<p>Estuaries interfacing with the land, atmosphere and open oceans can be influenced in a variety of ways by anthropogenic activities. Centuries of overexploitation, habitat transformation, and pollution have degraded estuarine ecological health. Key concerns of public and environmental managers of estuaries include water quality, particularly the enrichment of nutrients, increased chlorophyll a concentrations, increased hypoxia/anoxia, and increased Harmful Algal Blooms (HABs). One reason for the increased nitrogen loading over the past two decades is the proliferation of concentrated animal feeding operations (CAFOs) in coastal areas. This dissertation documents a study of estuarine eutrophication modeling, including modeling of major source of nitrogen in the watershed, the use of the Bayesian Networks (BNs) for modeling eutrophication dynamics in an estuary, a documentation of potential problems of using BNs, and a continuous BN model for addressing these problems.</p><p>Environmental models have emerged as great tools to transform data into useful information for managers and policy makers. Environmental models contain uncertainty due to natural ecosystems variability, current knowledge of environmental processes, modeling structure, computational restrictions, and problems with data/observations due to measurement error or missingness. Many methodologies capable of quantifying uncertainty have been developed in the scientic literature. Examples of such methods are BNs, which utilize conditional probability tables to describe the relationships among variables. This doctoral dissertation demonstrates how BNs, as probabilistic models, can be used to model eutrophication in estuarine ecosystems and to explore the effects of plausible future climatic and nutrient pollution management scenarios on water quality indicators. The results show interaction among various predictors and their impact on ecosystem health. The synergistic eftects between nutrient concentrations and climate variability caution future management actions.</p><p>BNs have several distinct strengths such as the ability to update knowledge based on Bayes' theorem, modularity, accommodation of various knowledge sources and data types, suitability to both data-rich and data-poor systems, and incorporation of uncertainty. Further, BNs' graphical representation facilitates communicating models and results with environmental managers and decision-makers. However, BNs have certain drawbacks as well. For example, they can only handle continuous variables under severe restrictions (1- Each continuous variable be assigned a (linear) conditional Normal distribution; 2- No discrete variable have continuous parents). The solution, thus far, to address this constraint has been discretizing variables. I designed an experiment to evaluate and compare the impact of common discretization methods on BNs. The results indicate that the choice of discretization method severely impacts the model results; however, I was unable to provide any criteria to select an optimal discretization method.</p><p>Finally, I propose a continuous variable Bayesian Network methodology and demonstrate its application for water quality modeling in estuarine ecosystems. The proposed method retains advantageous characteristics of BNs, while it avoids the drawbacks of discretization by specifying the relationships among the nodes using statistical and conditional probability models. The Bayesian nature of the proposed model enables prompt investigation of observed patterns, as new conditions unfold. The network structure presents the underlying ecological ecosystem processes and provides a basis for science communication. I demonstrate model development and temporal updating using the New River Estuary, NC data set and spatial updating using the Neuse River Estuary, NC data set.</p> / Dissertation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Present study had examined the influence of the sympathetic modulation of the hypoxic respiratory pattern in patients with chronic obstructive pulmonary disease (COPD). 12 were evaluated with the disease and, for comparative purposes, 10 healthy patients also participated. The subjects were assessed for Heart Rate Variability (HRV) through Finapress, and concurrently monitored oxygen saturation and respiratory rate, for 5 minutes at rest. After this evaluation, spirometry was performed on each patient, resulting in the forced expiratory volume values in one second (FEV1) and the FEV1 and the Forced Vital Capacity (FVC). When correlated the levels of Oxygen Saturation (SpO2) and Tiffeneau index, there is direct variation between spirometric index and SpO2 (ρ = 0.45). The reverse occurs between systolic blood pressure (SBP) and SpO2 and FEV1 and band Lf SAP (ρ = -0.65). When analyzed HRV in the time domain, a statistically significant difference between groups for the band Lf (p = 0.01) and LF / HF ratio (p = 0.02). It was seen that, when compared to healthy subjects, patients with COPD had lower values of HRV increased SBP and decrease in oxygen saturation (SpO2) and spirometric indices. Still, the drop in autonomic function seems to be more impaired in patients where FEV1 indexes and the FEV1 / FVC are lower. Apparently, the drop in SpO2, due to the loss in gas exchange, seems to increase the sympathetic modulation, increasing the SBP levels. / O presente estudo analisou os determinantes da modulação simpática nos pacientes com Doença Pulmonar Obstrutiva crônica (DPOC). Foram avaliados 12 pacientes portadores da doença e, para fins comparativos, 10 pacientes saudáveis. Os sujeitos foram submetidos à avaliação da Variabilidade da Frequência Cardíaca (VFC) através do aparelho finapress, sendo concomitantemente monitorada a saturação de oxigênio e frequência respiratória, durante 5 minutos, em seu estado de repouso. Após esta avaliação, foi realizada a espirometria de cada paciente, obtendo-se os valores de Volume Expiratório Forçado no primeiro segundo (VEF1) e a relação entre VEF1 e a Capacidade Vital Forçada (CVF). Quando correlacionados os índices de Saturação de Oxigênio (SatO2) e índice de Tiffenau, observa-se variação direta entre o índice espirométrico e a SatO2 (ρ = 0,45). O inverso ocorre entre a Pressão arterial sistólica (PAS) e SatO2 e VEF1 e banda de baixa frequência (Lf) da PAS (ρ = -0,65). Ao analisar-se a VFC no domínio da frequência, houve diferença estatisticamente significante entre os grupos para a banda Lf (p = 0,01) e no balanço simpato-vagal (p=0,02). Quando comparados aos sujeitos saudáveis, pacientes com DPOC apresentaram menores valores de VFC, aumento da PAS, bem como queda na Saturação de Oxigênio (SatO2) e nos índices espirométricos. Ainda, a queda na função autonômica parece estar relacionada aos índices de VEF1 e a relação VEF1/CVF, quando do decréscimo destes. Ainda, ao que parece, a queda na SatO2, decorrente do prejuízo nas trocas gasosas, parece aumentar a modulação simpática, elevando os níveis de PAS.

A anóxia neonatal, considerada problema clínico mundial, é importante causa de lesão encefálica em neonatos que pode apresentar consequências graves e permanentes, como déficits cognitivos e comportamentais. O objetivo deste estudo foi analisar longitudinalmente possíveis alterações na morte, proliferação e diferenciação neuronais no hipocampo de ratos submetidos à anóxia neonatal. Para tanto, utilizamos modelo adaptado e validado em nosso laboratório. Os resultados mostraram que a anóxia neonatal causa morte neural em CA1 e CA2-3, detectadas pela maior quantidade de células TUNEL+ em CA1 e CA2-3 e FJB+ em CA2-3, além de diferentes tipos de morte neuronal em CA1 e GD, 24 horas após a anóxia,observadas por microscopia eletrônica. Houve aumento do volume de CA1 em P14 no grupo anóxia, porém o padrão de proliferação na zona subgranular não foi alterado. Enfim, a anóxia neonatal promoveu diminuição da neurogênese em animais adultos, o que poderia estar associado aos déficits de memória espacial e aprendizagem descritos em literatura para modelos similares. / Neonatal anoxia, considered a worldwide clinical problem, is a major cause of brain injury in neonates and may present serious and permanent consequences such as cognitive and behavioral deficits. The aim of this study was to analyze possible changes longitudinally in neural death, proliferation and neuronal differentiation in the hippocampus of rats submitted to neonatal anoxia. We used an adapted model validated in our laboratory. The results showed that neonatal anoxia cause neural death in CA1 and CA2-3 detected by the TUNEL+ cells in CA1 and CA2-3 and FJB+ in CA2-3, and different types of neuronal death in CA1 and GD 24 hours of anoxia, observed by electron microscopy. There was an increase in the volume of CA1 in the P14 anoxia group but the pattern of proliferation in the subgranular zone was not changed. Anyway, neonatal anoxia caused decrease in neurogenesis in adult animals, which could be associated with deficits in spatial memory and learning described in the literature in similar models.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-26T14:14:15Z No. of bitstreams: 2 Pâmela_Buratti2017.pdf: 1971935 bytes, checksum: 016f66981771eed7ce5ec50ed236c207 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-26T14:14:15Z (GMT). No. of bitstreams: 2 Pâmela_Buratti2017.pdf: 1971935 bytes, checksum: 016f66981771eed7ce5ec50ed236c207 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-05-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cerebral palsy (CP) corresponds to a motor disorder, caused by non-progressive injury in the immature brain, resulting in changes in muscle tone, movement and postureThis study aimed to investigate the morphofunctional characteristics of the plantar muscle in an animal model of CP. To obtain the litters were used for paired adult Wistar rats (11 females and 06 males). The pregnant females were separated in: rats injected intraperitoneally with vehicle (100 μL of sterile saline) and rats injected intraperitoneally with lipopolysaccharide (LPS; 200 μg / kg LPS in 100 μL sterile saline). The injections were performed every 12 hours, from the 17th gestational day until the end of gestation (21st gestational Day). The male offspring were separated in: Control group (CG, n = 8) - pups of rats injected with saline solution during pregnancy, and CP group (CPG, n = 8) - pups of rats injected with LPS during pregnancy, submitted to perinatal anoxia and sensorimotor restriction. For perinatal anoxia, the pups were placed in a closed chamber, partially immersed in water at 37 °C ± 1, with a flow of 9 L/min of nitrogen (100%) for 20 minutes on the day of birth (postnatal day 0, P0). From the 1st to the 30th postnatal day (P1 to P30), the CPG animals were submitted to sensorimotor restriction for 16 hours/day. At 29 and 45 days of age, the animals were evaluated in the open-field test, being collected time of displacement, crossing, rearing and grooming frequency. At the 48th postnatal day the animals were weighed and the collection, measurement of weight and muscle length were performed. The muscle fibers were evaluated in histological sections submitted to Hematoxylin-Eosin (HE), NADH-TR reaction (Nicotinamide Adenine Dinucleotide - Tetrazolium Redutase) and transmission electron microscopy; intramuscular collagen was studied with Masson's trichrome staining; and the neuromuscular junctions (NMJs) ascertained by the Nonspecific Esterase reaction. The data were statistically evaluated using the parametric Student t-test and the non-parametric Mann-Whitney test, according to the Kolmogorov-Smirnov normality test.It was verified that when compared to CG the CPG presented: reduction in time of displacement, number of crosses and rearing at 29 days of age; in the time of displacement and rearing at 45 days of age; of body parameters (body weight, weight and length soleus muscle); of the nuclei/fiber and capillary/fiber relations; increased percentage of collagen; reduction of cross-sectional area of muscle fibers type I; increased counts of fibers type I; in the number of myofibrillar disorganization, of the Z line and dissolution of the Z line; and reduction in area, larger and smaller diameters of NMJs. It is concluded that the PC model used altered the morphological characteristics of the skeletal striated muscle, causing deficits in the locomotor activity of the animals, as evidenced by the results of the evaluation in the open field. / A paralisia cerebral (PC) corresponde a uma desordem motora, ocasionada por lesão não progressiva no cérebro imaturo, por conseguinte, acarreta alterações no tônus muscular, movimento e postura. Este estudo objetivou investigar as características morfofuncionais do músculo plantar em um modelo animal de PC. Na obtenção das ninhadas, ratos Wistar adultos foram utilizados para o pareamento (11 fêmeas e 06 machos) e as fêmeas prenhas foram agrupadas em: ratas injetadas intraperitonealmente com veículo (100 μL de solução salina estéril); e ratas injetadas intraperitonealmente com lipopolissacarídeo (LPS; 200 μg/kg de LPS em 100 μL de solução salina estéril), a cada 12 horas, a partir do 17º dia gestacional até o final da gestação (21º dia gestacional). Os filhotes machos foram separados em: Grupo controle (GC, n= 8), filhotes de mães injetadas com solução salina durante a gestação; e Grupo PC (GPC, n = 8), filhotes de mães injetadas com LPS na gestação, submetidos à anóxia perinatal e restrição sensório-motora. Para a anóxia perinatal, os filhotes foram colocados em uma câmara fechada, parcialmente imersa em água a 37°C±1, com fluxo de 9 L/min de nitrogênio (100%) durante 20 minutos no dia do nascimento (dia pós-natal 0, P0). Do 1º ao 30º dia pós-natal (P1 até P30), os animais do GPC foram submetidos à restrição sensório-motora durante 16 horas/dia. E, aos 29 e 45 dias de vida, os animais foram avaliados no teste de campo aberto, coletados os dados de tempo de deslocamento, cruzamento, frequências de erguidas (rearing) e de autolimpeza (grooming). No 48º dia pós-natal, os animais foram pesados e foi realizada a coleta, além das mensurações do peso e do comprimento muscular. As fibras musculares foram avaliadas em secções histológicas submetidas às técnicas de Hematoxilina-Eosina (HE), reação da NADH-TR (Nicotinamida Adenina Dinucleotídeo – Tetrazolium Redutase) e microscopia eletrônica de transmissão. O colágeno intramuscular foi estudado com a coloração de Tricrômico de Masson e as junções neuromusculares (JNMs) foram averiguadas pela reação Esterase Inespecífica. Os dados foram avaliados estatisticamente com o teste paramétrico t de Student e teste não paramétrico Mann-Whitney, de acordo com o teste de normalidade Kolmogorov-Smirnov. Verificou-se que, quando comparado ao GC, o GPC apresentou: redução significativa no tempo de deslocamento, no número de cruzamentos e no rearing aos 29 dias de idade; no tempo de deslocamento e no rearing aos 45 dias de idade; nos parâmetros corporais (peso corporal, peso e comprimento do músculo sóleo); nas relações núcleo/fibra e capilar/fibra; aumento na porcentagem de colágeno; redução da área de secção transversal das fibras musculares tipo I; aumento na contagem de fibras do tipo I; no número de desorganização miofibrilar, da linha Z e dissolução da linha Z; e redução na área, diâmetros maior e menor das JNMs. Conclui-se, portanto, que o modelo de PC utilizado alterou as características morfológicas do músculo estriado esquelético e causou déficits na atividade locomotora dos animas, comprovado pelos resultados da avaliação em campo aberto.

The late Cambrian-early Ordovician transition contains several trilobite extinctions. The first of these extinctions (the end-Marjuman) is thought to coincide with the Steptoean Positive Carbon Isotope Excursion or SPICE, a large and rapid excursion in the marine carbon isotope record. This excursion, which is expressed in sedimentary successions globally, is thought to represent a large perturbation to the carbon cycle during this time. Additionally, a limited amount of carbon isotope data from the Deadwood Formation in the Black Hills of South Dakota suggests the possibility of a small negative ẟ¹³C excursion near the extinction and preceding the SPICE. Previous high-resolution biostratigraphy has identified an expanded record of extinction event within the Nolichucky Formation of the Southern Appalachians making it an excellent candidate for the study of the precise relationship between the extinction and changes in the carbon cycle. This investigation confirms the onset of the SPICE occurs at the extinction boundary however no negative ẟ¹³C excursion occurs at the extinction boundary. Further there is no systematic relationship between local facies changes and ẟ¹³C or the extinction interval across the basin, which suggests that global environmental changes were responsible for both the ẟ¹³C record and the extinction event. / Master of Science

Dong Yingying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 99-125). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgement --- p.ii / Publication list --- p.iii / Abstract (English) --- p.ix / Abstract (Chinese) --- p.xii / Abbreviations --- p.xiv / List of figures / tables --- p.xvi / Chapter Chapter 1. --- General Introduction / Chapter 1.1 --- The role of endothelium in regulating vascular tone --- p.1 / Chapter 1.1.1 --- Nitric oxide (NO) --- p.2 / Chapter 1.1.2 --- Endothelium-derived hyperpolarizing factor (EDHF) --- p.7 / Chapter 1.1.3 --- Prostacyclin (PGI2) --- p.20 / Chapter 1.2 --- EDHF-mediated endothelial function in coronary circulation --- p.22 / Chapter 1.2.1 --- Role of EDHF in coronary microarteries --- p.23 / Chapter 1.2.2 --- Role of EDHF in cardiac veins --- p.24 / Chapter 1.3 --- Effect of ischemia-reperfusion on endothelial function in coronary circulation --- p.25 / Chapter 1.3.1 --- Ischemia-reperfusion injury --- p.26 / Chapter 1.3.2 --- Effect of ischemia-reperfusion on endothelial function in coronary microarteries --- p.28 / Chapter 1.3.3 --- Effect of ischemia-reperfusion on endothelial function in cardiac veins --- p.29 / Chapter 1.4 --- Alteration of endothelial function during cardiac surgery / Chapter 1.4.1 --- Cardioplegia and organ preservation solutions --- p.31 / Chapter 1.4.2 --- Combined effects of hypoxia-reoxygenation and ST solution on endothelial function in coronary microarteries/cardiac veins --- p.34 / Chapter 1.4.3 --- Effect of nicorandil on endothelial function --- p.34 / Chapter Chapter 2. --- Materials and Methods --- p.37 / Chapter 2.1 --- Isometric force study in micro arteries/veins --- p.37 / Chapter 2.1.1 --- Preparation of vessels --- p.37 / Chapter 2.1.1.1 --- Preparation of porcine coronary microarteries --- p.37 / Chapter 2.1.1.2 --- Preparation of porcine cardiac veins --- p.37 / Chapter 2.1.2 --- Technique of setting up --- p.39 / Chapter 2.1.2.1 --- Mounting of microvessels --- p.39 / Chapter 2.1.2.2 --- Normalization procedure for microvessels --- p.39 / Chapter 2.1.3 --- EDHF-mediated vasorelaxation --- p.40 / Chapter 2.1.3.1 --- Precontraction and stimuli of EDHF --- p.40 / Chapter 2.1.3.2. --- “Truéحresponse of EDHF --- p.40 / Chapter 2.1.4 --- Data acquisition and analysis --- p.41 / Chapter 2.2 --- Hypoxia and reoxygenation --- p.41 / Chapter 2.2.1 --- Calibration of 02-special electrode --- p.41 / Chapter 2.2.2 --- Measurement of --- p.02 / Chapter 2.3 --- Statistical analysis --- p.42 / Chapter 2.4 --- Chemicals --- p.43 / Chapter Chapter 3. --- Hypoxia-Reoxygenation in Coronary Microarteries: Combined Effect with St Thomas Cardioplegia and Temperature on the Endothelium- derived Hyperpolarizing Factor and Protective Effect of Nicorandil --- p.44 / Chapter 3.1 --- Abstract --- p.44 / Chapter 3.2 --- Introduction --- p.45 / Chapter 3.3 --- Experimental design and analysis --- p.47 / Chapter 3.3.1 --- Vessel Preparation --- p.47 / Chapter 3.3.2 --- Normalization --- p.48 / Chapter 3.3.3 --- Hypoxia --- p.48 / Chapter 3.3.4 --- Effect of H-R on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.5 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.6 --- Effect of addition of nicorandil Krebs or ST solution under H-R on EDHF-mediated relaxation in coronary microarteries --- p.49 / Chapter 3.3.7 --- Data analysis --- p.50 / Chapter 3.4 --- Results --- p.51 / Chapter 3.4.1 --- Resting force --- p.51 / Chapter 3.4.2 --- U46619-induced contraction force --- p.51 / Chapter 3.4.3 --- Partial pressure of oxygen in hypoxia --- p.51 / Chapter 3.4.4 --- EDHF-mediated relaxation in coronary microarteries --- p.51 / Chapter 3.4.4.1 --- Effect of H-R --- p.51 / Chapter 3.4.4.2 --- Combined effects ofH-R and ST solution on EDHF-mediated relaxation --- p.52 / Chapter 3.4.4.3 --- Effects of addition of nicorandil to Krebs or ST solution under H-R on EDHF-mediated relaxation --- p.52 / Chapter 3.5 --- Discussion --- p.53 / Chapter 3.5.1 --- EDHF-mediated relaxation after exposure to H-R --- p.53 / Chapter 3.5.2 --- EDHF-mediated relaxation after H-R in ST solution at different temperature --- p.54 / Chapter 3.5.3 --- Effect of addition of nicorandil to Krebs or ST solution during H-R on EDHF-mediated relaxation --- p.55 / Chapter 3.5.4 --- Clinical implications --- p.56 / Chapter Chapter 4. --- Hypoxia-Reoxygenation in Cardiac Microveins: Combined Effect with Cardioplegia and Temperature on the Endothelial Function --- p.68 / Chapter 4.1 --- Abstract --- p.68 / Chapter 4.2 --- Introduction --- p.69 / Chapter 4.3 --- Experimental design and analysis --- p.73 / Chapter 4.3.1 --- Vessel Preparation --- p.73 / Chapter 4.3.2 --- Normalization --- p.73 / Chapter 4.3.3 --- Hypoxia --- p.73 / Chapter 4.3.4 --- Effect of H-R on EDHF-mediated relaxation in cardiac micro veins --- p.74 / Chapter 4.3.5 --- Combined effects of H-R and ST solution on EDHF-mediated relaxation in cardiac microveins --- p.74 / Chapter 4.3.6 --- Data analysis --- p.75 / Chapter 4.4 --- Results --- p.75 / Chapter 4.4.1 --- Resting force --- p.75 / Chapter 4.4.2 --- U46619-induced contraction force --- p.76 / Chapter 4.4.3 --- Partial pressure of oxygen in hypoxia --- p.76 / Chapter 4.4.4 --- EDHF-mediated relaxation after H-R in Krebs solution at 37°C --- p.76 / Chapter 4.4.5 --- EDHF-mediated relaxation after exposure to H-R in ST solution at different temperatures --- p.77 / Chapter 4.5 --- Discussion --- p.78 / Chapter 4.5.1 --- Effect of H-R on EDHF-mediated relaxation --- p.78 / Chapter 4.5.2 --- Combined effects of H-R with ST solution on EDHF-mediated relaxation --- p.80 / Chapter 4.5.3 --- Clinical implications / Chapter Chapter 5. --- General Discussion --- p.89 / Chapter 5.1 --- EDHF-mediated endothelial function in porcine coronary circulation --- p.89 / Chapter 5.1.1 --- EDHF in porcine coronary microarteries --- p.92 / Chapter 5.1.2 --- EDHF in porcine cardiac veins --- p.90 / Chapter 5.2 --- Alteration of EDHF-mediated function after exposure to H-R --- p.91 / Chapter 5.2.1 --- In coronary microarteries --- p.91 / Chapter 5.2.2 --- In cardiac veins --- p.92 / Chapter 5.3 --- Alteration of EDHF-mediated function after exposure to ST solution under H-R --- p.92 / Chapter 5.3.1 --- In coronary microarteries --- p.93 / Chapter 5.3.2 --- In cardiac veins --- p.93 / Chapter 5.4 --- EDHF-mediated function in nicorandil-supplemented ST solution under H-R in coronary microarteries --- p.93 / Chapter 5.5 --- Clinical implications / Chapter 5.5.1 --- H-R injury --- p.94 / Chapter 5.5.2 --- H-R injury and cardioplegic solution --- p.95 / Chapter 5.5.2 --- Nicorandil-supplementation in cardioplegic solution --- p.95 / Chapter 5.6 --- Limitation of the study --- p.96 / Chapter 5.7 --- Future investigations --- p.96 / Chapter 5.8 --- Conclusions --- p.97 / References --- p.99

Chau, Kin Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 106-119). / Abstract also in Chinese. / Abstract --- p.II / 摘要 --- p.VI / Acknowledgements --- p.IX / List of figures --- p.X / List of Abbreviations --- p.XII / Table of Contents --- p.XV / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Incidence and etiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Structure of Hepatitis B Virus (HBV) --- p.2 / Chapter 1.3 --- Hepatitis B X protein (HBx) and HCC --- p.5 / Chapter 1.4 --- HBx and Apoptosis --- p.8 / Chapter 1.5 --- The role of Bcl-2 family in apoptosis and cell survival --- p.10 / Chapter 1.6 --- "Bid, the BH3-domain only protein" --- p.14 / Chapter 1.7 --- Dual Functions of Bid --- p.16 / Chapter 1.8 --- The relationship between Bid and HBx --- p.19 / Chapter 1.9 --- Hypoxia and HCC --- p.21 / Chapter 1.10 --- Hypoxia and HBx --- p.25 / Chapter 1.11 --- Hypoxia and Bid --- p.28 / Chapter 1.12 --- Aim of study --- p.29 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Confirmation of the culture of the stable cell lines --- p.30 / Chapter 2.2 --- Doxorubicin treatment to the cell lines --- p.34 / Chapter 2.3 --- Culture of the cell lines under hypoxic conditions --- p.35 / Chapter 2.4 --- Protein sample preparations --- p.37 / Chapter 2.5 --- Determination of protein samples --- p.38 / Chapter 2.6 --- Sodium dodecyl sulfate 226}0ؤ polyacrylamide gel electrophoresis (SDS- PAGE) --- p.39 / Chapter 2.7 --- Transfer of protein to nitrocellulose membranes --- p.39 / Chapter 2.8 --- Western blot analysis of proteins --- p.41 / Chapter 2.8.1. --- Antibodies --- p.41 / Chapter 2.8.2. --- Determination of expression profiles of desired proteins by immunoblotting --- p.45 / Chapter 2.9 --- "Measurement of cell viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay" --- p.46 / Chapter 2.10 --- Determination of cell proliferation by BrdU proliferation assay --- p.47 / Chapter 2.11 --- Detection of apoptosis of the cell lines by TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) --- p.50 / Chapter 2.12 --- Determination of the involvement of p38 MAPK in the generation of truncated Bid by p38 MAPK inhibitor SB203580 --- p.52 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Confirmation of plasmids and the stable cell lines --- p.53 / Chapter 3.2 --- Morphology and the basic parameters of the cells with full-length HBx or mutant HBx --- p.53 / Chapter 3.3 --- Cell viability under doxorubicin treatment with or without hypoxia --- p.59 / Chapter 3.4 --- Determination of cell proliferation under stress --- p.70 / Chapter 3.5 --- Expression profiles of various proteins in the stable cell lines under doxorubicin treatment with or without hypoxia --- p.74 / Chapter 3.5.1. --- Verification of hypoxia --- p.74 / Chapter 3.5.2. --- Pro-apoptotic proteins --- p.74 / Chapter 3.5.3. --- Anti-apoptotic proteins --- p.74 / Chapter 3.6 --- Determination of apoptosis of various cell lines under stress --- p.82 / Chapter 3.7 --- "p38 MAPK, but not Akt, was activated by doxorubicin" --- p.87 / Chapter 3.8 --- The p38 MAPK inhibitor SB203580 could attenuate the cleavage of Bid --- p.89 / Chapter Chapter 4: --- Discussion --- p.92 / Chapter Chapter 5: --- Conclusion and future prospective --- p.103 / Chapter Chapter 6: --- References --- p.106

Xie, Hui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 91-116). / Abstracts in English and Chinese. / CONTENTS --- p.I / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 中文摘要 --- p.v / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Overview of the Study --- p.1 / Chapter 1.2 --- Obstructive Sleep Apnea --- p.4 / Chapter 1.2.1 --- Epidemiology --- p.5 / Chapter 1.2.1.1 --- Prevalence --- p.5 / Chapter 1.2.1.2 --- Risk Factors --- p.6 / Chapter 1.2.2 --- Pathogenesis --- p.8 / Chapter 1.2.3 --- Pathophysiologic Consequences --- p.9 / Chapter 1.2.4 --- Diagnosis --- p.12 / Chapter 1.2.5 --- Treatment --- p.13 / Chapter 1.3 --- Memory and Long-term Potentiation --- p.15 / Chapter 1.3.1 --- Memory --- p.15 / Chapter 1.3.1.1 --- Classification of Memory --- p.15 / Chapter 1.3.1.1 --- Physiology of Memory --- p.17 / Chapter 1.3.2 --- Hippocampus --- p.18 / Chapter 1.3.2.1 --- Anatomy --- p.18 / Chapter 1.3.2.2 --- Hippocampus and Memory --- p.20 / Chapter 1.3.3 --- Long-term Potentiation (LTP) --- p.20 / Chapter 1.3.3.1 --- Discovery of LTP --- p.21 / Chapter 1.3.3.2 --- Types of LTP --- p.22 / Chapter 1.3.3.3 --- Properties of NMDA-LTP --- p.23 / Chapter 1.3.3.4 --- Early Phase LTP and Mechanism --- p.24 / Chapter 1.3.3.5 --- Late Phase LTP and Mechanism --- p.28 / Chapter 1.3.3.6 --- Important Factors in Long-term Potentiation --- p.29 / Chapter 1.4 --- Brain-derived Neurotrophic Factor (BDNF) --- p.33 / Chapter 1.4.1 --- Neurotrophins --- p.33 / Chapter 1.4.2 --- Structure and Expression of BDNF --- p.36 / Chapter 1.4.3 --- BDNF and Synaptic Plasticity --- p.37 / Chapter 1.4.3.1 --- BDNF and E-LTP --- p.38 / Chapter 1.4.3.2 --- BDNF and L-LTP --- p.39 / Chapter CHAPTER 2 --- METHODS --- p.43 / Chapter 2.1 --- Animal model of Obstructive Sleep Apnea --- p.43 / Chapter 2.1.1 --- Chronic Intermittent Hypoxia --- p.43 / Chapter 2.1.2 --- Bodyweight During Hypoxia Treatment --- p.47 / Chapter 2.2 --- Electrophysiological Experiments --- p.47 / Chapter 2.2.1 --- Brain Slice Preparation --- p.47 / Chapter 2.2.2 --- Multi-electrode Recording Setup (MED64) --- p.48 / Chapter 2.2.3 --- Slice Superfusion --- p.51 / Chapter 2.3.4 --- Field Potential Recordings --- p.53 / Chapter 2.3.5 --- LTP Induction Protocol --- p.55 / Chapter 2.3 --- Stereotaxic Surgery --- p.57 / Chapter 2.4 --- Drugs and Data Analysis --- p.58 / Chapter CHAPTER 3 --- RESULTS --- p.59 / Chapter 3.1 --- Validation of the OSA model --- p.59 / Chapter 3.2 --- Optimization for Studies of Early and Late-phase LTP by MED64 --- p.60 / Chapter 3.2.1 --- Optimization of Brain Slices --- p.60 / Chapter 3.2.2 --- Optimization of Field Potential Recording --- p.62 / Chapter 3.2.3 --- Optimization for LTP Study --- p.65 / Chapter 3.3 --- Effect of Intermittent Hypoxia on Hippocampal LTP --- p.68 / Chapter 3.3.1 --- Early-phase LTP (E-LTP) --- p.68 / Chapter 3.3.2 --- Late-phase LTP (L-LTP) --- p.71 / Chapter 3.4 --- Effect of BDNF on Intermittent Hypoxia-induced LTP Impairment --- p.75 / Chapter 3.4.1 --- BDNF Rescues LTP Impairment --- p.75 / Chapter 3.4.2 --- BDNF prevents LTP Impairment --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.80 / Chapter 4.1 --- Chronic Intermittent Hypoxia Model of OSA in Mice --- p.80 / Chapter 4.2 --- Impairment of LTP Induced by Intermittent Hypoxia --- p.82 / Chapter 4.3 --- The role of BDNF in IH-induced Impairment in Hippocampal Synaptic Plasticity --- p.84 / Chapter 4.4 --- Future Studies --- p.89 / REFERENCE --- p.91

Na atualidade, uma das causas importantes de lesão encefálica em neonatos é a anóxia neonatal. Este é um problema grave nos serviços de perinatologia dos hospitais em todo o mundo sendo ainda pior em países subdesenvolvidos, devido à carência de precauções e cuidados requeridos. Modelos animais de anóxia vêm sendo empregados para avaliar seus efeitos, tanto em nível neurológico, como em nível comportamental. A anóxia neonatal tem sido estudada pelo laboratório de Neurociências do Instituto de Ciências Biomédicas da Universidade de São Paulo, com modelos de estudo já desenvolvidos, adaptados e validados. Para investigar se a anóxia neonatal afeta o desenvolvimento motor somático e sensorial, ratos foram submetidos a um modelo não invasivo de anóxia global (Takada et. al., 2011). Ratos Wistar com 30 h de idade (6-8 gramas), machos e fêmeas, foram expostos por 25 minutos a gás nitrogênio 100% num fluxo de 3L/min, pressão 101.7 kPa e temperatura de 37ºC em câmara semi-hermética de policarbonato. O grupo controle foi submetido às mesmas condições porem com o ar ambiente normal. Os animais foram avaliados durante o período de aleitamento (P2 a P21) quanto a parâmetros do desenvolvimento somático; desenvolvimento ontogenético e quanto a reflexos sensório motores. Os resultados indicaram que o grupo Anoxia macho(AM) apresentou aumento no peso corporal {AM(42.25±3.62);CM(38.76±5.60);AF(40.64±5.08);CF(41.33±5.45)}e diminuição do eixo longitudinal do corpo {AM(10.15±0.27);CM(10.39±0.50);AF(9.82±0.44);CF(10.82±0.46)} em relação ao grupo Controle macho(CM) e Anoxia fêmea(AF), AF foi menor em relacao ao Controle fêmea (CF). AM apresentou maior eixo látero-lateral do crânio em relação CM e AF {AM (3.18 ±0.10); CM (3.17 ±0.13); AF(3.06 ±0.16); CF(3.00 ±0.15)} No desenvolvimento ontogenético houve retardo na abertura do canal auditivo {AM (13.79± 0.58); CM (13.75±0.83); AF(14.21±1.01); CF(13.36±0.50)} e abertura dos olhos {AM (14.00± 0.88); CM (14.64±1.28); AF(15.14±0.86); CF(13.79±0.42)} no grupo AF em relação a CF e AM, mas no grupo AM não houve diferença significante. Na erupção dos incisivos superiores {AM (10.79± 0.43); CM(11.71±1.68); AF(11.43±0.65); CF(10.07±0.27)} o grupo AM adiantou enquanto o AF atrasou em relação ao grupo controle. A avaliação dos reflexos sensóriais mostrou que a anoxia adiantou a colocação pelas vibrissas {AM (8.80± 1.21); CM (9.50±1.56); AF (9.93±1.14); CF(10.14±1.28) no AF e AM. Apenas o AM adiantou {AM (10.93± 2.09); CM(13.43±0.94); AF(10.50±0.85); CF(9.57±0.76)} no reflexo de aversão ao precipício. Nos relfexos de geotaxia negativa {AM (14.87± 1.30); CM (13.57±2.34); AF(14.57±1.40);CF (12.00±2.11)} e sobressalto ao susto {AM (14.00±0.53); CM (13.21±1.31); AF (13.29±0.61); CF (11.93±0.27)} e preensão palmar {AM (6.60±0.83); CM (4.71±0.47); AF(10.14±0.83); CF(4.71±0.47)} a anóxia provocou atraso tanto em macho quanto em fêmeas motores. Houve atraso na ontogênese da maioria dos testes de reflexos dos filhotes do grupo Anóxia. Os resultados deste estudo demonstraram que a anóxia causa danos persistentes na maioria dos parâmetros avaliados em relação aos grupos controle, e diminuição no número de neurônios do córtex sensóriomotor {M2: AM (46.84±1.72); CM (52±1.66); AF (45.55±1.80); CF (52±1.55)M1: AM (23.70±1.33); CM (41.89±1.49); AF (25.69±0.83); CF (43.88±1.46) S1HL: AM (27.93±2.69); CM (30.19±1.31); AF(23.42±2.38); CF (38.88±1.48) S1FL: AM (31.85±1.09); CM (33.88±0.48); AF(27.66±1.36); CF(32.28±1.70)}, com diferença de gênero o que evidencia a importância de que estratégias e procedimentos para minimizar os efeitos desse estímulos sejam consideradas em relação ao gênero / At present, one of the important causes of brain injury is the neonatal anoxia. This is a serious problem in the perinatology services of hospitals around the world being even worse in underdeveloped countries because of the lack of precautions and care required. Animal models of anoxia have been employed to assess their effects, both at the neurological level and at the behavioral level. Neonatal anoxia has been studied by the Neuroscience Laboratory of the Biomedical Sciences Institute of the University of São Paulo, with animal models already developed, adapted and validated. To investigate whether neonatal anoxia affects somatic and sensory motor development, rats were subjected to a non-invasive model of global anoxia (Takada et al., 2011). Male and female 30-h old (6-8 grams) Wistar rats were exposed for 25 minutes to 100% nitrogen gas in a flow of 3 L/min, pressure 101.7 kPa and temperature of 37ºC in a semi-hermetic chamber of polycarbonate. The control group was subjected to the same conditions but with normal ambient air. The animals were evaluated during the lactation period (P2 to P21) for parameters of somatic development; Ontogenetic development and for sensorimotor reflexes. The results indicated that the male Anoxia (AM) group presented increase in body weight (AM (42.25 ± 3.62), CM (38.76 ± 5.60), FA (40.64 ± 5.08), CF (41.33 ± 5.45)) and decrease in the longitudinal (10.82 ± 0.46), in relation to the male control group (CM) and the female Anoxia (AF), AF was lower in relation to the control group (AM) (10.15 ± 0.27), CM (10.39 ± 0.50), AF (9.82 ± 0.44) Female control (CF). AM increase in the cranio-lateral axis in relation to CM and AF (AM (3.18 ± 0.10); CM (3.17 ± 0.13); AF (3.06 ± 0.16); CF (3.00 ± 0.15). Concerning the ontogenetic development there was delay in opening the (13.79 ± 0.58), and the eyes {AM (14.00 ± 0.88); CM (14.64 ± 1.28), AF (15.14 ± 0.86), CF (13.79 ± 0.42)} in the AF group in relation to CF and AM, but in the AM group there was no significant difference. In the eruption of maxillary incisors (AM (10.79 ± 0.43), CM (11.71 ± 1.68), AF (11.43 ± 0.65), CF (10.07 ± 0.27), the AM group advanced while the AF delayed in control ration. The evaluation of the sensory reflexes showed that anoxia improved the placement of vibrissae (AM (8.80 ± 1.21), CM (9.50 ± 1.56), AF (9.93 ± 1.14), CF (10.14 ± 1.28) in AF and AM. Only AM advanced (AM (10.93 ± 2.09), CM (13.43 ± 0.94), AF (10.50 ± 0.85), CF (9.57 ± 0.76) in the reflex of aversion to the precipice. In negative geotaxia relays (AM (14.87 ± 1.30); CM (13.57 ± 2.34), AF (14.57 ± 1.40), CF (12.00 ± 2.11)} and startle reflex {AM (14.00 ± 0.53); CM (13.21 ± 1.31); AF (13.29 ± 0.61); CF (11.93 ± 0.27) and palmar grip (AM (6.60 ± 0.83); CM (4.71 ± 0.47), AF (10.14 ± 0.83), CF (4.71 ± 0.47)), anoxia caused delay in both male and female groups. There was a delay in the ontogenesis of most of the reflex tests of the puppies of the anoxia group. The results of this study demonstrated that anoxia causes persistent damage in most of the parameters evaluated in relation to the control groups, and a decrease in the number of sensory motor cortex neurons (M2: AM (46.84 ± 1.72), CM (52 ± 1.66), AF 1.80), CF (52 ± 1.55) M1: AM (23.70 ± 1.33), CM (41.89 ± 1.49), AF (25.69 ± 0.83), CF (43.88 ± 1.46) S1HL: AM (27.93 ± 2.69), CM (30.19 (31.88 ± 1.48), FA (27.66 ± 1.36), CF (32.28 ± 1.70), , which shows that strategies and procedures to minimize the effects of such stimuli should be considered in relation to gender

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.