Os escorpiões são um dos grupos de animais mais antigos da Terra. Eles são artrópodes e pertecem a classe Arachinida e Ordem Scorpionida. A família Buthidae compreende as espécies responsáveis pelos acidentes graves em humanos, incluindo a espécie Tityus serrulatus, o maior responsável por esses acidentes no Brasil. A peçonha do T. serrulatus contém diversas neurotoxinas que agem especificamente em canais para sódio, potássio e cálcio da membrana plasmática de células excitáveis, causando massiva liberação de neurotransmissores.As toxinas escorpiônicas podem ser usadas como ferramentas nos estudos de estrutura e função desses canais iônicos sensíveis a voltagem e também no estudo de liberação e captação de neurotransmissores. As toxinas escôrpionicas específicas para canais para sódio sensíveis a voltagem são as principais responsáveis pelos efeitos do envenenamento por estes artrópodes e podem ser classificadas em duas classes: toxinas e . As -toxinas retardam a inativação desses canais induzindo assim um prolongamento na fase de repolarização do potencial de ação. As - toxinas alteram a dependência de voltagem de ativação dos canais para sódio para potenciais mais negativos provocando potenciais de ação espontâneos e repetitivos. As toxinas específicas para canais para potássio (KTx) são geralmente peptídeos pequenos e de caráter básico, formados por 23-43 aminoácidos estabilizados por 3-4 pontes dissulfeto. As KTx são classificadas em 4 subfamílias:, , , . Neste trabalho, uma nova neurotoxina do escorpião T. serrulatus foi isolada e caracterizada bioquímica e funcionalmente. A toxina foi testada em ampla variedade de canais incluindo 5 subtipos de canais para sódio (Nav1.4; Nav1.5; Nav1.6; Nav1.8 e DmNav1) e 12 diferentes tipos de canais para potássio (Kv1.1 a Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR e hERG). A peçonha bruta solúvel foi fracionada em cromatografia de troca iônica em coluna CM-Celulose-52 (2,5 cm x 63 cm), previamente equilibrada e eluída com tampão NH4HCO3 (pH 7,8). Essa primeira etapa cromatográfica permitiu a separação de 13 frações nomeadas de I XIII. A fração X foi submetida à cromatografia de fase reversa em sistema de cromatografia líquida de alta eficiência em que a toxina pura Ts15 pode ser obtida. Seu sequenciamento amino-terminal demonstrou que esse peptídeo possui 36 resíduos de aminoácidos estabilizados por 3 pontes dissulfeto. A massa molecular obtida por espectrometria de massa foi de 3956 e o pI predito pelo programa ProtParam foi de 8,86, no entanto, o pI determinado por focalização isolelétrica foi maior que 9,3. Os experimentos de eletrofisiologia utilizando as técnicas patch clamp e two microelectrode voltage clamp mostraram que a toxina Ts15 bloqueia preferencialmente os subtipos de canais para potássio Kv1.2 e Kv1.3 com IC50 de 196 ± 25 nM e 508± 67 nM respectivamente. Os ensaios de captação de neurotransmissores em sinaptosomas de cérebro de rato foram realizados adicionando 3H-GABA e 3H-Glu na presença e ausência de diferentes concentrações da toxina Ts15. Não foram observados efeitos nos canais para sódio em todas as concentrações testadas assim como na captação do GABA. Porém, foi observado aumento significante na captação do glutamato em todas as concentrações testadas, provavelmente como resultado de efeito secundário da ação da Ts15 em canais para potássio sensível a voltagem. Em conclusão, a Ts15 pode ser considerada um autêntico novo tipo de toxina escorpiônica, com afinidade para canais para potássio Kv1.2 e Kv1.3 e capaz de aumentar a captação de glutamato. Essa toxina é o único membro da nova subfamília -Ktx21 e portanto nomeada -Ktx21.1 / Scorpions are one of the most ancient groups of animals on earth. They are arthropods and belong to the class Arachinida and Order Scorpionida. The Buthidae family comprises the species that are really dangerous for human, including Tityus serrulatus that is responsible for most severe accidents in Brazil. T. serrulatus venom contains several neurotoxins that specifically act on sodium, potassium or calcium channels in excitable membranes, causing a massive release of neurotransmitters and leading to the stimulation of the autonomic nervous system. Since ion channels play important roles in many physiological processes, scorpion toxins have been used as tools for studies of the neurophysiological mechanisms involving voltage-gated ion channels and neurotransmitter release/uptake. Voltage-gated Na+ channel (Nav channel) toxins are mainly responsible of the harmful effects of scorpion venom and can be classified into two classes: and -neurotoxins. The -toxins retard Nav channel inactivation and induce a prolongation of the repolarization phase of the action potential. The -toxins shift the voltage dependence of Nav channel activation to more negative potentials that result in an increased tendency of the cell to fire spontaneously and repetitively. Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: , , and . These peptides display varying selectivity and affinity for different Kv channel subtypes. In this work, a novel toxin from the T. serrulatus venom was isolated, biochemistry and pharmacologically characterized using a wide electrophysiological screening on 5 different subtypes of Nav channels (Nav1.4; Nav1.5; Nav1.6; Nav1.8 and DmNav1) and 12 different subtypes of Kv channels (Kv1.1 - Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR and hERG). The crude soluble T. serrulatus venom was fractionated by ion exchange chromatography on a CM-cellulose-52 column (2.5 cm x 63.0 cm), which was equilibrated and eluted with NH4HCO3 buffer (pH 7.8). This chromatography allowed the separation of 13 fractions which were named I to XIII. Fraction X was submitted to a reverse-phase C18 (0.46 cm x 25 cm) high performance liquid chromatography (RP-HPLC) and the pure toxin, Ts15, could be obtained. The amino acid sequence of this novel peptide showed that it contains 36 amino acids and is cross-linked by 3 disulfide bridges. The molecular mass of Ts15 (3956) was obtained by electrospray (ESI) triple-quadrupole mass spectrometry and its pI value (8,86) was predicted by ProtParam program. However, the pI determined by isoeletric focusing was greater than 9,3. Electrophysiological experiments using patch clamp and the two electrode voltage clamp technique, showed that Ts15 preferentially blocks Kv1.2 and Kv1.3 channels with IC50 value of 196 ± 25 and 508 ± 67 nM, respectively. Uptake assays were performed by adding 3H-GABA and 3H-Glu, in the absence (controls) or presence of different concentrations of Ts15, on isolated rat brain synaptosomes. No effect on Nav channels was observed, in all tested concentrations, as well as for GABA uptake. However, Ts15 induced a significant increase of the glutamate uptake, probably as a secondary effect of its action on Kv channels. In conclusion, Ts15 can be considered a bonafide novel type of scorpion toxin that presents high affinity by Kv1.2 and Kv1.3 channels and was able to increase the glutamate uptake. It is the unique member of the new -Ktx21 subfamily and therefore was named -Ktx21.1

Introdução. A estimulação do córtex motor (ECM) tem sido utilizada para o tratamento de pacientes com síndromes neuropáticas dolorosas crônicas e resistentes a tratamentos farmacológicos convencionais. O córtex motor primário pode ser a estrutura mais rostral do neuroeixo relacionada ao sistema de modulação da dor, e a ECM provoca ativação neuronal na substância cinzenta periaquedutal (PAG). A PAG é um dos principais centros do sistema descendente supressor de dor e recebe aferências de diferentes regiões do encéfalo. Esse estudo investiga o efeito da estimulação do córtex motor sobre a liberação de neurotransmissores na PAG em modelo de dor neuropática, com o objetivo de investigar os mecanismos neuroquímicos responsáveis pelo feito terapêutico. Métodos. No primeiro experimento, ratos Wistar machos foram aleatoriamente divididos em três grupos. No primeiro grupo, os animais foram submetidos à indução de dor neuropática através da constrição crônica do nervo ciático, no segundo grupo, os animais foram submetidos apenas à exposição do nervo ciático e no terceiro grupo, nenhuma intervenção para indução de dor neuropática foi realizada. Todos os animais foram submetidos a implante unilateral epidural de eletródios de estimulação sobre a área do córtex motor correspondente a pata posterior e implante de cânula guia direcionada à PAG utilizando coordenadas estereotáxicas. Os animais foram avaliados no teste de hiperalgesia mecânica e uma sonda de microdiálise foi introduzida em direção a PAG. As amotras de microdiálise foram coletadas e a análise dos neurotransmissores foi feita em um sistema de cromatografia líquida de alta eficiência (HPLC). No segundo experimento, ratos Wistar machos com dor neuropática induzida na pata posterior foram submetidos a implante estereotáxico de cânula guia direcionada à PAG, e foi realizada micro-injeção de antagonista de glicina e/ou GABA na PAG, previamente a ECM, para avaliar a influência desses antagonistas no efeito analgésico induzido pela estimulação cortical. Resultados. Animais submetidos à indução de dor neuropática apresentaram reversão da hiperalgesia mecânica após ECM. A estimulação cortical induziu um aumento significativo nos níveis de glicina durante (aumento de 153%) e após MCS (134%). A concentração de GABA aumentou 145% durante a estimulação epidural. Os níveis de glutamato não mostraram alteração no microdialisado da PAG após ECM. Houve uma correlação estatisticamente significativa entre o posicionamento da sonda de microdiálise nas colunas lateral e dorsolateral da PAG e o aumento na liberação do neurotransmissor glicina nos animais do grupo CCI. A administração de antagonista de glicina na PAG reverteu o efeito antinociceptivo da estimulação cortical. A micro-injeção de antagonista de GABA na PAG reverteu parcialmente o efeito da ECM. Conclusões. Nossos resultados sugerem que os neutransmissores glicina e GABA, liberados na PAG durante ECM, contribuem para o efeito antinociceptivo da via analgésica descendente. Os resultados desse projeto poderão contribuir para a elucidação dos mecanismos do efeito antinociceptivo da ECM / Introduction. Motor cortex stimulation (MCS) has been used for the treatment of patients with chronic neuropathic pain syndromes that are resistant to conventional pharmacological treatment. The motor cortex may be the most rostral structure in the neuroaxis responsible for pain modulation, and MCS increase the neuronal activation of periaqueductal gray (PAG). The PAG is one of the main subcortical centers of the descending pain suppressor system, and receives inputs from several brain areas. This study investigates the effects of MCS on the release of neurotransmitters in the PAG in neuropathic pain model, in order to investigate the possible neurochemical mechanisms responsible for this effect. Methods. In the first experiment, Wistar male rats were randomly subdivided into three surgical groups. In the first group, induction of neuropathic pain was performed through chronic constriction injury of the right sciatic nerve, in the second group, the animals were submitted just to exposure of the sciatic nerve and in the third group, no intervention for induction of neuropathic pain was performed. All the rats underwent implantation of unilateral epidural electrodes on the motor area corresponding to the right hind paw. The animals were evaluated for mechanical hyperalgesia test and a microdialysis guide cannula was stereotaxically implanted into the PAG. The microdialysate samples were collected and the neurotransmitters analysis was performed by a high- performance (HPLC). In the second experiment, animals with induced neuropathic pain in the hind paw were submitted to a stereotaxic implantation of a guidewire directed to PAG, and a microinjection of glycine and/or GABA antagonist in the PAG before the ECM was performed, to evaluate the influence of these antagonists on the analgesic effect induced by the cortical stimulation. Results. Animals subjected to induction of neuropathic pain showed reversal of mechanical hyperalgesia after motor cortex stimulation. Cortical stimulation induced a significant increase in glycine levels during (153 % increase) and after MCS (134%). The GABA concentration increases 145 % during transdural stimulation. Glutamate levels showed no change in PAG microdialysate after MCS. There was a statistically significant correlation between the positioning of the microdialysis probe in the lateral and dorsolateral columns of the PAG and the increase in the release of the neurotransmitter glycine in the animals of the CCI group. Administration of glycine antagonist in PAG reversed the antinociceptive effect of cortical stimulation. Microinjection of GABA antagonist in PAG partially reversed the effect of MCS. Conclusions. Our results suggest that the neurotransmitters glycine and GABA, released in PAG during MCS, contribute to descending antinociceptive actions. The results of this project will contribute for the elucidation of the mechanisms of the antinociceptive effect of MCS, a phenomenon that has not been fully understood currently

It is of interest to understand how new neurons incorporate themselves into the existing circuitry of certain neuronal populations. One such population of neurons is that which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where they differentiate into granule cells. Another area of interest is the role of brain-derived neurotrophic factor (BDNF) on the survival and overall health of these neurons. This study aimed to test whether or not BDNF is a survival factor for adult-born granule cells. Here were utilized a transgenic mouse model over-expressing BDNF under the α- calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its effect on olfactory granule cells under sensory deprived conditions. Results from this experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a survival factor for adult-born granule cells. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Establishing appropriate animal models for the study of human memory is paramount to the development of memory disorder treatments. Damage to the hippocampus, a medial temporal lobe brain structure, has been implicated in the memory loss associated with Alzheimer’s disease and other dementias. In humans, the role of the hippocampus is largely defined; yet, its role in rodents is much less clear due to conflicting findings. To investigate these discrepancies, an extensive review of the rodent literature was conducted, with a focus on studies that used the Novel Object Recognition (NOR) paradigm for testing. The total amount of time the objects were explored during training and the delay imposed between training and testing seemed to determine hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete time points in the task. The results suggest that the rodent hippocampus is crucial to the encoding, consolidation and retrieval of object memory. Next, it was determined that there is a delay-dependent involvement of the hippocampus in object memory, implying that other structures may be supporting the memory prior to the recruitment of hippocampus. In addition, when the context memory and object memory could be further dissociated, by altering the task design, the results imply a necessary role for the hippocampus in the object memory, irrespective of context. Also, making the task more perceptually demanding, by requiring the mice to perform a two-dimensional to three-dimensional association between stimuli, engaged the hippocampus. Then, in the traditional NOR task, long and short training exploration times were imposed to determine brain region activity for weak and strong object memory. The inactivation and immunohistochemistry findings imply weak object memory is perirhinal cortex dependent, while strong object memory is hippocampal-dependent. Taken together, the findings suggest that mice, like humans, process object memory on a continuum from weak to strong, recruiting the hippocampus conditionally for strong familiarity. Confirming this functional similarity between the rodent and human object memory systems could be beneficial for future studies investigating memory disorders. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como β3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal. / Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as β3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies.

Objetivos: Avaliar a implicação da doença periodontal (DP) avançada, e da expressão da NOS e sP dos tecidos gengivais inflamados, na intensidade de dor e na qualidade de vida de pacientes com dor crônica crânio-facial. Casuística e Métodos: foram avaliados e tratados 20 pacientes com queixas de dores crônicas crânio-faciais e DP (Grupo de Estudo), comparativamente com 20 pacientes que tinham somente DP (Grupo Controle). Todos os pacientes receberam tratamento cirúrgico periodontal. A avaliação foi realizada pré e pós-tratamento periodontal (7, 30 e 180 dias). Instrumentos de avaliação: ficha clínica EDOF-HC, Escala Visual Analógica (EVA), questionário de dor McGill, Índices de Placa (IP), Sangramento (IS), Profundidade Clínica de Sondagem (PCS) e de Inserção (PCI); questionário de qualidade de vida WHOQOL-bref e análise imunohistoquímica para a expressão da óxido nítrico sintase (NOS) e da substância P do tecido gengival inflamado. A avaliação da dor foi realizada por investigador independente. Resultados: Os grupos apresentaram evolução clínica semelhante pelos parâmetros da DP: IP (p=0,0934), IS (p=0,8657), PCS (p=0,1728) e PCI (p=0,7406) nos três momentos avaliados. Houve redução da EVA no Grupo de Estudo aos 30 dias (p<0,05) e 180 dias (p<0,05), comparativamente ao pré-operatório. Aos 180 dias houve melhora significativa das queixas iniciais (p=0,005 para o Grupo de Estudo e p = 0,027 para o Grupo Controle), comparativamente ao pré-operatório, entretanto, houve diferença significativa entre os dois grupos nos três momentos da avaliação, seja para o percentual de melhora (p<0,001), seja para os descritores de melhora Edof-HC (p=0,004 aos 30 dias e p=0,001 aos 180 dias). No Grupo de Estudo, houve redução significativa dos descritores afetivos de dor (questionário de dor McGill) aos 180 dias (p = 0,014). Os escores de qualidade de vida do Grupo de Estudo foram inferiores aos do Grupo Controle nos domínios psicológico e físico (p<0,001 e p=0,007, respectivamente). As expressões da nNOS e da iNOS foram maiores nas gengivas inflamadas do Grupo de Estudo (p<0,001 e p= 0,003, respectivamente) e a expressão da sP foi semelhante nos dois grupos (p=0,363). Conclusões: Houve redução da intensidade da dor crônica crânio-facial dos pacientes que receberam tratamento para doença periodontal avançada. A expressão da substância P foi semelhante nos tecidos gengivais inflamados dos dois grupos, mas a expressão da nNOS e iNOS foi superior na gengiva inflamada dos pacientes com dor crônica crânio-facial. Pelos dados deste estudo a Doença Periodontal avançada pode ter implicação nas queixas de dor dos pacientes com dor crônica crânio-facial. / Aims: To assess the implications of advanced periodontal disease (PD), and the expression of NOS and sP of inflamed gingival tissue, in the intensity of pain and the quality of life in patients with chronic craniofacial pain. Casuistic and Methods: Were evaluated and treated 20 patients with complaints of chronic craniofacial pain and DP (Study Group), compared with 20 patients who had only DP (Control Group). All patients received surgical periodontal treatment. The evaluation was performed at pre-operative and post-operative periodontal treatment (7, 30 and 180 days). Tools for evaluation: clinical record EDOF-HC, Visual Analogic Scale (VAS), McGill Pain Questionnaire, Scores of Plaque (IP), Bleeding (IS), Clinical Probing Depth (CPD) and Insertion (CPI), the quality of life questionnaire WHOQOL-Bref and immunohistochemical analysis for the expression of nitric oxide synthase (NOS) and substance P from inflamed gingival tissue. The assessment of pain was conducted by independent researcher Results: The groups had similar clinical evolution by parameters of the PD: IP (p=0.0934), IS (p=0.8657), PCS (p=0.1728) and PCI (p=0.7406) in the three moments evaluated. There was reduction in the VAS of Study Group at 30 days (p<0.05) and 180 days (p<0.05), compared to preoperative. At 180 days there was significant improvement of the initial complaints (p=0.005 for the Study Group and p=0.027 for the Control Group), compared to preoperative, but there was significant difference between the two groups in the three moments of assessment, for the percentage of improvement (p<0.001), and either for the improvement descriptors Edof-HC (p=0.004 at 30 days and p=0.001 for 180 days). In Study Group, there was significant reduction of pain affective descriptors (McGill Pain Questionnaire) to 180 days (p=0.014). The scores of quality of life of the Study Group were lower than the Control Group in the psychological and physical (p<0.001 and p=0.007, respectively). The nNOS and iNOS expressions were higher in inflamed gingival Study Group (p<0.001 and p=0.003, respectively) and the expression of sP was similar in the two groups (p=0.363). Conclusions: There was reduction in the intensity of chronic craniofacial pain of patients receiving treatment for advanced periodontal disease. The expression of substance P was similar in inflamed gingival tissue of the two groups, but the expression of iNOS and nNOS was higher in inflamed gingival of patients with chronic craniofacial pain. The data of this study show that advanced periodontal disease can have involvement in complaints of pain in patients with chronic craniofacial pain.

Serotonin (5-HT) is a neurotransmitter in the central nervous system. Decrease in the brain 5-HT level could induce depression, showing a state of low mood, aversion to motion and feeling of worthlessness. About 12 million adults in the United States have depression. Antidepressants, such as monoamine oxidase inhibitors and selective serotonin reuptake inhibitors, can alleviate the depressive mood by increasing the brain's 5-HT activity, however they can also induce a potentially life-threatening side effect, namely 5-HT syndrome. This syndrome is manifested by neuromuscular hyperactivities, mental disorders and autonomic dysfunctions. Clinical studies have demonstrated that 5-HT2A receptor antagonists could effectively block severe symptoms of patients with the 5-HT syndrome. To understand the underlying mechanisms, in this study we examined the activity of the 5-HT2A receptor in rats with the 5-HT syndrome evoked by a combined injection of clorgyline, a monoamine oxidase inhibitor , and paroxetine, a selective 5-HT reuptake inhibitor. The major findings from my study were that: (1) Chronic clorgyline treatment significantly exacerbated 5-HT2A receptor-mediated symptoms of the 5-HT syndrome animals; (2) The 5-HT2A receptor-mediated symptoms were also aggravated when the 5-HT syndrome animals were housed in warm (32 ÀC) ambient temperature; (3) Blocking 5-HT2A receptors in the medial prefrontal cortex alleviated the 5-HT syndrome through a circuit between raphe serotonergic neurons and medial prefrontal cortex glutamatergic neurons. Taken together, my data demonstrate that the activity of 5-HT2A receptors may be enhanced by chronic antidepressant treatment and warm environmental temperature. / The sensitized 5-HT2A receptor in the medial prefrontal cortex may exacerbate the syndrome through a positive-feedback circuit between medial prefrontal cortex and raphe nuclei, which would result in excessive 5-HT in the brain. This study casts a new light on the underlying mechanisms of the 5-HT syndrome. / by Gongliang Zhang. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Homeostasis is an indispensable phenomenon in the maintenance of living organisms. Genetic defects which disrupt negative feedback processes can impact homeostatic regulation, potentially resulting in disease. To uncover the molecular mechanisms governing these and other diseases potentially related to defective homeostasis, I used the Drosophila neuromuscular junction as a model system. I characterized two potential mechanisms that regulate homeostasis within the nervous system. First, in Drosophila larval motor neurons, ligand activation of Drosophila metabotropic glutamate receptor A (DmGluRA) mediates a Phosphoinositide 3-kinase (PI3K)-dependent downregulation of neuronal activity, but the mechanism by which mGluR activates PI3K remains incompletely understood. Here, I identified Ca 2+ /Calmodulin-dependant protein kinase II (CaMKII) and the Focal adhesion kinase (DFak) as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. I found that transgene-induced CaMKII inhibition or the DFak CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. I conclude that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak. Second, I observed that Push, a putative E3-ubiquitin ligase and Ca 2+ /Calmodulin binding protein, regulates both neurotransmitter release and retrograde signaling in the Drosophila neuromuscular junction. I found that RNAi-mediated Push inhibition in the neuron increases but, in the muscle decreases, neurotransmitter release. Similar results were obtained from RNAi knock down of PLCβ and IP3R, which mediates Ca 2+ release from the endoplasmic reticulum. I conclude that Push mediation of the ubiquitin proteasome system may be important in the regulation of PLCβ/IP3R-mediated intracellular Ca 2+ release, and that this Ca 2+ release in the neuron inhibits neurotransmitter release, but in the muscle activates neurotransmitter release via a retrograde signal.

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Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird. / Biogenic amines are small organic compounds that act as neurotransmitters, neuromodulators and/or neurohormones in vertebrates and in invertebrates. They form an important group of messenger substances and mediate their diverse effects by binding to membrane receptors that primarily belong to the large gene-family of G protein-coupled receptors. In insects, the group of biogenic amine messengers consists of five members: dopamine, tyramine, octopamine, serotonin, and histamine. Besides many other effects, some of these biogenic amines were shown, for example, to modulate gustatory sensitivity to sucrose stimuli in the honeybee (Apis mellifera). I have investigated various aspects of the aminergic signal transduction in the “model organisms” honeybee and American cockroach (Periplaneta americana). So far, I have characterized two dopamine receptors, a tyramine receptor, an octopamine receptor and a serotonin receptor of the honeybee, which is well-known for its learning and memory capacities. The receptors where expressed in cultivated mammalian cells in order to analyze their pharmacological and functional (i.e., second messenger coupling) properties. The spatiotemporal expression patterns of the respective receptor mRNA were investigated in the honeybee brain by using different techniques (RT PCR, Northern blotting, in situ-hybridization). The salivary glands of the American cockroach were used as a model object in order to investigate the cellular effects of biogenic amines. Both dopamine and serotonin trigger salivary secretion in isolated salivary glands. The quality of the secreted saliva is, however, different. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Serotonin-induced protein secretion is mediated by an increase in the intracellular concentration of cAMP. The pharmacological properties of dopamine receptors associated with cockroach salivary glands were investigated and the molecular characterization of putative aminergic receptors of the cockroach was initiated. Furthermore, I have characterized the ebony gene of the cockroach. This gene encodes an enzyme that is probably involved in the inactivation of biogenic amines in the cockroach (as in other insects). The ebony gene is expressed in the brain and in the salivary glands of the cockroach.