Tang Chi-Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 89-112). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- History and prevalence of food allergy --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as a major shellfish allergen --- p.13 / Chapter 2.4 --- Use of animal model in the studies of food allergy --- p.22 / Chapter 2.5 --- Future approaches for treatment of food allergy --- p.27 / Chapter Chapter 3. --- Cloning and expression of recombinant tropomyosin --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and Methods --- p.31 / Chapter 3.2.1 --- Design of PCR primers for amplification of tropomyosin gene --- p.31 / Chapter 3.2.2 --- Cloning of PCR-amplified cDNA into vector --- p.32 / Chapter 3.2.3 --- Transformation of competent E. coli Ml5 cells --- p.34 / Chapter 3.2.4 --- Confirmation of DNA sequence of the cloned vector --- p.34 / Chapter 3.2.5 --- Induction of the recombinant protein --- p.35 / Chapter 3.2.6 --- Purification and storage of the recombinant protein under native condition --- p.36 / Chapter 3.2.7 --- Concentration measurement and storage of the recombinant protein --- p.37 / Chapter 3.2.8 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.38 / Chapter 3.2.9 --- Regeneration of the Ni-NTA column --- p.40 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- DNA sequence of the cloned vector --- p.42 / Chapter 3.3.2 --- Expression of the recombinant protein --- p.42 / Chapter 3.3.3 --- Sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) --- p.43 / Chapter Chapter 4. --- Induction of hypersensitive response to shrimp tropomyosin in mice --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Mice and reagents --- p.52 / Chapter 4.2.2 --- Animal sensitization and challenge --- p.53 / Chapter 4.2.3 --- Morphological and behavioral changes --- p.54 / Chapter 4.2.4 --- Tropomyosin-specific IgE level --- p.55 / Chapter 4.2.5 --- Passive cutaneous anaphylaxis (PCA) reaction --- p.56 / Chapter 4.2.6 --- Tropomyosin-specific cellular proliferation level of splenocytes --- p.56 / Chapter 4.2.7 --- Cytokine profiles of splenoctyes --- p.58 / Chapter 4.2.8 --- Histological examination of small intestine --- p.59 / Chapter 4.2.9 --- Statistical analysis --- p.59 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Morphological and behavioral changes after challenge --- p.63 / Chapter 4.3.2 --- Tropomyosin-specific IgE level --- p.63 / Chapter 4.3.3 --- Passive cutaneous anaphylaxis (PCA) --- p.64 / Chapter 4.3.4 --- Tropomyosin-specific cellular proliferation level of splenocytes --- p.68 / Chapter 4.3.5 --- Cytokine profiles of splenocytes --- p.70 / Chapter 4.3.6 --- Histology of small intestines --- p.76 / Chapter 4.4 --- Discussion --- p.79 / Chapter Chapter 5. --- General conclusion --- p.88 / References --- p.89

Objectives: To establish a mouse orthotopic bladder cancer model with consistent tumor-take rate. This orthotopic model was subsequently used to evaluate small animal imaging techniques and investigate new therapeutic agents for bladder cancer treatment. / Materials and Methods: Different orthotopic implantation techniques have been tested. MBT-2 cells and syngeneic C3H/He mice were used in all experiments. Chemical bladder pre-treatment with different agents (saline, hydrochloric acid, trypsin and poly-L-lysine) and different concentration of instilled tumor cells (1 x 10⁶ or 2 x 10⁶) were investigated. In the second part of the experiment, trans-abdominal micro-ultrasound imaging (MUI) technique was investigated and validated. Bladder tumor growths were monitored with longitudinal measurement. Mice were killed at every MUI session. Bladder tumor volumes were measured and correlated with gross stereomicroscopy. Using the optimized orthotopic bladder cancer model, targeted contrast enhanced micro-ultrasound imaging has been investigated. VEGFR2 targeted contrast agent was prepared and injected intravenously before imaging sessions. The intra-tumoral perfusion, VEGFR2 expression and blood volume in real time were quantified. Contrast enhanced MUI was performed on Days 14 and 21. The feasibility of targeted contrast enhanced micro-ultrasound imaging was confirmed. After the establishment of orthotopic model and in vivo molecular imaging techniques, this robust platform was used for investigating new treatment agent in localized bladder cancer. Tumor-bearing mice were randomized into control and sunitinibtreated (40 mg/kg) groups. Tumor volume, intra-tumoral perfusion, and in vivo VEGFR2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by CD31 and Ki-67 immunohistochemistry. The clinical outcomes including total bladder weight, tumor stage, and survival were evaluated. / Results: A consistent tumor take-rate of over 90% was achieved by using poly-L-lysine pretreatment with 2 x 10⁶ MBT-2 cells in all of the experiments. MUI identified all tumors that were present on final histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). Measurements of intra-tumoral perfusion and in vivo VEGFR2 expression were also proved to be feasible. After the technical refinement and modification, complete measurements could be performed in all mice (n = 10) at 2 consecutive imaging sessions. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. This is the first report of applying targeted contrast enhanced MUI in orthotopic bladder cancer model. Finally, sunitinib was found to have significant tumor growth inhibition in both in vitro and in vivo experiments. In the orthotopic model, tumors in sunitinib-treated mice had reduced tumor volume and stage, lower proliferation index and micro-vessel density. Sunitinib prolonged survival in tumor-bearing mice as compared to control group. / Conclusions: The development of reliable orthotopic animal models assists in the discovery of novel therapeutic agents. The establishment in the methods of implantation with improved tumor-take rate and the advances in imaging technology form the important foundation of basic research in bladder cancer. Trans-abdominal MUI is proven to be a valuable tool for translational studies involving orthotopic mouse bladder cancer models. Furthermore, the first report of the application of targeted contrast enhanced MUI in deep-seated tumor in bladder has been published. It enables investigators to monitor tumor angiogenesis and vascular changes after treatment. It will be useful for direct, noninvasive, in vivo evaluation of anti-angiogenesis therapeutic agents. The preclinical study has demonstrated the activities of a new class of targeted therapy against localized bladder cancer in an orthotopic mouse model. Sunitinib inhibits tumor growth and thus decreases the tumor burden and prolongs survival compared with placebo. These results provide a rationale for future clinical trials using VEGFR-targeted treatments of localized bladder cancer in the neo-adjuvant and adjuvant settings. / Chan, Shu Yin Eddie. / Thesis (M.D) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 189-212).

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease in which motor neurons selectively degenerate, leading to paralysis and death. Rare causal mutations in FUS and TARDBP implicated RNA binding proteins and RNA metabolism in ALS disease mechanisms. The absence of faithful animal models has impeded precise understanding of the impact of ALS mutations on all functions of ALS-associated proteins. In my graduate studies, I used a novel, animal model of FUS-ALS to explore gain of function disease mechanisms and observed specific, aberrant interactions between mutant FUS and other RNA binding proteins including hnRNP U. Genetic experiments indicate loss of hnRNP U is toxic to motor neurons, suggesting mutant FUS toxicity may result from hnRNP U sequestration and loss of function. In a parallel series of experiments, I also generated novel knock-in mouse models of ALS expressing pathogenic TARDBP mutations to address the flaws of existing model systems and to study the functional consequences of disease-related mutations. We demonstrate that the ALS mutant alleles TDP-43M337V and TDP-43G298S are fully functional and are insufficient to cause age-dependent motor neuron pathology, indicating that physiological levels of mutant TDP-43 are alone insufficient to initiate disease. This model enables future exploration of the interaction between genetic and environmental factors that lead to TDP-43 toxicity in ALS and related disorders. Collectively, our findings suggest a gain of function mechanism of toxicity in which mutations and aging, with other factors, alter the local concentration of RNA binding proteins, leading motor neurons to degenerate.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A plant that has stood out for presenting great therapeutic potential is the species Aniba riparia (Nees) Mez, where from the unripe fruit substances were extracted, as Riparina III (RipIII). This study aimed to investigate the analgesic effect of Riparina III in mice and pharmacological mechanisms involved, being developed after approval by the CEPA/UFC (n 22/12). RipIII was administered orally in male Swiss mice (25-32g). Writhing test induced by acetic acid were used; the formalin test; inflammatory hyperalgesia induced by carrageenan; nociception test induced by specific stimuli such as capsaicin (2.2 mg/paw), cinnamaldehyde (10 nmol/paw), menthol (1.2 mmol/paw), acid salt 2% (pH 2.04, 20μl/paw), PMA (500 pmol/paw), 8-Br-cAMP (10 nmol/paw), bradykinin (10mg/paw) or glutamate (10 nmol/paw), and testing to verify the systems that could be involved in antinociceptive mechanism. RipIII results showed that the doses 25 and 50 mg/kg exhibited antinociceptive effect in the model of visceral nociception induced by acetic acid 0.6%. Time curve was performed and the selected dose of 50mg/kg in previous studies provide the best answer. It was observed that the effects of RipIII 50mg/kg appeared and persisted after 30 minutes about 360 minutes. Pretreatment with RipIII antinociceptive effect manifested only in the inflammatory phase induced by intraplantar formalin injection and showed a decrease of mechanical hyperalgesia induced by carrageenan at both doses. In the investigation of antinociceptive mechanism of RipIII (25 and 50 mg/kg, po) showed a decrease significantly associated with TRPV1 channels (43.25% and 41.71%, respectively), TRPM8 (47.27% and 94.99%, respectively) glutamate receptors (44.64% and 43.57%, respectively), PKA (87.65%) and PKC (38.04%). Investigating possible neurotransmitter pathways involved in the antinociceptive potential RipIII (50 mg/kg, po), a reversal of its effect on cholinergic systems (38,26%) was identified, dopaminergic (54.86%), opioid (47, 69%), serotonergic, related to availability of 5-HT (72.30%) and 5-HT1A receptors (65.57%), and oxidonitrÃrgico (72.07%). According to the results, one can conclude that RipIII presented antinociceptive effect in mice, and these effects are related to modulation of intracellular mediators (PKA and PKC), the molecular mechanisms of TRPV1 channel TRPM8, glutamate receptors, serotoninergic (synthesis 5-HT and 5-HT1A), muscarinic, dopamine, and opioid oxidonitrÃrgico system. / Uma planta que vem se destacando por apresentar grande potencial terapÃutico à a espÃcie Aniba riparia (Nees) Mez, no qual a partir do fruto nÃo maduro foram extraÃdas substÃncias, como a Riparina III (RipIII). Este trabalho teve o objetivo investigar o efeito antinociceptivo da Riparina III em camundongos e os mecanismos de aÃÃo farmacolÃgicos envolvidos, sendo desenvolvido apÃs aprovaÃÃo pela CEPA/UFC (nÂ22/12). RipIII foi administrada, via oral, em camundongos machos Swiss (25-32g). Foram utilizados testes de contorÃÃes abdominais induzidas por Ãcido acÃtico; teste da formalina; hipernocicepÃÃo inflamatÃria induzida pela carragenina; teste da nocicepÃÃo induzida por estÃmulos especÃficos, como capsaicina (2,2 Âg/pata), cinamaldeÃdo (10 nmol/pata), mentol (1,2 Âmol/pata), salina Ãcida 2%, (pH 2,04, 20Âl/pata), PMA (500 pmol/pata), 8-Br-AMPc (10 nmol/pata), bradicinina (10Âg/pata) ou glutamato (10 Âmol/pata), alÃm de testes para verificar os sistemas que poderiam estar envolvidos no mecanismo antinociceptivo. Resultados mostraram que RipIII, nas doses de 25 e 50 mg/kg, apresentaram efeito antinociceptivo no modelo de nocicepÃÃo visceral induzida por Ãcido acÃtico a 0,6%. Foi realizada curva de tempo e selecionada a dose de 50mg/kg por apresentar em estudos anteriores a melhor resposta. Observou-se que os efeitos da RipIII 50mg/kg apareceram apÃs 30 minutos e persisti por 360 minutos. O prÃ-tratamento com RipIII manifestou efeito antinociceptivo apenas na fase inflamatÃria induzida pela injeÃÃo intraplantar de formalina, e mostrou diminuiÃÃo da hipernocicepÃÃo mecÃnica induzida por carragenina em ambas as doses. Na investigaÃÃo do mecanismo antinociceptivo da RipIII (25 e 50 mg/kg,v.o.), apresentou uma reduÃÃo de forma significativa relacionado com os canais TRPV1 (43,25% e 41,71%, respectivamente), TRPM8 (47,27% e 94,99%, respectivamente), receptores glutamatÃrgicos (44,64% e 43,57%, respectivamente), PKA (87,65%) e PKC (38,04%). Investigando as possÃveis vias neurotransmissoras envolvidas com o potencial antinociceptivo da RipIII (50 mg/kg, v.o.), foi identificado uma reversÃo do seu efeito nos sistemas colinÃrgico (38,26%), dopaminÃrgico (54,86%), opiÃide (47,69%), serotonÃrgico, relacionado a disponibilidade de 5-HT (72,30%) e receptores 5-HT1A (65,57%), e oxidonitrÃrgico (72,07%). De acordo com os resultados, pode-se concluir que RipIII apresentou efeito antinociceptivo em camundongos, e estes efeitos estÃo relacionados à modulaÃÃo de mediadores intracelulares (PKA e PKC), à mecanismos moleculares dos canais de TRPV1, TRPM8, receptores glutamatÃrgicos, serotonÃrgicos (sÃntese de 5-HT e 5-HT1A), muscarÃnicos, dopaminÃrgicos, opiÃide e sistema oxidonitrÃrgico.

Déficits na regulação serotoninérgica dos circuitos catecolaminérgicos tem sido propostos como um mecanismo relacionado à etiologia dos transtornos de humor. Projeções do núcleo mediano da rafe (MnR) modulam a atividade dopaminérgica no prosencéfalo e são também parte de um sistema de inibição/desinibição comportamental que se assemelha às variações nos níveis de atividade apresentados durante os polos do transtorno bipolar. O objetivo do presente estudo foi avaliar se as alterações comportamentais induzidas pela inativação do MnR podem ser um modelo animal para estudo da mania humana. No Capítulo I, o procedimento de lesão eletrolítica do MnR foi realizado em ratos Wistar machos, tendo como controles os grupos lesão fictícia e intacto. Os resultados confirmaram a capacidade desta manipulação experimental em reproduzir hiperatividade e estereotipia crônicas, aumento na frequência de respostas positivamente reforçadas por solução de sacarose e padrão comportamental de dominância social. Ainda, foi demonstrada a potencialidade do tratamento crônico com lítio em reduzir a hiperatividade. No Capítulo II foram realizadas lesões eletrolíticas do MnR em camundongos C57BL/6J machos, tendo novamente como controles os grupos lesão fictícia e intacto. Os resultados demonstraram desenvolvimento de hiperatividade, estereotipia e maior frequência de exposição a situações aversivas/de risco nos testes do labirinto em cruz elevado e do claro/escuro. O tratamento crônico com lítio atenuou ou reverteu parte destas alterações comportamentais. Análises do tecido encefálico demonstraram níveis terapêuticos equivalentes de LiCl em todos os grupos submetidos ao tratamento e a histologia confirmou o sítio de lesão. O conjunto dos dados obtidos sugere a adequação do modelo proposto pelo atendimento aos critérios de validade de face e preditiva apresentando como vantagens a mimetização de diversos sintomas do transtorno e a cronicidade destes. Ainda, a reprodutibilidade dos efeitos da lesão em diferentes espécies sugere a existência de homologia evolutiva, acrescentando fundamentos à validade de constructo hipotetizada. Por fim, destaca-se que este modelo de mania parece ser heurístico pela possibilidade de contribuir para a investigação dos mecanismos de ação do lítio e para a compreensão das relações de oposição entre os sistemas neurotransmissores excitatórios e inibitórios como parte da neurobiologia dos transtornos de humor / Deficits in serotoninergic regulation of catecholaminergic circuits have been proposed as a mechanism related to the etiology of mood disorders. Projections from the median raphe nucleus (MnR) modulate the dopaminergic activity in the forebrain and are also part of a behavioral disinhibition/inhibition system that resembles the variations in activity levels shown during the poles of bipolar disorder. The aim of the present study was to evaluate if the behavioral effects induced by the inactivation of the MnR can be considered an animal model for studying the human mania. In Chapter I, the MnR electrolytic lesion was performed in male Wistar rats, having as control groups sham operated and intact animals. The results confirmed the capacity of this experimental manipulation to reproduce chronic hyperactivity and stereotypy, increased response frequency positively reinforced by sucrose solution and a social dominant behavioral pattern. Furthermore, it was shown the potentiality of the lithium treatment in reducing the hyperactivity. In Chapter II MnR electrolytic lesions were performed in C57BL/6J mice, having as control groups sham operated and intact animals. The results demonstrated the development of hyperactivity, stereotypy and increased frequency of exposure to aversive situations/\"risk taking\" in the elevated plus maze and light/dark box tests. Chronic treatment with lithium attenuated or reversed some of these behavioral alterations. Encephalic tissue analysis showed equivalent lithium therapeutic levels in all treated groups and the histology confirmed the lesion site. The set of data obtained suggests the suitability of the proposed model for attending criteria for face and predictive validities presenting advantages such as the mimicking of several disorder symptoms and the chronicity of them. Also, the reproducibility of the effects of the lesion in different species suggests the existence of evolutionary homology and adds basis to the construct validity hypothesized. Finally, it is emphasized that this model of mania seems to be heuristic by the possibility to contribute to the investigation of lithium mechanisms of action and for understanding the opposite relations between excitatory and inhibitory neurotransmitter systems as part of the neurobiology of mood disorders

A recaída ao uso de drogas é um dos principais obstáculos para o tratamento do adicto. Um dos modelos animais mais utilizados para estudar a recaída no laboratório é o chamado de cue-induced. Embora esse modelo tenha se mostrado útil para o estudo de processos neurofisiológicos envolvidos na recaída, seu valor para a análise do controle de estímulos ambientais na dependência tem sido limitado por não distinguir a função dos estímulos discriminativos e dos reforçadores condicionados que controlam a reinstalação da resposta que foi reforçada por droga. O principal objetivo do presente estudo foi analisar os controles estabelecidos sobre as respostas de pressão à barra de ratos submetidos ao procedimento cue-induced. Foram realizados três experimentos. No Experimento 1, os animais passaram pelas três fases experimentais que caracterizam esse modelo. Na primeira fase, a resposta de pressão à barra foi treina em dois componentes distintos. Em um deles, as respostas emitidas na presença de um odor de laranja (SD1) tiveram como conseqüência a apresentação de um estímulo luminoso (Sr1) e liberação de uma solução de etanol (grupo ET) ou de sacarose (grupo SAC). Enquanto na outra, pressões à barra na presença de um odor de erva-doce (SD2) tiveram como conseqüência a apresentação um estímulo sonoro (Sr2) e liberação de água. Na segunda fase, foram realizadas sessões de extinção na ausência dos estímulos utilizados no treino. Na terceira fase os estímulos discriminativos e reforçadores condicionados foram reintroduzidos, mas as respostas de pressão à barra não foram reforçadas por etanol, sacarose ou água. Finalizado o procedimento padrão, foram realizados testes adicionais, nos quais cada estímulo utilizado no treino foi apresentado separadamente. O procedimento do Experimento 2 foi similar ao do Experimento 1, porém foram controladas duas variáveis irrelevantes para o modelo, mas associadas com as contingências experimentais: a maravalha da bandeja da caixa experimental e o acionamento do bebedouro. No Experimento 3, foi adicionada sacarose à solução de etanol e a água. Os resultados dos Experimentos 1 e 2 mostraram que Sr1 foi efetivo em reinstalar a resposta de pressão à barra nos testes de reinstalação realizados, enquanto SD1 foi inconsistente em reinstalar essa resposta. A apresentação de SD2 e Sr2 não reinstalou a resposta. Os dados obtidos no Experimento 3 foram inconclusivos quanto ao controle exercido pelos estímulos olfativos, luminosos e sonoros. Os resultados parecem sugerir que o modelo animal cue-induced, tipicamente usado para estabelecer linhas de base de controle discriminativo sobre a auto-administração de drogas, pode não ser adequado para tal finalidade. No entanto, ainda são necessários refinamentos experimentais para a obtenção de resultados mais acurados. / The relapse into drug use is one of the key obstacles for addict treatment. One of the animal models most used for relapse studies in laboratory is the so called cue-induced. Even though this model has been proven to be useful for neurophysiologic processes related to relapse, its value for analyzing environment stimulus control on addiction is considered to be limited because it does not distinguish the function of discriminative stimulus and of conditioned reinforcer which control the reinstatement of the response that was reinforced by the use of drugs. The main objective of this study was to analyze the established controls over the lever pressure responses of rats submitted to the cue-induced procedure. They were subjected to three different experiments. On the first one the animals were exposed to the three different experimental stages that characterize a cue-induced procedure. At the first experimental phase, lever press response was trained over two different components. In one of them, the response to an orange odor (SD1) had as a consequence the appearance of a luminous stimulus (Sr1) and the release of ethanol (ET group) or a sucrose solution (SAC group). While in the other one, lever press in the presence of a anise odor had as a consequence the appearance of a sonorous stimulus accompanied by water release. At the second phase, there were conducted extinction sessions in the lack of the stimuli used on the training phase. And finally, at the third phase, the discriminative stimulus and conditioned reinforcer were reintroduced. Nevertheless, the lever press response was not reinforced by ethanol, sucrose or water. By the time the standard procedure was over, additional tests were run, where each stimuli used on the training phase were presented to the rats separately. The second experiment procedure was similar to the one employed at the first experiment, however two irrelevant variables for the model were controlled for, but these were associated with the experimental contingencies: the sawdust on the experimental box trail and the drinking fountain when put into action. At the third experiment, sucrose was added to the ethanol solution and also to the water. The results from the two first experiments showed that Sr1 was effective in reinstate the lever pressure response verified at the reinstatement tests, whereas SD1 was not successful in doing so. Neither the introduction of SD2 nor Sr2 reinstate the response. The data obtained by Experiment 3 was not conclusive in regards to the control imposed by the olfactory, luminous and sonorous stimuli. The results suggest that the cue-induced procedure, typically used to establish baselines for discriminative control over drugs self-administration, may not be in fact the most suitable one for this purpose. Nonetheless, there is still a need to refine the experiment in order to reach more accurate and conclusive results.

Em 2006, a Diálise Peritoneal representou, no Brasil, o método de tratamento de 9,3% dos pacientes com Insuficiência Renal Crônica Terminal. A terapia dialítica provoca uma lesão na membrana peritoneal de característica inflamatória, com grave desbalanço do processo oxidativo. Associa alto transporte de solutos e perda da capacidade de ultrafiltração da membrana. Vários fatores contribuem para a lesão, tais como a bioincompatibilidade das soluções de diálise peritoneal e sua elevada concentração de glicose. O objetivo do presente estudo é avaliar o efeito protetor da N-acetilcisteína na lesão provocada pelo uso de solução dialítica hipertônica em modelo experimental de diálise peritoneal. Foram estudados 22 ratos Wistar, machos, não-urêmicos, divididos em quatro grupos, sendo: um grupo-controle, não submetido à infusão de SDP; o grupo RL, que recebeu infusão diária de solução ringer lactato; o grupo SDH, que recebeu infusão de SDP e, finalmente, o grupo SDH+NAC, que recebeu infusão de SDP e tratamento com N-acetilcisteína via oral, diariamente (600mg/L). Após seis semanas, foi realizada avaliação funcional da membrana peritoneal pela relação uréia do dialisato, com uma hora de permanência na cavidade peritoneal, e plasma (D/P uréia), assim como pela razão entre a concentração de glicose no líquido peritoneal após uma hora de permanência na cavidade e a concentração de glicose na solução de diálise infundida (G1/G0). As substâncias reativas do ácido tiobarbitúrico (TBARS) foram utilizadas como marcadores no processo oxidativo. A concentração de TBARS foi avaliada em amostra de urina 24hs e dosagem no plasma. Comparada ao grupo-controle, a avaliação funcional do grupo SDH apresentou aumento do transporte peritoneal com D/P uréia de 0,67±0,1 vs 0,46 ± 0,05 (p: 0,03) e G1/G0 de 0,27±0,07 vs 0,44±0,08 (p: 0,01), demonstrando lesão funcional importante. Quando avaliado o grupo SDH+NAC, tratado com N-acetilcisteína e o grupo não tratado SDH, foi observado maior transporte de solutos do grupo não tratado, com D/P uréia 0,67±0,1 vs 0,51 ± 1(p: 0,03) e G1/G0 de 0,27± 0,07 vs 0,35 ± 0,06 (p:0,01). Portanto, foi constatada maior lesão de membrana comparada ao grupo tratado. Comparados ao controle, os grupos SDH e SDH+NAC apresentaram maiores concentrações de TBARS, seja pela dosagem urinária (p:0,002), seja pelo TBARS plasmático (p:0,0001). O grupo tratado SDH+NAC apresentou menores concentrações de TBARS, comparado ao grupo não tratado SDH, provavelmente por um efeito protetor da Nacetilcisteína. Quando avaliada a membrana peritoneal parietal, o grupo tratado apresentou menor espessamento de membrana, medido em micrômetro, em relação ao grupo não tratado (p:0,01) O presente estudo sugere que a lesão da membrana peritoneal provocada pelo uso de solução dialítica hipertônica sofre uma forte influência do processo oxidativo e que o uso contínuo de N-acetilcisteína, por via oral, pode ter efeito redutor dessa lesão. / Peritoneal Dialysis represents the modality of treatment for 9.3% of end stage renal disease patients in Brazil. During the period of treatment, the solution induced a peritoneal membrane lesion of inflammatory characteristic. Oxidative stress has been implicated in the development of endothelial damage. It associates the high transport of solutes and loss of ultrafiltration capacity of the membrane. The lesion is caused by several factors as follows: peritoneal dialysis solution incompatibility (PDS) and its increased concentration of glucose. The aim of the present study was to evaluate the N-acetylcysteine protecting effect on lesion aggravated by the use of hypertonic dialytic solution in peritoneal dialysis experimental model. Twenty two male, non-uremic Wistar rats were divided in four groups as follows: (I) control group, not submitted to PDS infusion; (II) RL group receiving daily infusion of ringer lactate; (III) HDS group, receiving PDS infusion, and; (IV) HDS+NAC group receiving PDS infusion and treated with N-acetylcysteine (600mg/L) orally. The peritoneal membrane functional evaluation was performed six weeks later by the dialisate-to-plasma urea, ratio (D/P), and glucose reabsorption determined by the ratio of glucose concentration in peritoneal fluid after one hour of solution permanence in the cavity (G1/GO). The thiobarbituric acid reactive substances (TBARS) were used as markers on studying the oxidative process. The TBARS concentration was assessed in 24h sampling urine and plasmatic dosage. The functional evaluation for the HDS group showed increased peritoneal transport compared to the control group with urea D/P of 0.67±0.1 versus 0.46±0.05 (p:0.03), and G1/GO 0.27±0.07 versus 0.44±0.08 (p:0.01), demonstrating functional lesion. When evaluating the HDS+NAC group, treated with N-acetylcysteine and the HDS non-treated group, a greater transport of solutes was observed for the non-treated group, with urea D/P 0.67±0.1 versus 0.51 ± 1 (p: 0.03), and G1/GO 0.27±0.07 versus 0.35±0.06 (p: 0.01), therefore greater lesion present on the membrane than for the treated group. Compared to the control group, the HDS and HDS+NAC presented greater TBARS concentrations either for urine dosage (p: 0.002) or for plasmatic TBARS (p: 0.0001). The HDS+NAC treated group showed lower TBARS concentrations compared to the non-treated HDS group, a probable protective effect of Nacetylcysteine on the treatment. In the treated group, peritoneal membrane demonstrate lower trickiness compared with untreated group.(p:0,01) The present study suggests that the peritoneal membrane lesion induced by the use of hypertonic dialytic solution undergoes strong influence from the oxidative process and that the continuous use of N-acetylcysteine can preserve these alterations by inhibiting the oxidative stress within the peritoneal membrane.

Abnormality of dopamine D2 receptor (D2R) function, often observed as D2R supersensitivity (D2RSS), is a commonality of schizophrenia and related psychiatric disorders in humans. Moreover, virtually all psychotherapeutic agents for schizophrenia target D2R in brain. Permanent D2RSS as a feature of a new animal model of schizophrenia was first reported in 1991, and then behaviorally and biochemically characterized over the next 15–20 years. In this model of schizophrenia characterized by production of D2RSS in ontogeny, there are demonstrated alterations of signaling processes, as well as functional links between the biologic template of the animal model and ability of pharmacotherapeutics to modulate or reverse biologic and behavioral modalities toward normality. Another such animal model, featuring knockout of trace amine-associated receptor 1 (TAAR1), demonstrates D2RSS with an increase in the proportion of D2R in the high-affinity state. Currently, TAAR1 agonists are being explored as a therapeutic option for schizophrenia. There is likewise an overlay of D2RSS with substance use disorder. The aspect of adenosine A2A-D2heteroreceptor complexes in substance use disorder is highlighted, and the association of adenosine A2Areceptor antagonists in discriminative and rewarding effects of psychostimulants is outlined. In summary, these new animal models of schizophrenia have face, construct, and predictive validity, and distinct advantages over earlier models. While the review summarizes elements of D2RSS in schizophrenia per se, and its interplay with substance use disorder, a major focus is on presumed new molecular targets attending D2RSS in schizophrenia and related clinical entities.

Mice with human immune systems, generated by transplanting human CD34+ cells into immunodeficient mice, are essential tools for studying phenomena unique to the human immune system or poorly reproduced in existing mouse models. Human immune tolerance induction, function and autoimmunity have been poorly modeled in conventional murine models, which often have poor predictive value for preclinical development. Models that allow the study of human immune cells with the reproducibility and flexibility of small animal models are required. In our lab, humanized mouse models have been used to study preclinical protocols for human xenotolerance induction and to better understand the immunological underpinnings of human autoimmunity. These are each areas of critical unmet medical need. Xenotolerance-inducing protocols may be necessary to allow long-term survival of a transplanted pig organ in a human patient, and, with more than 113,000 Americans currently waiting for a life-saving organ, the need to expand the pool available for transplantation is urgent. Additionally, clinical options for patients with autoimmune diseases are limited. Currently, most patients with autoimmunity are only diagnosed after significant immune damage of target organs. Predicting who will develop autoimmunity – and who will not – before damage occurs would be very useful but is currently very difficult. Small animal models that can better help us understand how human autoimmunity develops could help us develop protocols for early detection and even prevention. We have developed a personalized immune model to study the development of an individual patient’s immune system in a transplanted mice to better understand immune abnormalities that underlie autoimmunity. We have used existing humanized mouse models to answer important questions related to human xenotolerance induction and autoimmunity, but in the studies described here we have worked to extend our capacity to use these models to study human T cell development and peripheral function. We would like to be able to study both the initial selection of T cell receptors (TCRs) in the thymus based on their ability to recognize antigen in the context of presenting MHC without reacting unduly to self-antigen, as well as in the periphery, where T cells interact with peripheral antigen-presenting cells (APCs) to maintain homeostasis and respond to antigen. First, we have incorporated TCR transgenesis into our humanized mouse models to allow greater precision in studying thymic selection in our humanized mice. Developing a system for this would allow us to study in greater detail mechanisms of human xenotolerance induction, including confirming that a swine thymus can support positive selection of T cells with human-restricted TCRs to allow a future xenotransplantation patient to maintain immune competence, while also robustly tolerizing human T cells expressing pig-reactive TCRs. We will also expand this system to study the thymic selection of human T cells with autoreactive TCRs to better understand mechanisms of central tolerance and understand how they fail in autoimmunity. Finally, while processes of thymic selection are critical for human T cell development and function, peripheral interactions also have a large impact on human T cell function and homeostasis and may contribute to the development of autoimmunity. For these interactions to occur appropriately requires robust engraftment and reconstitution of APCs, especially of myeloid and B cell lineages, in transplanted immunodeficient mice. APC reconstitution tends to be suboptimal in humanized mice and is even more so in mice transplanted with patient-derived CD34+ cells. Better characterization of human APC populations and their progenitors could allow us to develop approaches to improve long-term human APC reconstitution in patient-derived humanized mice, allowing us to more fully model patient peripheral T cell function.

Frequency selectivity is one of the most important functions of the mammalian hearing organ – the cochlea. The interaction of fluid mass and organ of Corti compliance sets a traveling wave along the basilar membrane (BM), which is longitudinally tuned to different frequencies. Beyond this passive tuning process, cochlear amplification locally enhances the vibration of the best frequency peak by factors of hundreds to boost the frequency selectivity and sensitivity of the cochlea. This amplification is achieved by a positive feedback loop between BM motion and outer hair cell (OHC) electrical-mechanical response. However, this active mechanism is vulnerable to damage and cannot be fully recovered in vivo. As the instruments of cochlear amplification, the frequency response of BM and OHCs are of great importance to understand cochlear tuning process. This thesis used animal models, aimed to understand cochlear tuning and investigate possibilities to manipulate the cochlear amplifier, by testing the cochlear amplifier’s sensitivity to operating conditions. The first project tested whether the cochlear amplification can adjust to a lower endocochlear potential (EP), which controls OHC electromechanical force by providing part of the voltage source to drive OHC transduction current. To investigate this possibility, we use intraperitoneal (IP) and intravenous (IV) injection of furosemide to reversibly reduce EP, while monitoring the EP and cochlear amplification simultaneously. Cochlear amplification was monitored by measuring the local cochlear microphonic (LCM) and distortion product emission (DPOAE). With IV injection, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the 2nd harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with re-centering of the operating point. The second project studied the physiological and anatomical effects of perfusing the cochlea with a viscous fluid, for better understanding cochlear fluid mechanics. Perilymphatic perfusion was applied with artificial perilymph and viscous sodium hyaluronate (Healon, HA) in four different concentrations. Using compound action potential (CAP) thresholds as an indicator of cochlear condition, our results and analysis indicated that the cochlea can sustain, without a significant CAP threshold shift, up to a 1.5 Pa shear stress. Histology of the cochleae perfused with higher shear stress showed the Reissner's membrane was torn. These data also indicated that the cochlea mechanics remains normal within increased perilymphatic fluid viscosity up to an increase of a factor of 50. Beside these findings, a temporary CAP threshold shift was observed, perhaps due to the presence and then clearance of viscous fluid within the cochlea, or to a temporary position shift of the organ of Corti. The last project was to test the effect of OHC intracellular Cl- concentration on cochlear amplification. Chloride is known to enable the electromotility of the OHC by binding its motor protein, prestin. By locally perfusing high chloride perilymph and the chloride ionophore tributyltin, this study investigated whether increasing intracellular chloride concentration can restore cochlear sensitivity in a cochlea that was slightly damaged. This had been shown by others in guinea pig. However, we did not observe recovery in several attempts in gerbil.