Com objetivos de validar o modelo de anóxia neonatal apresentado e analisar a distribuição de neurônios imunorreativos à proteína Fos (IR-Fos) no encéfalo de ratos neonatos submetidos ao insulto anóxico, foram utilizados 24 ratos wistar neonatos machos divididos em dois grupos: Anóxia (n=12) e Controle (n=12). O grupo Anóxia apresentou decréscimo de 75% no valor da saturação periférica de oxigênio durante a exposição ao nitrogênio, enquanto o Controle não apresentou alterações nos valores (97%±0,5). A análise do número de células IR-Fos mostrou ativação de áreas encefálicas relacionadas ao controle da respiração, estruturas límbicas e núcleos da rafe. Tais dados sugerem que o modelo experimental de anóxia neonatal utilizado é eficiente em produzir privação temporária de oxigênio em ratos neonatos, levando a respostas condizentes com a anóxia. Espera-se, com estes resultados, facilitar a compreensão dos eventos relacionados à neurodegeneração e neurorregeneração após anóxia neonatal e, se possível, abrir caminho para novas abordagens e perspectivas terapêuticas. / The aim of this study is to validate the experimental model of neonatal anoxia and evaluate by Fos imunoreactivity (Fos-IR) the effects of anoxic insult. Twenty-four male wistar neonates (weighting 6-8g) were divided in two groups: Anoxia (n=12) and Control (n=12). The anoxic group presented decrease of 75% in the value of the peripheric saturation of oxygen during exposure to the nitrogen while control group did not present alterations in the values of the peripheric saturation of oxygen during the studied time (97 % ± 0,5). These data suggest that the experimental model of neonatal anoxia presented is efficient in producing temporary deprivation of oxygen in neonates rats, leading to responses that characterize anoxia. Fos-IR neurons analisis showed important activation of respiratory regions, limbic strutures and raphe nuclei in anoxic group when compared to control group. We hope facilitate the understanding of neonatal anoxia neurodegeneration and neuroregeneration and possibly contribute for new approaches and therapeutic perspectives.

A anóxia neonatal é uma das causas mais importantes de lesão encefálica em neonatos. Várias espécies de mamíferos têm contribuído no estudo dos mecanismos patogênicos envolvidos no prejuízo da anóxia/hipóxia, em particular roedores tem sido utilizado com sucesso como modelo da anóxia em humanos. Fatores como a maturidade do encéfalo no momento da deficiência de oxigênio, a duração da asfixia, vulnerabilidade regional e celular específica, apresentam grande influência no surgimento de problemas neurológicos, déficit sensoriomotor, prejuízo no aprendizado e memória. Alterações em processos atentivos e ansiedade também podem afetar as funções cognitivas. Em roedores, tem-se observado marcada hiperatividade na idade infantil, acompanhada por significativa hipoatividade na idade adulta. Os objetivos deste estudo foram avaliar as possíveis alterações comportamentais, tais como memória de referência espacial, memória operacional, alterações sensoriais, ansiedade e aquisição do medo condicionado ao som e ao contexto, em ratos, submetidos à anóxia neonatal. Foram utilizados 25 ratos albinos (Rattus norvegicus, linhagem Wistar) machos, neonatos, divididos em dois grupos experimentais: Anóxia (n = 13) e Controle (n = 12). Nos testes envolvendo a memória de referência espacial e memória operacional, o grupo experimental evidenciou que a anóxia neonatal causa prejuízo na capacidade de aprendizagem e navegação espacial no labirinto aquático de Morris, porém verificou-se que ambos os grupos conseguiram valer-se das suas experiências prévias para melhorarem seu desempenho na medida em que passavam pelos diferentes testes (memória de referência espacial, memória operacional com intervalo entre as tentativas de 10, 30 e 0) demonstrados por meio da diminuição na latência e no comprimento do trajeto, sendo esta melhora no desempenho inferior nos animais anóxia. Os animais anóxia, também necessitaram de mais dias de testes, para atingirem desempenho próximo ao encontrado nos animais do outro grupo. Observou-se variação na velocidade do nado ao longo dos dias de testes em ambos os grupos, porém verificou-se que os animais anóxia demonstraram velocidade inferior aos animais controle. No teste do labirinto em cruz elevado os animais anóxia mostraram-se menos ansiosos, tendo permanecido menor tempo em congelamento no teste do medo condicionado ao contexto, porém não ao som. Portanto, estes dados indicam que o modelo de anóxia neonatal utilizado promove alterações neuronais que modificam o comportamento do animal na vida adulta, verificados por meio das alterações comportamentais. Espera-se, com estes resultados, favorecer a compreensão das alterações comportamentais após anóxia neonatal e se possível, abrir caminho para novas abordagens e perspectivas terapêuticas / Neonatal anoxia is a major cause of brain injury in newborns. Several species of mammals have contributed to the study of pathogenic mechanisms involved in the injury caused by anoxia / hypoxia; particularly rodents have been used successfully as a model for the study of anoxia in humans. Factors such as maturity of the brain when oxygen deficiency, the duration of asphyxia, specific regional and cell vulnerability, have great influence on the rise of neurological disorders, sensory motor deficit, and impaired learning and memory. Changes in attentional processes and anxiety can also affect cognitive functions. In rodents, there has been marked hyperactivity at young age, accompanied by a significant hypoactivity in adult life. The objectives of this study were to evaluate the possible behavioral changes, such as spatial reference memory, working memory, sensory disturbance, anxiety and acquisition of conditioned fear to the sound and context in rats subjected to neonatal anoxia. We used 25 albino rats (Rattus norvegicus, Wistar) male neonates, divided into two groups: Anoxia (n = 13) and control (n = 12). In tests involving spatial reference memory and working memory, the experimental group showed that neonatal hypoxia adversely affects the ability of spatial learning and navigation in the Morris water maze, but it was found that both groups were able to draw on their experiences prior to improve its performance as they passed through various tests (spatial reference memory, working memory at an interval timing between attempts of 10, 30 and 0) shown by the decrease in latency and path length, and this improvement in underperform on anoxic animals. Anoxic animals also required more days of testing to achieve performance close to that found in animals of the other group. There was variation in the speed of swimming during the days of testing in both groups, but it was found that the anoxic animals showed lower speed than the control animals. In the elevated plus-maze test, the anoxic animals were less anxious and reminded less time freezing in the conditioned fear test to the context but not to the sound. Therefore, these data indicate that the used neonatal anoxia model promotes neuronal alterations that modify the behavior of the animal in adult life, verified through behavioral changes. It is expected, with these results, to promote understanding of the behavioral alterations after neonatal anoxia and, if possible, to make way for new approaches and therapeutic perspectives

The inter-tissue metabolic responses to hypoxia were determined in lungfish (Protopterus aethiopicus), and trout (Salmo gairdneri). Lungfish respond to hypoxia with a reduction in metabolic rate. It was intended to determine which tissue, or tissues exhibit decreased metabolic rates during hypoxia, and then compare the results with the metabolic reactions observed in trout, which are not reported to reduce metabolic rate during hypoxia. The metabolic potentials of the heart, brain, white muscle and liver in the African lungfish were estimated using enzymatic data. Metabolic effects of a 12 hr submergence were monitored using metabolite measurements. Heart was the most oxidative tissue, but also showed the greatest anaerobic potential. The brain displayed relatively low oxidative capabilities. White muscle remained almost inert. Although high energy phosphate concentrations in brain and heart did not fall during submergence, glycolysis was activated as indicated by cross-over plots, depletion of endogenous glycogen stores, and lactate accumulation. Blood-tissue lactate and glucose gradients indicated (1) that the heart and brain released lactate throughout submergence, (2) that after 12 hr of submersion the brain and heart were probably obtaining all their required glucose from the blood (3) that the liver released glucose throughout submergence, and (4) the white muscle was metabolically isolated from the rest of the body during submergence. The lack of measurable changes in white muscle metabolite concentrations coupled with the low enzyme activities leads to the suggestion that the most significant adaptation to hypoxia in these fishes may not be the capacity for increased anaerobic energy production. Instead, it is likely that the ability of the muscle to prevent the activation of glycolysis during hypoxic dysoxia is the key to the animal's survival. Histochemical and ultrastructural studies were done on the axial musculature of the lungfish. The small wedge of red coloured muscle evident upon gross examination was shown by histochemical demonstrations of lactate and succinate dehydrogenases, of adenosine triphosphatases, and of lipid to be composed of a mosaic of red and intermediate fibres. Respectively, these fibres measured 23.6 and 34.3 microns in average diameter. The bulk of the myotome is composed of white fibres having an average diameter of 67.3 microns. Mitochondrial density, capillarity and lipid content were very low for all fibres. These data suggest that the axial musculature is geared primarily for anaerobic function. The relatively large percentage of white muscle indicates that the overall metabolic rate of the axial muscle is low. The capacity of the muscle to exist with a reduced rate of ATP turnover (as was suggested above) may be related to the large proportion of white fibres present in the myotome. Tissue metabolites were measured in a hypoxia sensitive organism, the Rainbow trout (Salmo qairdneri), before and after exposure for 3 hr to inspired oxygen tensions of 20 torr (at 4°C). There were small changes in the brain but the energy status was maintained. The red muscle was the least affected. White muscle creatine phosphate was depleted. Various data indicate that the white muscle is the major user of glycolytic substrates and the major producer of lactate. The heart is stressed as indicated by a decline in glycogen, ATP, CrP, and the total adenylate pool. The liver exhibited declines in every indicator of metabolic homeostasis. The liver concentrations of glycogen did not decline. The fact that anaerobic glycolysis has been activated in the white muscle, while the muscle remains in metabolic communication with the other tissues via the blood, supports the suggestion that the metabolism of the white muscle will have a pronounced effect on the metabolic status of the whole animal. The trout is maintaining its rate of oxygen uptake while activating anaerobic glycolysis in the attempt to maintain 'normal' rates of energy utilization. The turnover rates of glucose and lactate were measured in trout subjected to the same hypoxic stress as above. Glucose turnover did not change while lactate turnover increased from 2.8 ± 0.4 µmoles/min./kg to 20.6 ± 6.8 µmoles/min./kg. The lack of increase in glucose turnover was attributed to the observation that liver glycogen concentrations do not change and so there is no increase in glucose flux. The increase in lactate turnover emphasizes the fact that anaerobic glycolysis is activated and that some tissues are oxidizing lactate. The problem of when a cell becomes hypoxic and the reactions of the cell to that stress is addressed. The cell (tissue, organ, animal) has two options if oxygen supply drops to a level which prevents oxidative metabolism from supplying all of the requirements for ATP synthesis. The cell may exhibit a decline in requirements, in which case the rate of ATP production need not be as high as in the oxidative state or, conversely, anaerobic energy production may increase in the attempt to maintain ATP production rates. The lungfish muscle appears to be capable of the former, thus preserving substrates for other tissues and reducing the rate of end-product formation. The trout white muscle, on the other hand, exerts a major influence upon the other tissues when the animal is stressed with hypoxia. The term 'energy conformer' is applied to animals which do not maintain oxygen uptake in the face of a declining supply, and which allow ATP production to decline concomittantly by not activating glycolysis to a marked degree. An energy regulator would activate glycolysis in the attempt to maintain oxidative rates of ATP production. The trout is more of an energy regulator than is the lungfish with the main difference in this capacity being in the white muscle. / Science, Faculty of / Zoology, Department of / Graduate

Glicose e oxigênio desempenham um importante papel na regulação do metabolismo celular. Dada a importância de ambos no metabolismo - o primeiro como fonte de carbono preferencial da maior parte das células, e o segundo como aceptor final de elétrons na cadeia respiratória, em diversos organismos desenvolveram-se métodos adequados para detectar sua presença de modo a ajustar o metabolismo em função de sua disponibilidade. Neste trabalho foi realizado o estudo da expressão, no nível transcricional, dos genes envolvidos nas vias metabólicas primárias e genes envolvidos em morte celular, em células humanas, com o intuito de determinar as alterações no metabolismo energético em resposta a condições de hipóxia e anóxia, por meio da técnica de microarrays de cDNA. Utilizamos, inicialmente, células normais de fibroblasto humano ASl98 e células de fibroblasto humano MRC-5 imortalizadas por transfecção por SV40, e por fim células provenientes de ilhotas pancreáticas humanas, para a elaboração de um protocolo de cultura celular em que as mesmas crescessem aderidas a microcarregadores Cytodex I. Numa segunda etapa, células de ilhotas pancreáticas humanas foram cultivadas em suspensão, aderidas aos microcarregadores, num biorreator, sendo então realizada a análise do perfil transcricional dos genes escolhidos, frente às condições de baixa tensão de oxigênio. É apresentada a análise da expressão gênica de aproximadamente 160 genes na qual foram verificados um comportamento de indução daqueles envolvidos no metabolismo de lipídios e alguns na morte celular e um comportamento inicial de indução, e posterior inibição, do metabolismo primário como um todo. Em vista dos dados obtidos é de interesse ressaltar que essas células deveriam ser mantidas em saturações de oxigênio acima de 5% para evitar o efeito deletério observado na baixa concentração de oxigênio sobre a viabilidade celular, em termos da indução de alguns genes envolvidos na morte celular e da repressão geral dos relacionados ao metabolismo energético. Também foi verificado que, em saturações de oxigênio de até 10%, as células adotaram um padrão transcricional que indicou uma resposta ao estresse por falta de oxigênio, este por sua vez reflete-se na viabilidade celular, característica crucial para o sucesso do transplante clínico de ilhotas. / Glucose and oxygen have important roles on the regulation of cellular metabolism. Due to their importance in metabolism, the former as the preferential carbon source and the later as the final electron acceptor of the respiratory chain, many organisms have developed suitable processes to detect their presence in order to adjust the cellular metabolism to their availability. In this work, we have studied, in human cells and at the transcriptional level, the expression of genes involved in primary metabolism pathways and some of those related to cell death, aiming to resolve alterations in the energetic metabolism as a response to hypoxic and anoxic conditions, by means of cDNA microarrays. We initially used AS198 human fibroblastic normal cells and MRC-5 human fibroblastic cells immortalized by SV40, and later on cells from human pancreatic islets, to develop a cell culture protocol in which they would grow on the surface of Cytodex 1 microcarriers. As a second step, cells from human pancreatic islets were cultured on microcarriers in suspension inside a bioreactor. This culture was then used to carry out the transcriptional profile analysis of selected genes in response to low levels of oxygen. This work presents the analysis of gene expression of approximately 160 genes that can be divided into two distinct groups. The first group, the expression of which is induced, comprises genes involved in lipid metabolism and some of those related to cell death. The expression of the second group, consisting of diverse genes of the primary metabolism, suffers an initial induction followed by repression. Given the data acquired it is interesting to note that the human pancreatic islets should be maintained under at least 5% dissolved oxygen to avoid the deleterious effects on cell viability observed at lower oxygen concentrations, resulting in the induction of some genes involved in cell death and the repression of those related to energetic metabolism. It was also verified that, under oxygen saturation of at least 10%, these cells adopted a transcriptional profile that indicated a response to the stress created by the lack of oxygen, which would in turn reflect on cell viability, a crucial characteristic for success in clinical islet transplantation.

Intertidal mussels regularly experience emersion-induced anoxia, in contrast to normoxic conditions experienced during submersion. We therefore hypothesized that acclimation to a tidal rhythm, as opposed to a rhythm of constant submersion, preconditions the proteome of the California mussel, Mytilus californianus, to respond differently to emersion-induced anoxia. Following acclimation, mussels either continued to receive the acclimation conditions (control) or were exposed to 100% nitrogengas (anoxia) during aerial emersion. We collected gill tissue for subsequent analysis of protein abundance with 2D gel electrophoresis and protein identification with tandem mass spectrometry. Relative to subtidally-acclimated mussels, tidally-acclimated mussels showed a greater propensity to respond to distrupted protein homeostasis during emersion through higher levels of several small heat shock protein isoforms, while they showed lower levels of several chaperones involved in redox-sensitive protein maturation in the endoplasmic reticulum during acute anoxia. Several metabolic proteins showed elevated levels in tidally-acclimated mussels, suggesting a compensatory response to reduced feeding times. However, changes in the abundance of several tricarboxylic acid cycle enzymes (e.g. aconitase, succinate dehydrogenase) suggest that tidally-acclimated mussels are also primed to sense reactive oxygen species (ROS) and limit their production, respectively. These findings are further supported by higher abundances of several aldehyde dehydrogenases and thioredoxin peroxidase, which function as scavengers of aldehydes and ROS, common products of lipid peroxidation. Finally, tidally-acclimated mussels are also more responsive to changes in cytoskeletal and vesicular trafficking dynamics in response to acute anoxia. Together, our analysis showed that proteostasis, energy metabolism, oxidative stress and cytoskeletal and trafficking processes are all involved in priming tidally-acclimated mussels to respond more dynamically to acute emersion-induced anoxia in Mytilus gill.

abstract: Anoxia tolerance is strongly correlated with tolerance to heat, desiccation, hyperosmotic shock, freezing, and other general stressors, suggesting that anoxia tolerance is broadly related to stress tolerance. Age affects the capacity of many animals to survive anoxia, but the basis to this ontogenic variation is poorly understood. We exposed adult Drosophila, 1, 3, 5, 7, 9, and 12 days past eclosion, to six hours of anoxia and assessed survival 24-hours post-treatment. Survival of anoxia declined strongly with age (from 80% survival for one-day-old flies to 10% survival for 12 day-old-flies), a surprising result since adult fly senescence in Drosophila is usually observed much later. In anoxia, adenosine triphosphate (ATP) levels declined rapidly (< 30 min) to near-zero levels in both 1 and 12-day old adults; thus the higher anoxia-tolerance of young adults is not due to a better capacity to keep ATP elevated. Relatively few physiological parameters are reported to change over this age range in D. melanogaster, but gut bacterial content increases strongly. As a partial test for a causal link between bacterial load and anoxia tolerance, we replaced food daily, every third day, or every sixth day, and assayed survival of six hours of anoxia and bacterial load at 12 days of age. Anoxia tolerance for 12-day old flies was improved by more food changes and was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age-related decline of anoxia tolerance in Drosophila. / Dissertation/Thesis / Masters Thesis Biology 2020

Paratetramitus jugosus, a vahlkampfiid amoebomastigote, was isolated into monoprotist/monobacterial (Bacillus sp.), cultures from laminated microbial mats (Laguna Figueroa, Baja California Norte, Mexico) and muds (Eel Pond, Woods Hole, Massachusetts). Chromidia, roughly spherical (2-4 µm in diameter) were released from both walled spherical cysts (10-12 µm) and phagocytotic amoebic forms. Desiccation-resistant walled chromidia, at first spherical, resorb their walls and develop into small pleiomorphic phagocytotic amoeba. Small amoebae feed and mature into typical monopodial vahlkampfiid adults confirming previous work (Dobell, 1913, and especially the analysis of a larger encysting vahlkampfiid amoeba associated with Long Island oyster disease studied at Woods Hole by Mary Jane Hogue, 1914). I show here that P. jugosus reproduces and develops through its life history stages of chromidia, mature monopodial amoebae, and cysts as rapidly and abundantly under low oxygen levels as at ambient atmospheric oxygen concentrations. Anoxia was achieved in the laboratory by incubation of entirely desiccated inocula from old mat or mud samples in Brewer jars with or without gas packs to control atmospheric conditions. Three sets of experiments yielded the same results: vigorous growth on bacillus food occurred on manganese acetate media by two weeks on the surface of agar plates under ambient oxic or hypoxic to anoxic conditions. Preliminary investigations of similar amoeba from geographically distinct field sites in Europe, North America, and the Caribbean were made. From them, I suggest it is likely these coastal amoebomastigotes that propagate by small desiccation resistant, oxygen-independent, manganese tolerant chromidia are genuinely cosmopolitan in its distribution.

Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia response phenotypes and the identification of human orthologs for the detoxification enzymes and provides insight into the gene expression pattern.

Indiana University-Purdue University Indianapolis (IUPUI) / The evolution of land plant root systems occurred stepwise throughout the Devonian, with the first evidence of complex root systems appearing in the mid-Givetian. This biological innovation provided an enhanced pathway for the transfer of terrestrial phosphorus (P) to the marine system via weathering and erosion. This enhancement is consistent with paleosol records and has led to hypotheses about the causes of marine eutrophication and mass extinctions during the Devonian. To gain insight into the transport of P between terrestrial and marine domains, presented here are geochemical records from a survey of Middle and Late Devonian lacustrine and near lacustrine sequences that span some of these key marine extinction intervals. Root innovation is hypothesized to have enhanced P delivery and results from multiple Devonian sequences from Euramerica show evidence of a net loss of P from terrestrial sources coincident with the appearance of early progymnosperms. Evidence from multiple Middle to Late Devonian sites (from Greenland and northern Scotland/Orkney), reveal a near-identical net loss of P. Nitrogen and Carbon isotopes from a subset of these lakes confirm elevated input of terrestrial plant material concurrent with P perturbations. Terrestrial P input appears to be episodic in nature, suggesting land plant expansion was driven by an external catalyst in the study region. All sites analyzed are temporally proximal to significant marine extinctions, including precise correlation with the Kačák extinction event and the two pulses associated with the Frasnian-Famennian (F/F) mass extinction. The episodic expansion of terrestrial plants appears to be tied to variations in regional and global climate, and in the case of the F/F extinction, also to atmospheric changes associated with large scale volcanism. Using P data presented here as an input into an Earth system model of the coupled C-N-P-O2-S biogeochemical cycles shows that globally scaled riverine phosphorus export during the Frasnian-Famennian mass extinction generates widespread marine anoxia consistent with the geologic record. While timing precludes land plants as an initiating mechanism in the F/F extinction, these results suggest they are implicated in every marine extinction event in the Mid to Late Devonian.

No description available.