Uma das causas mais importantes de lesão encefálica em neonatos na atualidade é a anóxia neonatal. Este é um dos problemas mais graves e comuns nos serviços de perinatologia dos hospitais no mundo, sendo ainda pior em países subdesenvolvidos, devido à carência de precauções e cuidados requeridos. Há relativamente pouco tempo estudos têm indicado que a hipotermia promove um importante efeito neuroprotetor, podendo ser usada como tratamento alternativo promissor para danos causados pela anóxia neonatal. Porém, embora diversas pesquisas mostrem a ação neuroprotetora da hipotermia, não existem evidencias consistentes do seu papel preventivo em relação as alterações comportamentais decorrentes da anóxia neonatal. O objetivo deste trabalho foi avaliar se a hipotermia previne alterações comportamentais decorrentes da anóxia neonatal, incluindo funções de memória espacial, condicionamento aversivo e ansiedade. Foram incluídos no estudo 91 ratos Wistar machos organizados em 4 grupos: anóxia com hipotermia (AH), anóxia sem hipotermia (AC), controle (para anóxia) com hipotermia (CH) e controle sem hipotermia (CC). O protocolo de anóxia neonatal foi iniciado 24 horas após o nascimento dos ratos, usando uma câmara semi-hermética saturada com nitrogênio gasoso. A temperatura da câmera foi mantida a 37°C e o tempo de exposição à anóxia foi de 25 minutos. Animais controle para anóxia foram expostos à mesma câmera, exceto pelo nitrogênio que foi substituído por ar. O tratamento com hipotermia foi iniciado imediatamente após da anóxia em uma câmara a 30°C, onde os animais permaneceram durante 5 horas. O tratamento controle para hipotermia envolveu o mesmo protocolo, exceto pela temperatura da câmera que foi mantida a 37°C. No final do período, os neonatos foram colocados em uma câmara aquecida a 37°C por 40 minutos até se recuperarem. Quando os animais atingiram 70 dias de idade foram submetidos ao paradigma teste-reteste no labirinto em cruz elevado, para avaliar níveis de ansiedade, atividade locomotora e memória aversiva. Subsequentemente, quando os animais fizeram 75 dias, iniciou-se o teste de memória espacial no Labirinto Aquático de Morris. Finalmente, quando os animais atingiram 115 dias de idade, realizou-se o teste de condicionamento de medo ao som e ao contexto. A anóxia neonatal e a hipotermia não interferiram nos níveis de ansiedade no Labirinto em cruz elevado. Porém, a hipotermia aumentou a atividade locomotora e comportamentos de avaliação de risco. Os resultados obtidos no Labirinto Aquático de Morris indicam que a hipotermia previne prejuízos na memória espacial induzidos pela anóxia neonatal. Finalmente, a anóxia neonatal reduziu a taxa de extinção de memória aversivas, efeito que foi prevenido pela hipotermia. No conjunto, esses resultados mostram, por um lado, que a hipotermia previne alterações da memória espacial e de medo condicionado. Por outro lado, eles mostram que a hipotermia induz aumento da atividade locomotora e de comportamentos de avaliação de risco em ratos / Neonatal anoxia is one of the main causes of brain injury in newborns. This is among the most serious problems in many hospitals around the world and is even worse in developing countries due to the lack of required precautions and care. Recent studies have indicated that hypothermia promotes important neuroprotective effects. Thus, it could constitute a promising alternative treatment to dysfunctions caused by neonatal anoxia. Although there have been studies demonstrating that hypothermia promotes neuroprotection following neonatal anoxia, there is no solid evidence showing to which extent this neuroprotection prevents behavioral changes. This study aimed at evaluating to which extent behavioral changes induced by neonatal anoxia are prevented by hypothermia, focusing on anxiety, spatial memory and fear conditioning, in rats. The study included 91 male Wistar rats organized in 4 groups: anoxia with hypothermia (AH), anoxia without hypothermia (AC), control (for anoxia) with hypothermia (CH) and control without hypothermia (CC). Neonatal anoxia protocol started 24 hours after birth, using a semi-hermetic chamber saturated with nitrogen gas. The chamber temperature was maintained at 37°C and the time of exposure to anoxic conditions was 25 minutes. Hypothermia treatment started immediately after the anoxia protocol, within a chamber at 30°C, where the newborns remained for 5 hours. At the end of this period, newborns were transferred to a chamber at 37°C for 40 minutes until its recovery. Control treatment for anoxia involved the same protocol except for the nitrogen that was substituted for air. Control treatment for hypothermia involved to maintain the subjects in the same chamber at 37°C for 5 hours. When the animals were 70 days old, they were subjected to the elevated plus maze, in order to evaluate their anxiety, locomotor activity and aversive memory. Subsequently, when the animals were 75 days old, their spatial memory was evaluated in the Morris Water Maze. Finally, when the animals were 115 days old, they were subjected to an auditory and contextual fear conditioning task. Neonatal anoxia did not interfere with anxiety as evaluated in the elevated plus maze. In contrast, hypothermia by itself increased risk assessment behavior in the elevated plus maze. Performance in the Morris water maze task indicated that hypothermia prevents anoxia-induced disruption of spatial memory. Extinction of both auditory and contextual fear conditioning were slowed by anoxia, and this effect was prevented by hypothermia treatment. Therefore, the present experiments show that hypothermia prevents anoxia-induced (1) disruption of spatial memory and (2) slowing of extinction of fear conditioning; however, by itself, hypothermia increases risk assessment, in rats

Introdução: Pneumopatias que cursam com hipóxia crônica, como a doença pulmonar obstrutiva crônica, podem ser acompanhadas por alterações cognitivas secundárias a queda prolongada da saturação de oxigênio. Objetivo: 1) Averiguar se existem alterações cognitivas nos pacientes com fibrose pulmonar idiopática; 2) Caso estejam presentes, avaliar o seu perfil neuropsicológico e 3) Verificar se há correlação entre a gravidade da doença e desempenho cognitivo. Métodos: Vinte e um pacientes com diagnóstico de fibrose pulmonar idiopática foram selecionados do Ambulatório de Doenças do Interstício Pulmonar da Divisão de Pneumologia do Hospital das Clínicas da USP e foram submetidos a exame neurológico convencional, testes laboratoriais, neuroimagem, avaliação neuropsicológica abrangente e prova de função pulmonar. O grupo controle consistiu de vinte indivíduos não dementes clinicamente saudáveis equiparados por sexo, idade, escolaridade e nível sócio-econômico com o grupo anterior. Resultados: Os resultados do Mini-exame do estado mental, fluência verbal (categoria frutas), Teste de memória de figuras-aprendizado, Teste de dígito-símbolo e o Teste de trilhas parte B apresentaram diferença entre os dois grupos (p < 0,05). Conclusões: O estudo mostrou que os pacientes com fibrose pulmonar idiopática apresentam desempenho pior que o grupo controle em testes de funções executivas e velocidade de processamento mental, o que vai ao encontro com a hipótese de uma encefalopatia do tipo subcortical, semelhante àquela encontrada em pacientes com outras doenças pulmonares que cursam com hipóxia. É necessária a confirmação desses resultados com testes de tempo de reação mais precisos / Background: Lung disease with hypoxaemia as chronic pulmonary obstructive disease may be associated with cognitive impairment caused by continuous fall in oxygen saturation. Objective: 1) to identity if there is cognitive impairment in patients with idiopathic pulmonary fibrosis; 2) if present, to evaluate the neuropsychological profile of these alterations and 3) to verify if there is correlation of disease severity and cognitive performance. Methods: Twenty-one patients with idiopathic pulmonary fibrosis were selected from the Interstitial Lung disease Out-patient Clinic and were submitted throughout conventional neurological exam, blood lab, neuroimaging, broad neuropsychological and lung function evaluation. The control group consisted of twenty non-demented healthy subjects paired by gender, age, schooling and socio-economic level. Results: The Mini-mental state examination, Semantic verbal fluency (fruit category), learning item of the Brief Cognitive Battery, Digit symbol test and Trail making part B results were different between both groups (p < 0.05). Conclusions: This study showed that patients with IPF performed worse in tests of executive functions and mental speed, which is congruent with the hypotheses of encephalopathy of subcortical type similar to the one found in patients with other lung hypoxaemic disease. These results should be confirmed with more accurate reaction time tests

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

Intrashelf basins occurring on epeiric carbonate platforms are commonly associated with the presence of excellent carbonate source rocks, and because they often border potential carbonate reservoirs updip, they may form the core of rich petroleum systems. This is a common phenomenon of many Mesozoic hydrocarbon plays in the Middle East. Despite this fact, studies investigating intrashelf-basinal, fine-grained carbonates are rare, because it is assumed that little lithofacies variability is present in these successions as a consequence of their relatively homogeneous appearance where they are sampled in core or visited in exposures. Those that have been performed mostly lack process-detail analyses, and interpret the organic-carbon enrichment in these sediments to be simply a function of either occurrence of localised bottom-water anoxia or high primary organic production under low-energy conditions, dominated by suspension-settling events.

Studies were undertaken to determine the cardiac and ventilatory responses of restrained and unrestrained rainbow trout (Salmo gairdneri) to changes in inspired oxygen and carbon dioxide tensions. The role of blood oxygen carrying capacity in the control of ventilation was investigated, as well as the location and innervation of oxygen receptors activated by hypoxia. Ventilation volume (Vg) was measured directly in restrained fish using a ventilation chamber that separated inspired from expired water, the latter being collected in a graduated cylinder. In receptor localization experiments a wooden tongue depressor held vertically in the buccal cavity in the median plane divided water flows to the gills on each side of the fish. Thus, one set of gills could be irrigated with hyperoxic water to maintain arterial oxygen tension, while hypoxic water was passed over the other set of gills. Blood samples were obtained from cannulae implanted in both the dorsal aorta and right common cardinal vein. Vg increased in hypercapnea (inspired CO₂ tension (PICO₂) 0.5-2.0 kPa) due to increased "stroke volume (frequency remained constant), with higher levels of Vg recorded at higher C0₂ tensions. In fish exposed to PICO₂ levels of 0.5 and 0.9 kPa, raising the inspired oxygen tension (PIO₂) to 60.4 kPa eliminated the ventilatory response to hypercapnea. Hyperoxia had little or no effect on ventilatory responses to (PICO₂) levels of 1.5 and 2.0 kPa. Ventilation volume was inversely related to blood oxygen content (CaO₂) in trout. CaO₂ decreased and Vg increased during hypercapnea (PICO₂ 0.8 kPa), hypoxia (PIO₂12.4 kPa) and anaemia (haematocrit reduced from 22.3% to 14.3%), while CaO₂ increased and Vg decreased during hyperoxic hypercapnea (PIO₂ 60.4 kPa, PICO₂ 0.8 kPa). Increased Vg during hypercapnea is attributed to hypoxaemia produced by Bohr and Root off-shifts which result from increased blood CO₂ tension and reduced blood pH. Oxygen uptake remained constant during all experimental trials, indicating that the manoeuvre of increasing Vg is effective in relieving adverse effects of hypoxaemia. The significance of elevated Vg as a short-term adaptation to hypoxaemia is discussed, Heart rate decreased and ventilation increased in unrestrained fish exposed to gradual hypoxia (PIO₂ decreased from 20 kPa to 4 kPa) at 7°C and 16°C. The initial heart rate of fish acclimated to 16°C was higher than that of the 7°C group, but at the lowest level of PIO₂, heart rates of both groups dropped to the same level. Thus, the cardiac chronotropic response to hypoxia in trout is temperature independent. Receptors causing hypoxic bradycardia are located in the dorsal region of the first gill arch. Hypoxic bradycardia was eliminated by removing the first gill arch, or by sectioning the branches of cranial nerves IX and X innervating the arch. Blood flow through the arch does not appear to be necessary for this response, since ligation of the arch at its ventral insertion on the body wall did not affect hypoxic bradycardia. The pseudobranch has no role in cardiac control since interrupting the flow of blood through, and deafferentation of, the pseudobranch had no effect on the cardiac response to hypoxia. The biological significance of hypoxic bradycardia, and ventilatory-circulatory interaction during hypoxia, are discussed. Ventilatory responses to hypercapnea and hypoxia were unchanged after bilateral section of the nerves to the first gill arch. Receptors in the first gill arch thus have no role in control of ventilation during either hypercapnea or hypoxia. Possible locations for receptors responsible for control of ventilation are discussed. / Science, Faculty of / Zoology, Department of / Graduate

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19367号 / 医博第4044号 / 新制||医||1011(附属図書館) / 32381 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 佐藤 俊哉, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Compound specific isotope analysis of lake sediments is a powerful tool in deciphering evidence of changing climatic and paleoenvironmental conditions through time. Isotopic analysis of Lake El’gygytgyn pilot sediment cores, PG1351 and LZ1029, have contributed increased insight into paleoenvironmental interpretations regarding conditions of permanent ice cover and water column anoxia at the lake over the past 250 kyr. Bulk sediment δ15N was measured as a proxy for denitrification and a possible indicator for water column anoxia intensity. However, it appears that insufficient quantities of water column nitrate to fuel denitrification make its correlation with anoxia intensity ineffective. In pilot core LZ1029, compound-specific δ13C of alkanes, fatty acids, and alcohols were analyzed to determine the changing sources of organic matter as well as the source of a strong negative isotopic shift in the bulk sediment δ13C (-26‰ to -33‰) over the past 50 kyr. Results indicate that the majority of alkanes, fatty acids, and alcohols are long chain compounds consistent with a terrestrial plant origin, with increased aquatic contribution during the local last glacial maximum (LLGM). Among the compound classes examined, only the mid chain fatty acids display a strong LLGM depletion (δ13C = -43‰). Short chain fatty acids exhibit an LLGM depletion (δ13C = -35‰) similar to bulk sediment δ13C, while the δ13C trend of long chain alkanes, fatty acids, and alcohols differ from the bulk sediment δ13C trend, suggesting an autochthonous source of bulk isotope depletion. Evidence of methane cycling exists only in the presence and isotopic value of diplopterol (LLGM δ13C = -93.4‰), a biomarker for aerobic oxidation of methane. Two compounds indicative of archaeal lipids were present at considerable concentrations during the LLGM (394 and 668 µg/g TOC), but without the extreme negative δ13C associated with methanogenesis and methanotrophy. These results suggest insufficient generation of methane in the lake to have derived from such a large anaerobic archaeal methanogen community suggesting that archaea are not acting entirely as methanogens. Furthermore, it appears unlikely that a significant anoxic layer existed in the water column of Lake El’gygytgyn during the past ~50kyr. The results of this work will be applied to ongoing investigations on the newest cores from Lake El’gygytgyn, which represent the past 3.5 Myr.

Mainstream Partial Nitritation/Anammox, also known as Mainstream Deammonification, is a promising technology for future wastewater purification that aims to remove nitrogen from wastewater in order to prevent the eutrophication. It is less costly than the traditional nitrification/denitrification process and it heads towards the direction of converting the WWTPs from energy consuming into energy producing facilities.   This Master’s thesis is based on a study regarding the nitrogen removal from mainstream wastewater. It was conducted at Hammarby Sjöstadsverk that is a research facility in the area of the Henriksdal Waste Wastewater Treatment Plant in Stockholm. Three parts of the study were developed. The main one had the purpose to evaluate the process performances of a biological pilot-scale IFAS reactor used for Mainstream Deammonification that was operated from October 2017 to March 2018. This evaluation was addressed to comprehend how the pilot-scale reactor works at different operational conditions. The remaining studies analysed the progress of the pilot-scale reactor in relation to different factors and to the settling properties of the activated sludge used in the process.   It was found that the process performances improved by changing the aeration pattern from 40 to 50 minutes for non-aeration time and from 20 to 10 minutes for aeration time and by increasing the dissolved oxygen set-point from 0.6 to 1.0 mg/L. The enhancement of the performances consisted in an inhibition of nitrite oxidizing bacteria and rise of the total nitrogen removal efficiency. In addition, anammox biofilm was observed to grow on the carriers and it was observed that the activated sludge did not have good settling properties.

Many tropical aquatic environments worldwide are characterized by intermittent or prolonged hypoxia (low dissolved oxygen). Nevertheless, many tropical freshwater fishes are able to inhabit these challenging environments via a range of morphological, physiological and behavioral adaptations. Brachyhypopomus is a diverse genus of weakly electric fishes represented by 28 known species distributed from Panama to Argentina. 17 species are restricted to permanently normoxic habitats (blackwater rivers and terra firme streams), eight species are restricted to seasonally or perennially hypoxic habitats (whitewater floodplains of large tropical rivers or permanent swampy habitats), and three species are eurytopic (occur in both seasonally hypoxic and normoxic habitats). These habitat distributions offer the opportunity to explore both species- and population-level variation in adaptive responses to hypoxia. Across 25 of the 28 known species in the genus (for which specimens were available), one- and two-way ANOVA was used to correlate total gill filament length (a metric of gill surface area) with lifestyle-divided into four categories: 1) stenotopic species (i.e. species occurring in a narrow range of habitats) restricted to hypoxic habitats; 2) stenotopic species restricted to normoxic habitats; 3) populations of eurytopic species from hypoxic habitats, and; 4) populations of eurytopic species from normoxic habitats. One-way ANOVA revealed that populations of eurytopic species from hypoxic habitats had significantly larger total gill filament lengths than stenotopic species from the same habitat (P = 0.0169). Likewise, populations of eurytopic from normoxic habitats had significantly larger total gill filament lengths than stenotopic species from normoxic habitats (P[less than] 0.005). Two-way ANOVA showed that eurytopic species had significantly larger total gill filament lengths than stenotopic species, independent of the disparity in total gill filament length associated with either hypoxic or normoxic habitats. Results indicate a strong correlation between gill surface area and oxygen-habitat among species and populations, which supports the hypothesis that an enlarged gill surface area increases oxygen uptake and serves as an adaptive response to seasonal hypoxia.