VEV) e o herpesvírus bovino tipo 5 (BoHV-5). Na primeira etapa, foi avaliada a função das células residentes do SNC no tocante à expressão de quimiocinas durante a infecção pelo VEV em ratos-veadeiros. No presente estudo, durante a encefalite causada pelo vírus da estomatite vesicular sorotipo New Jersey (VEVNJ) em ratos-veadeiros, as quimiocinas RANTES e MCP-1 foram expressas somente no bulbo olfatório (BO), local onde o vírus estava restrito. A expressão de quimiocinas foi seguida de um influxo de células inflamatórias no BO tardiamente no curso da doença aguda. A quimiocina RANTES foi expressa por neurônios, astrócitos e micróglia, ao passo que MCP-1 foi expressa por neurônios e astrócitos. Embora os astrócitos e a micróglia tenham respondido à infecção pelo VEVNJ ao expressarem quimiocinas, os neurônios foram o principal tipo celular infectado pelo vírus. Portanto, os neurônios infectados podem exercer um papel crucial na geração da resposta imune no BO. A sinalização entre neurônios e outras células residentes do SNC muito provavelmente é o mecanismo pelo qual os astrócitos e micróglia são ativados durante a encefalite causada pelo VEVNJ. Os resultados aqui apresentados, também indicam que a expressão de RANTES e MCP-1 no BO de ratos-veadeiros infectados pelo VEVNJ pode ajudar a prevenir a disseminação do vírus para outras áreas do SNC. Na segunda etapa, foi avaliada a susceptibilidade de camundongos isogênicos BALB/c frente à infecção pelo BoHV-5 em camundongos isogênicos BALB/c em diferentes dias pós-inoculação (DPI). O BoHV-5 quando inoculado pela via intracraniana foi capaz de infectar e se replicar no SNC de camundongos BALB/c. Entretanto, até o momento avaliado (15 DPI), os animais sobreviveram a infecção sem apresentar sinais neurológicos evidentes. A infecção foi acompanhada de uma resposta imune do tipo Th1 importante, com expressão significativa das citocinas IFN-Y e TNF-α, e quimiocina CCL-2. A expressão das citocinas e quimiocinas se deu principalmente no início da infecção (3 e 4 DPI), a qual foi seguida por uma meningo-encefalite com manguitos perivasculares e periventriculite, compostas predominantemente por macrófagos e linfócitos. Após a expressão significativa das citocinas e quimiocina, os animais foram capazes de debelar a infecção aguda, uma vez que partículas virais viáveis não foram detectadas após o 6 DPI. Entretanto, o BoHV-5 foi capaz de infectar o gânglio trigeminal, uma vez que grande quantidade de DNA de BoHV-5 foi detectada no 3 DPI, o que foi confirmado pela presença de antígenos virais no citoplasma de neurônios do gânglio trigeminal de camundongos BALB/c infectados / The present work comprises the study of immune and inflammatory responses in the central nervous system (CNS) of mice or deer mice (Peromyscus maniculatus) during infection by two different neurotropic viruses, the vesicular stomatitis virus (VSV) and bovine herpesvirus type 5 (BoHV-5). In the first part, the role of CNS resident cells regarding chemokine expression in deer mice infected with VSV was evaluated. Here, we demonstrated that during vesicular stomatitis New Jersey virus (VSNJV) encephalitis in deer mice, chemokines RANTES and MCP-1 are expressed only in the olfactory bulb (OB), where the virus was restricted. This chemokine expression was followed by the influx of inflammatory cells to the OB later in the course of acute disease. Neurons, astrocytes and microglia expressed RANTES, whereas MCP-1 was expressed by neurons and astrocytes. Although astrocytes and microglia responded to VSNJV infection by expressing chemokines, neurons were the predominantly infected cell type. Therefore, infected neurons may have a critical role in initiating an immune response in the OB. The signaling between neurons and other CNS resident cells is most likely the mechanism by which astrocytes and microglia are activated during the course of VSV encephalitis. Our results also indicate that the expression of RANTES and MCP-1 in the OB of deer mice infected with VSNJV might help prevent the spread of VSNJV to other areas of CNS. In the second part of the study, the susceptibility of isogenic BALB/c mice to BoHV-5 infection was evaluated in different days post-inoculation (DPI). BoHV-5, when inoculated through intracranial route, was able to infect and replicate within the CNS of BALB/c mice. However, until the evaluated time (15 DPI), the mice was able to survive without showing prominent neurological signs. The infection was accompanied by an important Th1 immune response, with a significant expression of the cytokines IFN-Y and TNF-α, and chemokine CCL-2. The expression of these cytokines and chemokines was detected mainly on the early course of infection (3 and 4 DPI), and was followed by a meningoencephalitis with perivascular cuffing and periventriculitis, composed mainly by macrophages and lymphocytes. After the expression of cytokines and chemokine, the mice were able to curb BoHV-5 acute infection, since viable viral particles were not detected after 6 DPI. However, BoHV-5 was able to infect the trigeminal ganglia, since a large number of BoHV-5 DNA copies was detected on 3 DPI, which was confirmed by the presence of viral antigens within the cytoplasm of neurons in the trigeminal ganglia of infected BALB/c mice

A migrânea e a cefaléia tensional são cefaléias primárias que surgem de estruturas não-mastigatórias, porém, a presença de sintomas de DTM, como a dor, pode influenciar de modo excitatório tais condições e vice-versa, influenciando no resultado final do tratamento. Esta pesquisa tem o objetivo principal de avaliar o impacto da presença de cefaléias primárias no tratamento das Disfunções Temporomandibulares (DTMs), e testa a hipótese nula de que a presença de cefaléias primárias não interfere com o resultado do tratamento. Como objetivos secundários, de avaliar se existe diferença na presença de dor miofascial nos músculos mastigatórios e cervicais, se existe diferença entre a variação da dor medida pela Escala Analógica Visual (EAV) em relação ao gênero, estresse e hábitos parafuncionais, e se essa diferença também se apresenta entra as variáveis oclusão, tempo de dor, número de queixas e número de tratamentos indicados. Para isso foram selecionados 546 prontuários clínicos de pacientes, sendo 313 com DTM e 233 com DTM e cefaléias, e analisados segundo a EAV ao início e fim do tratamento para DTM, bem como a variação entre a dor inicial e final entre os grupos. Testes de Mann-Whitney, Correlação de Spearman e Qui-quadrado analisaram os dados, com 5% de significância. A presença de cefaléias primárias interferiu negativamente no índice de sucesso do tratamento da DTM (p<0,05) (redução de 38,70 e de 24,66 na EAV para os grupos de DTM e DTM associada a cefaleia, respectivamente). A presença de dor miofascial nas musculaturas mastigatória e cervical foi semelhante entre os grupos. A variação entre a dor inicial e final não foi afetada pela diferença entre os gêneros, assim como pelo auto-relato da presença de hábitos parafuncionais e de estresse. Da mesma forma, a presença de má-oclusão, o tempo de experiência de dor, o número de queixas relatadas e o número de tratamentos indicados pelo profissional não influenciaram os resultados finais. Conclui-se que presença de cefaléias primárias parece interferir negativamente na melhora do quadro sintomático de pacientes tratados para DTM. / Migraine and tension-type headaches are primary headaches that arise from non-masticatory structures, however, the presence of TMD symptoms, like pain, may have a excitatory effect in these conditions and vice versa, influencing the outcome of treatment. This research has the main objective of evaluating the impact of the presence of primary headache in the treatment of Temoromandibulares Disorders (TMD), and tests the null hypothesis that the presence of primary headache does not interfere with treatment outcome. As secondary objectives, to evaluate whether there are differences in the presence of myofascial pain in the masticatory and cervical muscles, if there is a difference between the change in pain measured by visual analog scale (VAS) in relation to gender, stress, and parafunctional habits. The influence of malocclusion, duration of pain, number of complaints and number of treatments given were also evaluated. For this reason, 546 medical records of patients, 313 and 233 with TMD TMD and headaches were selected, and analyzed using a VAS at the beginning and end of treatment for TMD, as well as the variation between the initial and final pain between the groups. Mann-Whitney, Spearman correlation and chi-square test analyzed the data with 5% significance level. The presence of primary headaches interfered negatively with the rate of successful treatment of TMD (p <0,05) (reduction of 38.70 and 24,66 in the VAS for groups of TMD and headache associated with TMD, respectively). The presence of myofascial pain in the masticatory and cervical muscles was similar between groups. The variation between the initial and final pain was not affected by gender differences, as well as by self-report the presence of parafunctional habits and stress. Likewise, the presence of malocclusion, time of pain experience, the number of complaints reported and the number of treatments given by the professional did not influence the final results. It is concluded that the presence of primary headache seems to have a negative effect on symptomatic improvement in patients treated for TMD.

No description available.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.