Justificativa e Objetivos - A indução anestésica em indivíduos em choque hipovolêmico pode agravar a instabilidade hemodinâmica. O anestésico inalatório xenônio (Xe) é gás inerte com propriedades que mantêm a estabilidade hemodinâmica, durante a anestesia, em individuos normais. O objetivo deste estudo foi avaliar os efeitos hemodinâmicos, metabólicos e sobre o transporte de oxigênio, durante anestesia com Xe, em cães submetidos a choque hipovolêmico. Método - Vinte e um cães sem raça definida foram submetidos a hipnose e relaxamento muscular por infusão venosa contínua de etomidato e vecurônio e ventilação controlada mecânica com FiO2 21% (oxigênio + ar comprimido). Sob anestesia local, inseriu-se cateter em artéria pulmonar para monitorização hemodinâmica e cateteres em artérias femorais direita e esquerda para medida da pressão arterial média e indução do choque hipovolêmico. Após indução do choque hemorrágico até a pressão arterial média atingir 40mmHg por 2 minutos, os animais foram randomizados em grupos controle e xenônio. O grupo xenônio recebeu ventilação controlada mecânica com O2 21% + Xe 79% durante 20 min. Realizou-se coleta de gasometria arterial e venosa mista e coleta de dados hemodinâmicos, com posterior cálculo hemodinâmico e do transporte de O2 antes da indução do choque, imediatamente após a sangria, após 5 min e 20 min, com administração de Xe, e 20 minutos após a suspensão do Xe. O grupo controle foi submetido ao mesmo procedimento, sem a administração de Xe. Os dois grupos foram comparados, utilizando análise de variância para medidas repetidas, considerando-se significativo p<0,05. Resultados: Os grupos foram comparáveis em relação aos valores médios de peso corpóreo e volume de sangramento. As variáveis hemodinâmicas, metabólicas e de transporte de oxigênio não apresentaram diferenças significativas entre os grupos controle e xenônio. Após a indução do choque, no grupo Xe a freqüência cardíaca variou de 130,22±20,57 para 131,89±24,34 em 5min e 138,44±33,66 após 20 minutos. No grupo controle esta variação foi de 144,33±21,03 para 143,75±22,58 após 5min (p=0,82) e para 149,50±23,52 após 20min(p=0,16). Conclusão: A administração de xenônio em cães, em estado de choque hipovolêmico, não altera a condição hemodinâmica, metabólica e de transporte de oxigênio, permitindo sugeri-lo como anestésico seguro em individuos nestas condições / Rationale and objectives - Anesthesia induction in subjects with hypovolemic shock may worsen a baseline hemodynamic instability. Xenon (Xe), an inhalation anesthetics, is an inactive gas with properties that, in normal subjects, maintain hemodynamic stability during anesthesia. The aim of this study was to assess Xe effects on hemodynamics, metabolism, and oxygen delivery during anesthesia in dogs with induced hypovolemic shock. Method - Twenty-one hybrid dogs were submitted to hypnosis and muscle relaxation by continuous venous infusion of etomidate and vecuronium and mechanichal controlled ventilation with a 21% FiO2 (oxigen + compressed air). Under local anesthesia, catheters were introduced into the pulmonary artery for hemodynamic monitoring, and in the left and right femoral arteries for mean arterial blood pressure measurements and hypovolemic shock induction. After induction of an hemorrhagic shock until a mean arterial blood pressure of 40mmHg for 2 minutes was reached, the animals were randomized into the Xenon or the control group. Dogs from the Xenon group received controlled mechanichal ventilation with a 21% FiO2 + 79% Xe for 20 minutes. Blood samples were collected for arterial e mixed venous gas analyses; hemodynamic data were also collected, with further hemodynamic calculation of O2 delivery before shock induction, immediately after the artificial bleeding, 5 min and 20 minutes after starting Xe administration, and 20 minutes after stopping Xe. The control group was submitted to the same procedures, without Xe administration. Both groups were compared using variance analysis for repeated measurements, considering statistical significance where a p<0,05 was reached. Results: Both groups were comparable in terms of mean body weight values and bleeding volume. Hemodynamic and metabolic variables and oxygen delviery had no significant differences between both groups, control and Xenon. In the Xenon group, after shock was induced, heart rate changed from 130.22 ± 20.57 to 131.89 ± 24.34 after 5 minutes, and to 138.44 ± 33.66 after 20 minutes. In the control group, heart rate varied from 144.33 ± 21.03 to 143.75 ± 22.58 after 5 minutes (p=0.82), and to 149.50 ± 23.52 after 20 minutes (p=0.16). Conclusion: Xenon administration does not cause changes in hemodynamics, metabolism, and oxygen delivery in dogs with induced hypovolemic shock, supporting its recommendation as a safe anesthetic in such conditions

INTRODUÇÃO: A técnica de ESD (Endoscopic Submucosal Dissection) é um procedimento endoscópico de grande complexidade, com alto índice de complicações e dificuldades técnicas. Para superar este problema, muitos centros de treinamento em endoscopia vêm publicando a aplicabilidade dos modelos animais para a aquisição de competência em ESD. Em todas as publicações sobre o assunto, a habilidade do aluno é acompanhada pela evolução de variáveis como o tempo de ressecção, ressecção em bloco e complicações tais como sangramento e perfuração; entretanto a profundidade de ressecção nunca foi utilizada como parâmetro de aprendizagem, o que pode ser um fator relevante a ser ensinado, dado que atingir o plano de dissecção ideal é de suma importância para uma ressecção curativa e na prevenção de complicações intraoperatórias. O objetivo do estudo foi analisar o aprendizado em ESD em treinamentos de curta duração através da avaliação da profundidade de submucosa ressecada; e sua associação com complicações. MÉTODOS: estudo experimental; incluídos 25 endoscopistas com experiência em procedimentos terapêuticos ( > 5anos) e 75 peças ressecadas por ESD sendo uma média de 3 resseções por endoscopista. Os parâmetros de aprendizagem (tempo de ressecção, tamanho, taxa de ressecção em bloco, sangramento, perfuração e análise histológica da camada submucosa) foram prospectivamente avaliados. Antes, durante e ao final do treinamento os participantes foram submetidos a um questionário sobre a dificuldade e insegurança em realizar o procedimento. RESULTADOS: Todas as ressecções foram realizadas no corpo gástrico (n=75). O tamanho médio das peças ressecadas foi de 23,97 ± 7,2 mm. O número de complicações como sangramento, perfuração e morte foram respectivamente, 17 (22,67%), 3 (4%) e 0 casos. Na terceira dissecção, tempo médio do procedimento diminuiu de 28,44 ± 9,73 para 18,72 ± 8,81 minutos (p < 0,001). Quando comparada a primeira com a terceira dissecção houve uma diminuição significativa na taxa de sangramento (p=0,047) em contraste com a percentagem de submucosa ressecada que foi aumentando progressivamente quando comparada a primeira (53.5 ± 23.76%), segunda (61.8±26.47%) e terceira (69.82 ± 27.86) dissecção (p=0,073). O número de participantes que se sentiam inseguros diminuiu de 100(IC95%: 83,88-100) para 32(IC95%: 17,18-51,78), (p < 0,001). O grupo que teve sangramento durante o procedimento ressecou 37,97±21,13% da camada submucosa e o grupo sem sangramento ressecou 68,66±23,99%, demonstrando uma associação significante entre a profundidade de dissecção submucosa e a incidência de sangramento (p < 0,001). De acordo com a análise de curva ROC, o valor de corte da profundidade de submucosa ressecada para a ocorrência de sangramento é de 61%(64% sensibilidade, 94% especificidade), logo quando o ESD é realizado em uma profundidade maior do que 61% da camada submucosa o risco de sangramento durante o procedimento diminui (VPP=0,97; IC95%:0,85-0,99). CONCLUSÃO: O modelo de treinamento de curta duração possibilitou um aprendizado da técnica de ESD mostrando uma melhora cognitiva dos alunos já na terceira dissecção através de parâmetros como tempo de ressecção, diminuição dos casos de sangramento, um menor nível de insegurança e um maior percentual de submucosa ressecada comprovada na análise histológica. Existe uma associação significativa entre a profundidade de ressecção da submucosa com o risco de sangramento, ou seja, quanto mais profundo a ressecção na camada submucosa menor serão os episódios de sangramento / BACKGROUND: Endoscopic submucosal dissection is a complex endoscopic technique, with several technical difficulties to overcome and potentially high complication rates. To overcome that problem many endoscopic training centers in the west have been reporting the usefulness of animal models to achieve some expertise. In most of these reports the variables used to evaluate their learning curve are resection time, complete en-bloc resection rate and complications as bleeding and perforation; however the depth of the submucosal resection has never been analyzed. That might be a relevant factor since appropriate depth of submucosal dissection is important to a curative resection and prevent intraoperative complications which could be a very important concept to be taught during ESD training. The aim of this study is to evaluate the association between the depth of submucosal resection with the learning curve in ESD and their complications. METHODS: Twenty-five senior endoscopists with experience in therapeutic procedures ( > 5years) undergone seventy-five en bloc ESDs in live porcine models. Each participant did at least 3 endoscopic resections. The learning curve parameters (procedure time, specimen size, en-bloc resection rate, perforation, bleeding and histological analysis of the submucosal layer) were prospectively evaluated. During and after each procedure the participants were submitted into a questionnaire about difficulties and insecurities in doing this procedure. RESULTS: ESDs were all completed at the gastric body (n=75). Medium sizes of the specimens resected were 23.97 ± 7.2 mm. Complication as bleeding, perforation and death were seen respectively in 17 (22.67%), 3 (4%) and 0 cases. After the third ESD, procedure mean time has progressively reduced from 28.44 ± 9.73 to 18.72±8.81 minutes (p < 0.001). Bleeding rate were significantly lower when comparing the first to the third resection (p=0.047) and the depth of submucosal resection, in contrast, has increased when comparing the first (53.5±23.76%), second (61.8±26.47%) and third (69.82±27.86) dissection (p=0.073). Results of the questionnaire showed that participants felt increasingly less insecure from the first (95%CI: 100(83.88-100)) until the third (95%CI: 32(17.18-51.78)) ESD procedure (p < 0.001). The group that had bleeding during the procedure resected 37.97±21.13% of the submucosal layer and the non-bleeding group resected 68.66±23.99%, showing a significant association between the depth of submucosal dissection and the incidence of bleeding (p < 0.001). According to the ROC curve analysis, the resulting cutoff value of the submucosal dissection depth for bleeding is 61 %( 64% sensitivity, 94% specificity). When ESD is done deeper than 61% of the submucosal layer the risk of bleeding decreases during the procedure (PPV=0.97, 95%CI: 0.85-0.99). CONCLUSION: The short term ESD training course in live porcine models made a significant improvement on ESD skills regarding on resection time, bleeding rate, insecurity and increased depth of submucosal resection. Association between the depths of submucosal resection with the incidence of bleeding might be significant, which means that deeper in the submucosal layer undergoes the procedure; lower will be the risk of bleeding

INTRODUÇÃO: A lesão da medula espinal é um dos grandes desafios da medicina. Apesar de décadas de pesquisa sobre o assunto, seu tratamento ainda não é satisfatório. A padronização de modelos de lesão da medula espinal permite a reprodutibilidade e a análise dos resultados sendo importante para a pesquisa sobre o tema. OBJETIVO: Validar a padronização de um modelo de lesão da medula espinal e avaliação da lesão neurológica em camundongos. MÉTODOS: Submetemos 30 camundongos BalbC divididos em 4 grupos experimentais e um grupo controle à lesão da medula espinal torácica por queda de peso de diferentes alturas (gerando lesões de graus variados). O grupo controle (SHAM) foi submetido apenas à laminectomia. Os camundongos foram avaliados por seis semanas durante as quais foram aplicadas escalas de avaliação funcional motora. Após seis semanas os animais foram sacrificados para avaliação histológica das medulas espinais lesadas. Os achados foram correlacionados entre si para validar se a lesão foi efetiva e se os grupos diferenciaram-se entre os diferentes graus de lesão. Adicionalmente avaliamos se as escalas utilizadas são aplicáveis e se são fiéis aos achados histológicos. RESULTADOS: Seis dos trinta camundongos do experimentos evoluíram para óbito sendo um do Grupo 3, um do Grupo 4 e quatro do Grupo 5. Um camundongo do Grupo 4 apresentou autofagia. O Grupo 5 foi excluído do experimento por alta mortalidade e perda de dados. Todas as escalas funcionais estudadas foram estatisticamente diferentes entre si e demonstraram evolução durante o experimento. Os achados foram confirmados por histologia e apresentaram uma correlação forte com as escalas BBB e BMS e moderada a forte com a escala MFS. A Escada Horizontal apresentou forte correlação com a degeneração neurológica porém não apresentou correlação com os demais parâmetros histológicos estudados. CONCLUSÃO: O modelo de lesão da medula espinal em camundongos apresentado neste estudo é efetivo, confiável e reprodutível, com exceção da lesão causada por queda de peso (10g) de 50mm de altura, que traz mortalidade inaceitável. Das escalas estudadas, BBB e BMS são as mais confiáveis, enquanto que a Escada Horizontal tem seu uso discutível / INTRODUCTION: Spinal cord lesion is a great medical challenge. Even with many decades of research, no satisfactory treatment is available yet. The standardization of animal experimentation models makes the spinal cord lesion reproducible allowing a reliable analysis of the results. Hence, standardization is a major concern in spinal cord lesion research. OBJECTIVE: To validate the standardization of a spinal cord lesion model with neurologic evaluation using mice. METHODS: Thirty BalbC mice were divided in four experimental groups and one control group and submitted to spinal cord lesion produced by weight drop from different heights (producing different severity lesions). The control group (SHAM) was submitted to laminectomy only. Every mice was followed up for six weeks during which functional motor scales were applied. After six weeks the animals were sacrificed for histological examination. Findings were correlat-ed to confirm if the spinal cord lesion was effective and if the groups were dif-ferent between themselves. Additionally all functional motor scales were corre-lated with the histological findings to confirm if the scales are reliable and truly represented the spinal cord lesion. RESULTS: Six mice died during the experi-mentation period (one mouse from the Group 3, one mouse from the Group 4 and four mice from Group 5). One mouse from Group 4 presented autophagia and was excluded from the experiment. Group 5 was excluded from the exper-iment for high mortality rates and data loss. All functional motor scales applied demonstrated significant results with moderate or strong correlation with the histological findings. The Horizontal Ladder scale had strong correlation with neurologic degeneration but had weak or worse correlation with the rest of the histological parameters studied. CONCLUSION: The spinal cord lesion model using mice presented in this study is reliable and reproducible, excluding the lesion produced by a weight drop (10g) from 50mm, which brings unacceptable mortality rate. Of all fuctional motor scales studied, BBB and BMS scales are the most reliable. The use of the Horizontal Ladder scale, however, must be carefully evaluated

吸煙是引起骨質疏鬆症的因素之一。臨床研究清楚表明吸煙者的骨密度降低，但其他干擾因素可能掩蓋了吸煙對骨頭的不良效果。使用動物模型用以研究吸煙和骨質疏鬆症之間是否有直接的因果關係與它潛在的機制是有必要的。為此，我們使用年輕和雌激素耗盡的小鼠作被動吸煙模型以及小鼠成骨細胞和破骨細胞株來作研究。 / 年輕的Balb/c小鼠暴露於2%或4% (v/v)的香煙煙霧中，代表中度和重度吸煙的人。骨代謝生物標誌物明顯增加，4%吸煙組在14週後股骨的微觀結構4%顯著下降，這相等於人類吸煙12年。此外，雌性Balb/c小鼠進行4%吸煙和/或卵巢切除術(OVX)。吸煙+OVX組增加血清中骨轉換指標水平。4%吸煙組的股骨生長板較薄。μ-CT數據進一步表明，相對骨體積(BV / TV)和結構模型指數(SMI)在吸煙組有顯著影響，而且在吸煙+ OVX組上有更大程度的影響。 / 在細胞研究中使用氯仿(CSE)和乙醇的香煙提取物(ESE)。CSE抑制小鼠細胞株RAW 264.7形成破骨細胞，並刺激小鼠成骨細胞株的分化和功能。這個與體內研究矛盾的結果暗示直接從煙霧中提取的化學成分並不是引起骨質疏鬆的元兇。影響骨代謝的很可能是其他從煙霧中生成的活性代謝物和一些吸煙引起的內源性激素物質。在吸煙引起的骨質流失中，這些代謝物或內源性物質可能是非常重要的。 / 有見及此，4%吸煙小鼠的血清用以研究其對成骨細胞和破骨細胞活動的影響。吸煙小鼠血清顯著降低在成骨細胞中鹼性磷酸酶(ALP)活性和鈣沉積，一些成骨細胞標記基因和蛋白表達均下降，而且 Wnt/β-catenin信號通路下調。此外，吸煙小鼠血清顯著增加形成破骨細胞的數量，組織蛋白酶K的基因和蛋白表達增加，在NF-κB和p-38 MAPK信號傳導途徑的信號分子表達增加。 / 總而言之，大量吸煙可能影響年輕小鼠和雌激素耗竭小鼠的骨代謝和微結構，通過類似的行動機制，人類也可能有同樣的骨疾病風險。這項研究揭示了吸煙導致的骨質疏鬆症在青少年和絶經後婦女的發病機制。這也給我們線索如何預防和治療與吸煙有關的骨骼疾病。這項研究還傳達了一個明確的信息：在年輕時應開始應控制吸煙。 / Cigarette smoking is one of the risk factors for osteoporosis. Clinical studies clearly showed that smokers have lower bone mineral density, but other confounding factors could mask the adverse actions of smoking on bones. Animal models are warranted to study the direct causal relationship between cigarette smoking and osteoporosis, and also the underlying mechanisms. In this regard, we used a mouse passive smoking model in both young and estrogen depleted mice, and the mouse osteoblast and osteoclast cell lines. / Young Balb/c mice were exposed to 2 or 4% (v/v) of cigarette smoke, similar to moderate or heavy smoking respectively in humans. Biomarkers for bone turnover were increased and bone microstructure of femur was significantly deteriorated after 4% smoking for 14 weeks which is similar to cigarette smoking for 12 years in humans. Furthermore, the effects of heavy smoking on ovariectomized mice were also investigated. Female Balb/c mice were subjected to 4% cigarette smoking and/or ovariectomy (OVX). Cigarette smoking together with OVX further increased the levels of bone turnover markers in serum. Femur growth plate was thinner in the 4% smoking group when compared to those in the SHAM- and OVX-operated groups. Micro-CT data further indicated that the relative bone volume (BV/TV) and structural model index (SMI) were significantly affected in the smoking groups, and to a greater extent in the 4% smoking + OVX group. / Chloroform (CSE) and ethanol smoke extracts (ESE) were used in cell studies. CSE suppressed the formation of osteoclasts, and stimulated the differentiation and function of mouse osteoblasts. These findings are contradictory to those found in in vivo study implying that chemical components directly extracted from cigarette smoke are not the culprits in causing bone disorder in animals. It is likely that other active metabolites from cigarette smoke and some endogenous hormonal substances released by cigarette smoking could affect bone metabolism. These active metabolites together with the endogenous bone hormones are perhaps crucial in smoking-induced bone loss in the body. / In view of this hypothesis, sera from 4% smoking mice were used to investigate their effects on osteoblast and osteoclast activities. It was found that the alkaline phosphatase (ALP) activity and calcium deposition in osteoblast were reduced significantly by the sera from smoking mice. Gene and protein expressions of some osteoblast markers were also decreased. The downregulation of Wnt/β-catenin signaling pathway was observed after the treatment with the serum obtained from the 4% smoking group. Moreover, the number of osteoclasts being formed was increased significantly by the smoking mouse serum. Cathepsin K gene and protein expressions were also induced. The increased expressions of the signaling molecules including NF-κB and p-38 MAPK were also observed. / In conclusion, heavy cigarette smoking could deteriorate bone metabolism and microstructures in young female and also estrogen depleted mice. The same kind of risk in bone disease may also apply to humans through similar mechanisms of action. This study sheds light in understanding the pathogenesis of smoking-induced bone disorders in teenagers and also postmenopausal women. It also gives us clues how to prevent and treat smoking related bone diseases. This study also conveys a clear message that cigarette smoking control should be started in young ages. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Lok Yi Ruby. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 182-207). / Abstracts also in Chinese. / Chan, Lok Yi Ruby.

From the individual to species level, it is common for animals to have connections with one another. These connections can exist in a variety of forms; from the social relationships within an animal society, to hybridisation between species. The structure of these connections in animal systems can be depicted using networks, often revealing non-trivial structure which can be biologically informative. Understanding the factors which drive the structure of animal networks can help us understand the costs and benefits of forming and maintaining relationships. Multivariate modelling provides a means to evaluate the relative contributions of a set of explanatory factors to a response variable. However, conventional modelling approaches use statistical tests which are unsuitable for the dependencies inherent in network and relational data. A solution to this problem is to use specialised models developed in the social sciences, which have a long history in modelling human social networks. Taking predictive multivariate models from the social sciences and applying them to animal networks is attractive given that current analytical approaches are predominantly descriptive. However, these models were developed for human social networks, where participants can self-identify relationships. In contrast, relationships between animals have to be inferred through observations of associations or interactions, which can introduce sampling bias and uncertainty to the data. Without appropriate care, these issues could lead us to make incorrect or overconfident conclusions about our data. In this thesis, we use an established network model, the multiple regression quadratic assignment procedure (MRQAP), and propose approaches to facilitate the application of this model in animal network studies. Through demonstrating these approaches on three animal systems, we make new biological findings and highlight the importance of considering data-sampling issues when analysing networks. Additionally, our approaches have wider applications to animal network studies where relationships are inferred through observing dyadic interactions.

Chan Tai-po. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 94-101). / Abstracts in English and Chinese. / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 2 --- HYPOTHESIS --- p.3 / Chapter 3 --- OBJECTIVE --- p.4 / Chapter 4 --- CLINICAL IMPLICATIONS --- p.5 / Chapter 5 --- METHODOLOGY --- p.6 / Chapter 5.1 --- Materials --- p.8 / Chapter 5.2 --- Study method --- p.13 / Chapter 5.3 --- Venues of the research --- p.22 / Chapter 5.4 --- Data acquisition --- p.23 / Chapter 5.5 --- Data management and analysis --- p.24 / Chapter 5.6 --- Ethical considerations --- p.25 / Chapter 5.7 --- Participations of persons in the research --- p.28 / Chapter 6 --- RESULTS --- p.34 / Chapter 6.1 --- Problems and fate of rats in the model development group --- p.34 / Chapter 6.2 --- Morbidity and mortality after LEM administration --- p.38 / Chapter 6.3 --- Results of radiological findings --- p.39 / Chapter 6.4 --- Results of liver function tests --- p.48 / Chapter 6.5 --- Results of liver morphology --- p.52 / Chapter 6.6 --- Histological results --- p.53 / Chapter 7 --- DISCUSSION --- p.69 / Chapter 7.1 --- Problems encountered in the development group --- p.69 / Chapter 7.2 --- The pilot study group --- p.71 / Chapter 7.3 --- The need for the present study --- p.74 / Chapter 7.4 --- LEM in cirrhotic rat compared with the normal liver rat --- p.75 / Chapter 7.5 --- Liver function markers in cirrhotic liver --- p.76 / Chapter 7.6 --- Discussion on the assumptions of the research --- p.80 / Chapter 7.7 --- Assessment on measurement error --- p.82 / Chapter 7.8 --- Errors in the pilot study --- p.83 / Chapter 8 --- CONCLUSIONS --- p.84 / Chapter 9 --- Future experiments that may be performed using this model --- p.85 / Chapter 10 --- APPENDICES --- p.86 / Chapter 10.1 --- Appendix 1: Copy on the letter of ethics approval from the Animal Research Ethics Committee of the Chinese University of Hong Kong --- p.86 / Chapter 10.2 --- Appendix 2: Copy on the licences issued by the Department of Health of Hong Kong --- p.88 / Chapter 11 --- REFERENCES --- p.94

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).

Cheung Shu Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 124-137). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of Breast Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology of Breast Cancer in Females --- p.1 / Chapter 1.1.2 --- Incidence and Morality of Female Breast Cancer in Hong Kong --- p.2 / Chapter 1.1.3 --- Epidemiology of Breast Cancer in Males --- p.3 / Chapter 1.2 --- Risk Factors for Female Breast Cancer --- p.4 / Chapter 1.2.1 --- Genetic Risk Factors --- p.4 / Chapter 1.2.2 --- Hormonal Risk Factors --- p.6 / Chapter 1.2.2.1 --- Endogenous Hormonal Risk Factors --- p.7 / Chapter 1.2.2.2 --- Exogenous Hormonal Risk Factors --- p.8 / Chapter 1.2.3 --- Other Environmental Risk Factors --- p.9 / Chapter 1.3 --- Oncogenetic Basis of Female Breast Cancer --- p.10 / Chapter 1.4 --- Hormonal Basis of Female Breast Cancer --- p.12 / Chapter 1.4.1 --- Mechanisms of Hormone Action --- p.12 / Chapter 1.4.1.1 --- Estrogen and Progesterone --- p.12 / Chapter 1.4.1.2 --- Prolactin --- p.14 / Chapter 1.4.2 --- Hormonal Regulation of Normal Breast Development --- p.15 / Chapter 1.4.3 --- Hormonal Regulation of Breast Carcinogensis and Its Subsequent Progression --- p.17 / Chapter 1.4.3.1 --- Androgen --- p.17 / Chapter 1.4.3.2 --- Estrogen --- p.18 / Chapter 1.4.3.3 --- Progesterone --- p.20 / Chapter 1.4.3.4 --- Prolactin --- p.22 / Chapter 1.5 --- Animal Models for Breast Cancer --- p.23 / Chapter 1.5.1 --- Mouse Models --- p.24 / Chapter 1.5.2 --- Rat Models --- p.25 / Chapter 1.5.2.1 --- Carcinogen Induced Rat Models --- p.26 / Chapter 1.5.2.2 --- Hormone Induced Rat Models --- p.28 / Chapter 1.5.2.3 --- Spontaneously Developed Rat Models --- p.31 / Chapter 1.6 --- Aims of Study --- p.34 / Tables and Figures --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rats --- p.37 / Chapter 2.2 --- Supply of Materials --- p.37 / Chapter 2.3 --- Induction of Mammary Tumors by Singe Dose of Chemical Carcinogens in Female Rats --- p.38 / Chapter 2.3.1 --- Induction by 7，12-Dimethylbenz[a]anthracene in Female Noble Rats --- p.38 / Chapter 2.3.2 --- Induction by N-Methyl-N-Nitrosourea in Female Sprague- Dawley Rats --- p.38 / Chapter 2.4 --- Induction of Mammary Tumors by Long-Term Treatments with Steroid Hormone --- p.39 / Chapter 2.4.1 --- Preparation of Steroid Hormone-filled Silastic® Tubings --- p.39 / Chapter 2.4.2 --- Surgical Implantation of Silastic® Tubings --- p.40 / Chapter 2.4.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.5 --- Collection of Spontaneously Developed Mammary Tumors in Noble Rats --- p.41 / Chapter 2.6 --- Transplantation of Spontaneously Developed Mammary Tumors into Noble Rats --- p.41 / Chapter 2.7 --- Bilateral Ovariectomy of Female Noble Rats bearing Spontaneously Developed Mammary Tumors --- p.42 / Chapter 2.8 --- Measurement of Mammary Tumor Growth --- p.43 / Chapter 2.9 --- Whole Mount Preparation of the Hormone-Treated Mammary Glands in Noble Rats --- p.44 / Chapter 2.10 --- Histological Examination of Mammary Gland and Tumors in Noble Rats --- p.45 / Chapter 2.11 --- Detection of Protein Expression of Hormone Receptors in Normal Mammary Glands and Mammary Tumors of Noble Rats --- p.45 / Chapter 2.11.1 --- Antibodies --- p.45 / Chapter 2.11.2 --- Immunohistochemistry --- p.47 / Chapter 2.11.3 --- "Protein extraction, SDS-PAGE and western blotting analysis" --- p.48 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Gross Appearance of Mammary Tumors --- p.51 / Chapter 3.2 --- Incidence Rate of Mammary Tumors --- p.53 / Chapter 3.2.1 --- Spontaneously Developed Mammary Tumors in Noble Rats --- p.53 / Chapter 3.2.2 --- Hormone Induced Mammary Tumors in Female Noble Rats --- p.53 / Chapter 3.2.3 --- DMBA Induced Mammary Tumors in Female Noble Rats --- p.54 / Chapter 3.2.4 --- NMU Induced Mammary Tumors in Female SD Rats --- p.54 / Chapter 3.3 --- Histology of Normal and Lactating Mammary Glands in Female Noble Rats --- p.54 / Chapter 3.4 --- Histopathology of Mammary Tumors --- p.55 / Chapter 3.4.1 --- Histopathology of Spontaneously Developed Mammary Tumors in Noble Rats --- p.55 / Chapter 3.4.2 --- Histopathology of Hormone Induced Mammary Tumors in Female Noble Rats --- p.59 / Chapter 3.4.3 --- Histopathology of DMBA Induced Mammary Tumors in Female Noble Rats --- p.60 / Chapter 3.4.4 --- Histopathology of NMU Induced Mammary Tumors in Female SD Rat --- p.60 / Chapter 3.5 --- Whole Mount Preparation of Mammary Glands under Hormonal Treatments --- p.61 / Chapter 3.6 --- Effects of Bilateral Ovariectomy on the Growth of Spontaneously Developed Mammary Tumors --- p.61 / Chapter 3.7 --- Transplanability of the Spontaneously Developed Mammary Tumors in Noble Rats --- p.62 / Chapter 3.8 --- Examination of the Malignancy of Mammary Tumors by Immunohistochemical analysis of Epithelial Keratin Expression --- p.62 / Chapter 3.9 --- Immunohistochemical Analysis of Expression and Localization of Hormone Receptor Protein in Normal and Neoplastic Mammary Tissues of Female Noble Rats --- p.63 / Chapter 3.9.1 --- Expression and Localization of Hormone Receptors in Control Tissue --- p.63 / Chapter 3.9.2 --- Expression and Localization of Estrogen Receptor α --- p.64 / Chapter 3.9.3 --- Expression and Localization of Estrogen Receptor β --- p.65 / Chapter 3.9.4 --- Expression and Localization of Progesterone Receptor --- p.65 / Chapter 3.9.5 --- Expression and Localization of Androgen Receptor --- p.66 / Chapter 3.9.6 --- Expression and Localization of Prolactin Receptor --- p.66 / Chapter 3.10 --- Western Blot Analysis of Expression of Hormone Receptor Proteins in Normal and Neoplastic Mammary Tissues of Female Noble Rats - --- p.67 / Chapter 3.10.1 --- Expression of Estrogen Receptor α --- p.67 / Chapter 3.10.2 --- Expression of Estrogen Receptorβ --- p.68 / Chapter 3.10.3 --- Expression of Progesterone Receptor --- p.68 / Chapter 3.10.4 --- Expression of Androgen Receptor --- p.69 / Chapter 3.10.5 --- Expression of Prolactin Receptor --- p.69 / Figures and Tables --- p.71 / Chapter Chapter 4 --- Discussions / Chapter 4.1 --- Comparison of the Incidence Rate of Spontaneously developed Mammary Tumors in Noble Rats with the Previously Reported Incidence Rate --- p.102 / Chapter 4.2 --- Comparison of the Incidence rate of Spontaneously Developed Mammary Tumors in Noble Rats with the Incidence Rate in Other Rat Strains --- p.103 / Chapter 4.3 --- Crucial Factors Influencing the Incidence Rate of Spontaneously Developed Mammary Tumors in Noble Rats --- p.104 / Chapter 4.4 --- Comparison of the T+E2 Induced Mammary Tumors with the T+DES Induced Mammary Tumors in Female Noble Rats --- p.105 / Chapter 4.5 --- Comparison of the Incidence Rate & Latency Period of the Hormone Induced Mammary Tumors in Noble Rats with the Previously Reported Data --- p.106 / Chapter 4.6 --- Comparison of the Phenotypic Behaviors in Spontaneously Developed Mammary Tumors with the Hormone Induced Mammary Tumors in Female Noble Rats --- p.107 / Chapter 4.7 --- Comparison of the Behaviors of Carcinogen Induced Mammary Tumors with Spontaneously Developed & Hormone Induced Mammary Tumors in Female Noble Rats --- p.109 / Chapter 4.8 --- "Comparison of Expression Patterns of Hormone Receptor Proteins in Spontaneously Developed, Hormone Induced & Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.111 / Chapter 4.9 --- "Expressions of ERα & ERβ Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.112 / Chapter 4.10 --- "Expressions of PR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.115 / Chapter 4.11 --- "Expressions of AR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.116 / Chapter 4.12 --- "Expressions of PRLR Proteins in Spontaneously Developed, Hormone Induced and Carcinogen Induced Mammary Tumors in Female Noble Rats" --- p.120 / Chapter Chapter 5 --- Conclusions --- p.123 / References --- p.124

Submitted by LUCIANA ARTIOLI COSTA null (lu_artioli@hotmail.com) on 2017-02-01T16:07:56Z No. of bitstreams: 1 tese Luciana Artioli para impressão com ficha catalográfica.pdf: 2502803 bytes, checksum: 525c27910354db7a14fa6e2425b7e7aa (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-03T19:39:54Z (GMT) No. of bitstreams: 1 costa_la_dr_araca.pdf: 2502803 bytes, checksum: 525c27910354db7a14fa6e2425b7e7aa (MD5) / Made available in DSpace on 2017-02-03T19:39:54Z (GMT). No. of bitstreams: 1 costa_la_dr_araca.pdf: 2502803 bytes, checksum: 525c27910354db7a14fa6e2425b7e7aa (MD5) Previous issue date: 2016-12-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Objetivo: Avaliar os efeitos da movimentação dentária induzida (MDI) em molares de ratos em função do tempo de reparo pós luxação extrusiva (LE). Métodos: Sessenta e três ratos machos adultos jovens (Rattus norvegicus albinus, Wistar), com 45 dias de idade e faixa de peso entre 230-250, foram distribuídos em nove grupos (n=7): grupos controle (C), trauma de LE e acompanhamento por 3, 5 ou 7 dias (T3D, T5D, T7D), movimentação controle (MC) – MDI por 7 dias,dispositivo controle (DC) – dispositivo de MDI inativo por 7 dias, e grupos submetidos a LE, espera por 3, 5 ou 7 dias e, então, submetidos a MDI por 7 dias (T3D/M, T5D/M, T7D/M). Após o período experimental os animais foram eutanasiados e cortes seriados longitudinais de 4μm, corados com hematoxilina e eosina, foram obtidos. Foram realizadas análises descritivas, semi-quantitativas e histomorfométricas do primeiro molar superior direito. A distribuição dos escores nos grupos foi comparada por meio do teste Kruskal Wallis com comparações múltiplas pelo método de Bonferroni e as análises histomorfométricas avaliadas pelo teste Kruskal Wallis, ambos com nível de significância de 5%. Resultados: Alterações vasculares (hemorragia) no ligamento periodontal e reabsorções radiculares foram observadas com maior frequência nos animais do grupo T3D/M, na face mesial da raiz disto-vestibular, com diferença significante (p<0.05) em relação aos controles. Conclusão: Quando o tempo de reparo do ligamento periodontal pós LE não é respeitado para o inicio da MDI, observa-se uma maior frequência de reabsorção radicular e alterações vasculares. / Objective: To evaluate the effects of induced tooth movement (ITM) on rat molars related to the time of repair after extrusive luxation (EL) trauma Methods: Sixty three male young adults rats (Rattus norvegicus Albinus, Wistar), 45 days old and weighing range of 230-250 were divided into nine groups (n = 7): control (C), EL trauma and follow-up for, 3, 5 or 7 days (T3D, GT5D, T7D), control movement (CM) – MDI for seven days, control device (CD) - MDI device inactive for 7 days, and groups submitted to LE, hold for 3, 5 or 7 days and, then, ITM for 7 days (T3D/M T5D/M T7D/M). After the experimental period, animals were euthanized and longitudinal serial sectioning of 4μm, stained with hematoxylin and eosin, were obtained. Descriptive, semi-quantitative and histomorfometrics analyzes of the right upper first molar were made. The distribution of scores in the groups was compared using the Kruskal Wallis test with multiple comparisons by Bonferroni method and histomorphometric analysis evaluated by Kruskal Wallis test, both with 5% significance level. Results: Vascular changes (bleeding) in the periodontal ligament and root resorption were observed more frequently in animals of T3D/M group, on mesial side of distobuccal root, with a significant difference (p<0.05) in relation to the controls. Conclusion: When the repair time of the periodontal ligament after LE is not respected for MDI beginning, a greater frequency of root resorption and vascular alterations is observed.