Orientador: Selma Giorgio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T05:53:51Z (GMT). No. of bitstreams: 1 Degrossoli_Adriana_D.pdf: 32093196 bytes, checksum: 3de303f2f8a58bbae17d157ead572ede (MD5) Previous issue date: 2009 / Resumo: Diversas patologias provocam mudanças na pressão parcial de oxigênio, tornando o microambiente tecidual hipóxico. O interesse em analisar as alterações fenotípicas de células em hipóxia deve-se a necessidade de entender os mecanismos patológicos e a resistência aos tratamentos e ao desenvolvimento de terapias celulares. Células como os macrófagos adaptam-se a hipóxia modificando o metabolismo e a produção de citocinas. As lesões causadas pelo parasita intracelular Leishmania amazonensis são hipóxicas e o cultivo de macrófagos (células hospedeiras da Leishmania) em hipóxia induz redução da infecção com o parasita e modula a expressão de proteínas do choque térmico, indicando alterações funcionais e estruturais em ambiente hipóxico. Neste trabalho avaliamos os mecanismos responsáveis pela resistência destas células ao parasita em hipóxia e as modificações dos macrófagos causadas por este microambiente. Macrófagos cultivados em hipóxia não apresentam alterações na produção de óxido nítrico (NO) e na expressão da sua enzima produtora, óxido nítrico sintase (iNOS). Além disso, macrófagos knockout para enzima iNOS, que não produzem NO, são capazes de reduzir a infecção por L. amazonensis semelhante a macrófagos selvagens, o que sugere que o efeito leishmanicida da hipóxia não se deve ao NO. A liberação das citocinas TNF-a, IL-6, IL-12 e IL- 10 pelos macrófagos é alterada quando estes são cultivados em microambiente hipóxico. A produção destas moléculas pelos macrófagos infectados com L. amazonensis é semelhante em hipóxia e normóxia, indicando que estas citocinas não participam do efeito leishmanicida. O metabolismo energético dos macrófagos infectados, avaliado pela produção de ATP, não é modificado pela hipóxia, indicando que este fator não está envolvido na morte do parasita em macrófagos cultivados em hipóxia. Embora macrófagos fagocitem menos partículas inertes em hipóxia do que macrófagos em normóxia, a fagocitose do parasita vivo não é alterada pela hipóxia, sugerindo que o processo fagocítico também não está relacionado à diminuição da infecção pela hipóxia. Macrófagos cultivados em hipóxia aumentam a produção de ROS em relação a normóxia, mas a produção de ROS por macrófagos infectados com L. amazonensis em hipóxia não é alterada. Mas a inibição do efeito leishmanicida pelos antioxidantes N-acetilcisteína e Ebselen sugere que ROS tem papel na resistência dos macrófagos a L. amazonensis em hipóxia. A expressão de duas isoformas do fator transcricional hypoxia-inducible factor (HIF) (HIF-1alfa e 2alfa) é induzida em macrófagos cultivados em ambiente hipóxico e, interessantemente, em macrófagos infectados com L. amazonensis, mesmo quando estes são cultivados em normóxia. A inibição do HIF-1alfa pelo cloreto de cobalto impediu a sobrevivência do parasita dentro do macrófago, indicando que este fator é importante na manutenção do macrófago como célula hospedeira da L. amazonensis. Nossos resultados também demonstraram que a hipóxia alterou a capacidade proliferativa dos linfócitos T e o processamento e apresentação de antígeno de L. amazonensis pelos macrófagos. Assim, concluímos que a hipóxia induz alterações funcionais e estruturais nos macrófagos e que ROS são importantes para o efeito leishmanicida da hipóxia. Palavras-chave: Leishmania, macrófagos, hipóxia / Abstract: Regions of low oxygen tension (hypoxia) are common features of inflamed/infected tissues. The analyses of cell phenotypic alterations by hypoxia are helpful for understanding of the pathological mechanisms and treatment resistance and for the development of cellular therapies. Macrophages, cells involved in the clearance of microorganisms from infected tissues, are influenced by oxygen tension changing metabolism and cytokines production. Lesions caused by intracellular protozoan Leishmania amazonensis are hypoxic and macrophages (host cells for Leishmania) cultured in hypoxia are resistant to the infection and change heat shock proteins expression, suggesting functional and structural alterations of these cells in hypoxic microenvironment. In the present work we evaluated mechanisms involved in the macrophage resistance to the parasite as well as macrophages phenotypic alterations in hypoxia. Macrophages cultured in hypoxia did not show alterations in nitric oxide (NO) production and oxide nitric synthase (iNOS) enzyme expression. Furthermore, iNOS knockout macrophages lacking NO production are also able to reduce L. amazonensis infection as wild type macrophages, what suggests the leishmanicidal effect of hypoxia is not related to NO. The cytokines TNF-a, IL-6, IL-12 e IL-10 release is altered when macrophages are cultured in hypoxia. However, the production of these cytokines by L. amazonensis-infected macrophages in hypoxia is similar to normoxia, indicating these cytokines do not participate of the leishmanicidal effect of hypoxia. The energetic metabolism of infected macrophages, evaluated through ATP production, is not modified by hypoxia, suggesting this factor is not responsible for parasite death by macrophages cultured in hypoxia. Although the uptake of inert particles by macrophages in hypoxia is lower than in normoxia, living L. amazonensis phagocytosis by macrophages is not altered by hypoxia. This result suggests that phagocytic process is also not related to low infection in hypoxia. Cultured macrophages in hypoxia have shown higher ROS production than in normoxia, but the ROS production by L. amazonensis-infected macrophages is not altered by hypoxia. Indeed, the inhibition of leishmanicidal effect of hypoxia by antioxidants N-acetylcystein and Ebselen suggests ROS play a role in the macrophage resistance to L. amazonensis in hypoxia. The expression of two isoforms of the transcriptional factor hypoxia-inducible factor (HIF) (HIF- 1alpha e 2alpha) is induced in macrophages cultured in hypoxia and, interestingly, in L. amazonensis-infected macrophages in normoxia and hypoxia. The inhibition of HIF-1alpha by cadmium chloride impaired survival of intracellular parasite, suggesting this factor is important for macrophages as host cell for L. amazonensis. Our results also demonstrated hypoxia altered the proliferative capacity of T lymphocytes and the L. amazonensis antigen presentation by macrophages. We conclude hypoxia induces macrophages functional and structural alterations and ROS are important for the leishmanicidal effect of hypoxia. Keywords: Leishmania, macrophages, hypoxia / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

Disrupted sleep and nocturnal hypoxia are common in cystic fibrosis (CF). However, the predictors of nocturnal hypoxia in CF are still controversial. In order to identify the risk factors for nocturnal desaturation and sleep disturbances, we carried out a clinical and polysomnographic investigation of CF patients. We studied 30 clinically stable CF cases with clinical lung disease (mean age=12.8; mean forced expiratory volume in 1 second FEV1=65.2), 10 CF cases without significant lung disease (mean age=13.3; mean FEV1=99.8), and 20 controls (mean age=15.5). Patients were evaluated by spirometry, 6-min walk test (6MWT), the ShwachmanâKulczycki (SâK) score, and full overnight polysomnography. Cases with clinical lung disease had lower body mass index, forced vital capacity, and SâK scores. During sleep, five CF cases with clinical lung disease (15%) had SaO2 <90% during more than 30% of total sleep timeand 11 cases (36.6%) had a nadir SaO2 below 85%. FEV1 values for CF cases with clinical lung disease were related to nadir SaO2 (P<0.03) and to mean oxygen saturation SaO2 (P=0.02). A receiver operating characteristic (ROC) analysis determined FEV1 at 64% to be predictive of nocturnal desaturation as defined by minimum SaO2 <85% (sensitivity=92.3%; specificity=77.3%) or SaO2<90% for 30% of sleep time (sensitivity=81.8%; specificity=85.2%). Frequency of impaired sleep was not different in CF cases with (N=5) and without significant lung disease (N=2, P=0.53). Sleep architecture was not significantly different between the two groups. Sleep apnea was present in three CF cases with clinical lung disease and in one case without significant lung disease. In summary, desaturation during sleep can be predicted by FEV1<64%with good sensitivity and specificity. There are no significant differences in sleep architecture between clinically stable CF cases with and without significant lung disease. The recognition of biological markers that can predict clinical deterioration in cystic fibrosis (CF) is a key issue in everyday care of these patients. The (S-K) scores and (FEV1) have been considered the best independent predictors of impairment/disability. The aim of this study was to evaluate the role of high-resolution computed tomography of the chest (HRCT) and the use of the Bhalla score in the detection of functional disability in CF. Cases of both genders, aged older than six years, with CF clinically stable were studied with spirometry, basal oxygen saturation SpO2, the 6MWT, HRCT and the S-K score. Twenty-five patients (15 male, mean age 14.2Â5.6) with FEV1 (range 28.6-98.0; mean 62.5Â21.8) were studied. Nine patients had severe/moderate respiratory insufficiency (40<FEV1&#8804;59), nine had mild (59<FEV1&#8804;79) and six had normal function (FEV1>79). Bronchiectasis was the most frequent finding. Peribronchial thickening, mucus plugging and emphysema, despite being less severe, were also commonly observed. None of the cases presented bullae. Total scores of CT abnormalities varied from 7 to 25 (13.8Â4.4). The ROC curve showed the high sensitivity/specificity for Bhalla and S-K scores in the prediction of clinical disability as measured by the FEV1. By comparison, the Bhalla scores showed higher sensitivity than the S-K scores. SpO2 and the 6MWT were not good predictors of disability as measured by functional pulmonary tests. Melatonin, a natural hormone secreted by the pineal gland, has an important function in the synchronization of circadian rhythms, including the sleepâwake cycle, and has been shown to possess significant anti-oxidant properties. To evaluate the effects of exogenous melatonin on sleep and inflammation and oxidative stress markers in CF we conducted a randomized double-blind placebo controlled study initially involving 20 patients with CF. One case failed to conclude the study. All subjects were clinically stable when studied and without recent infectious exacerbation or hospitalization in the last 30 days. Groups were randomized for placebo (N= 10; mean age 12.10Â6.0) or melatonin 3.0 mg (N=9; mean age 16.62Â8.26) during 21 days. Actigraphy was performed during 6 days before start of medication and in the third week (days 14 to 20) of treatment. Isoprostane and nitrite levels were determined in exhaled breath condensate (EBC) at baseline (day 0) and after treatment (Day 21). Melatonin improved sleep efficiency (p=0.01) and tended to improve sleep latency (p= 0.08). Melatonin reduced EBC nitrite (p=0.01) but not isoprostane. In summary, melatonin administration reduces nitrite levels in EBC and improves sleep measures in clinically stable CF patients. / A Fibrose CÃstica (FC) à uma doenÃa crÃnica e progressiva acompanhada por episÃdios repetidos de infecÃÃes respiratÃrias. Neste trabalho, realizaram-se investigaÃÃes relacionadas aos aspectos polissonogrÃficos, de tomografia computadorizada de alta resoluÃÃo do tÃrax (TCAR) e um estudo sobre os efeitos da melatonina em pacientes com FC, que serÃo descritos a seguir. Na FC, as alteraÃÃes do sono e a dessaturaÃÃo noturna da oxi-hemoglobina sÃo comuns, no entanto, os preditores dessa dessaturaÃÃo ainda sÃo controversos e a indicaÃÃo para a realizaÃÃo de polissonografia ainda nÃo foi definida. Com o objetivo de identificar os fatores de risco associados com hipÃxia noturna e com as alteraÃÃes do sono, realizou-se uma investigaÃÃo clÃnica e polissonogrÃfica de pacientes com FC com e sem envolvimento pulmonar. Trata-se de um estudo transversal de pacientes clinicamente estÃveis com (N=30; mÃdia de idade = 12,8 anos; mÃdia de volume expiratÃrio forÃado no primeiro segundo (VEF1= 65,2%) e sem (N=10; mÃdia de idade =13,3; mÃdia de VEF1 = 99,8%) doenÃa pulmonar e controles (N=20; mÃdia de idade =15,5). Os pacientes foram avaliados por meio das provas de funÃÃo pulmonar (PFP), teste da caminhada de seis minutos (TC6min), pelo escore Swhachman-Kulczycki (S-K) e polissonografia de noite inteira. Os pacientes com doenÃa pulmonar apresentavam Ãndices mais baixos de massa corpÃrea, VEF1, capacidade vital forÃada e escore S-K. Durante o sono, entre os pacientes com FC e doenÃa pulmonar, cinco (15%) tinham SpO2 <90% durante mais de 30% do tempo total de sono e 11 (36,6%) tinham SpO2 mÃnima. Observou-se uma correlaÃÃo entre os nÃveis de VEF1 e os nÃveis mÃdios de SpO2 (p=0,02) e valores mÃnimos da SpO2 (p<0,03). A Receiver Operating Curve (ROC) mostrou que o VEF1 < 64% à um preditor da dessaturaÃÃo noturna ao se considerar o nadir, SpO2 menor que 85% (sensibilidade = 92,3% e especificidade = 77,3%) ou SpO2 < 90% durante mais de 30% (sensibilidade = 81,8% e especificidade = 85,2%). A frequÃncia das alteraÃÃes do sono, quando se considerou a qualidade subjetiva do sono (IQSP), nÃo foi diferente entre os casos de FC com (N=5) e sem comprometimento pulmonar (N=2, P=0.53). A arquitetura do sono nÃo foi significativamente diferente entre os casos de FC com e sem doenÃa pulmonar. Apneia obstrutiva do sono estava presente em trÃs casos com doenÃa pulmonar e em um caso sem doenÃa pulmonar. Em conclusÃo, a dessaturaÃÃo durante o sono pode ser prevista por um VEF1 < 64% com boa sensibilidade e especificidade. NÃo hà diferenÃas significantes entre os casos de FC clinicamente estÃveis com e sem envolvimento pulmonar. Sugere-se que a polissonografia pode ser Ãtil em casos selecionados de FC com e sem doenÃa pulmonar quando hà suspeita de apneia obstrutiva do sono. Em relaÃÃo ao estudo com TCAR do tÃrax, deve ser enfatizado que o reconhecimento de marcadores de gravidade, capazes de predizer a deterioraÃÃo clÃnica na fibrose cÃstica à de fundamental importÃncia para o manuseio terapÃutico dos pacientes. O escore de S-K e o VEF1 sÃo considerados os melhores preditores independentes do prognÃstico em FC. O objetivo desse estudo foi avaliar o papel da TCAR e o escore de Bhalla na avaliaÃÃo da gravidade de pacientes com FC. Casos de ambos os sexos, com idade superior a seis anos, clinicamente estÃveis, foram avaliados mediante espirometria, nÃveis basais de saturaÃÃo de oxigÃnio (SpO2), TC6min, TCAR e escores S-K e Bhalla. Vinte e cinco pacientes (15 homens, idade mÃdia 14,2  5,6) com VEF1 (variaÃÃo 28,6-98,0; mÃdia 62,5  21,8) foram estudados. Nove pacientes apresentavam insuficiÃncia respiratÃria moderada/grave (40 < VEF1 &#8804; 59), nove tinham insuficiÃncia respiratÃria leve (59 < VEF1 &#8804; 79) e seis tinham funÃÃo normal (VEF1 > 79). As bronquiectasias foram o achado tomogrÃfico mais frequente. Espessamento peribrÃnquico, rolha de muco e enfisema, apesar de menor gravidade, foram tambÃm comumente observados. Nenhum dos casos apresentava bolhas. Os escores totais das anormalidades tomogrÃficas variaram de 7 a 25 (13,8  4,4). A curva (ROC) mostrou alta sensibilidade/especificidade para o escore Bhalla na prediÃÃo da gravidade da doenÃa medida pelo VEF1. De forma comparativa, os escores Bhalla apresentaram maior sensibilidade do que os escores S-K. Os nÃveis basais de SpO2 e o TC6min nÃo foram bons preditores de gravidade avaliada pelos testes de funÃÃo pulmonar. Realizou-se um estudo sobre os efeitos da melatonina na FC. A melatonina à um hormÃnio natural secretado pela glÃndula pineal, tem uma funÃÃo importante na sincronizaÃÃo do ritmo circadiano, incluindo o ciclo vigÃlia-sono e tem propriedades antioxidantes. Com o objetivo de avaliar os efeitos da melatonina no sono, na inflamaÃÃo e no estresse oxidativo pulmonar realizou-se um estudo randomizado, duplo-cego e controlado por placebo. Vinte pacientes com FC foram inicialmente avaliados. Um paciente nÃo concluiu o estudo. Todos os indivÃduos estavam clinicamente estÃveis por ocasiÃo do estudo, ou seja, nÃo tinham apresentado exacerbaÃÃes infecciosas ou hospitalizaÃÃes nos Ãltimos 30 dias. Os grupos foram randomizados para o uso de placebo (N= 10; mÃdia da idade 12,10  6,0) ou melatonina 3,0 mg (N=9; mÃdia da idade 16,62  8,26) durante 21 dias. Um registro actigrÃfico foi realizado durante seis dias, antes do inÃcio da medicaÃÃo e na terceira semana (dias 14 a 20) do tratamento. Os nÃveis de isoprostano e nitrito foram determinados no condensado de ar exalado (CAE) no inÃcio do estudo (dia 0) e depois do tratamento (dia 21). A melatonina melhorou a eficiÃncia do sono (p=0,01) e nitrito do CAE, porÃm nÃo reduziu o isoprostano. Em conclusÃo, em pacientes com FC clinicamente estÃveis, a administraÃÃo de melatonina reduz os nÃveis de nitrito e melhora os parÃmetros de sono.

Adaptace na chronickou hypoxii zvyšuje toleranci srdce k ischemicko-reperfuznímu poškození. Tato adaptace je umožněna řadou fyziologických změn na buněčné úrovni. Jednou z nich je změna v buněčném energetickém metabolismu. Tento proces může být regulován proteinem FTO (z angl. fat mass and obesity associated), demetylázou epigeneticky regulující buněčnou syntézu proteinů. Srdeční metabolismus může být také modulován adipokiny leptinem a adiponektinem. Cílem tohoto projektu bylo proto studovat roli FTO a adipokinů v chronicky hypoxickém srdci. Dospělí samci potkanů kmene Sprague Dawley byli adaptováni na dva modely kontinuální normobarické hypoxie (CNH; 12 % O2 a 10 % O2; 3 týdny). CNH (10 % O2) redukovala u těchto zvířat rozsah infarktu myokardu o 20 %. CNH (12 % O2) nebyla kardioprotektivní. Hladina proteinu FTO byla měřena v tkáni levých (LV) a pravých (RV) komor, stejně jako v játrech a koncovém mozku hypoxických i normoxických zvířat. Za normoxie je hladina FTO v RV o 50 % vyšší než v LV. Ve vysoce metabolicky aktivních tkáních jater a koncového mozku jsou pak hladiny FTO vyšší dokonce 6krát a 11krát. CNH (12 % O2) vedla k signifikantnímu nárůstu hladiny proteinu FTO v srdci. Jednalo se o 21% nárůst v LV a 27% v RV. Hladiny v játrech a koncových mozcích nebyly CNH ovlivněny. Silnější CNH (10 % O2)...

Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation. / Science, Faculty of / Zoology, Department of / Graduate

Neutrophils are key effector cells of the innate immune system. They employ a number of powerful ‘weapons’ to eliminate pathogens, including an array of destructive proteins packaged into distinctive granule subsets. In addition to their microbicidal activity, these granule proteins are capable of causing substantial tissue damage if inappropriately deployed. To mitigate against this possibility, most physiological stimuli induce minimal extracellular degranulation. Sites of inflammation and infection are usually hypoxic, and it has been shown that oxygen depletion compromises neutrophil function by impairing the generation of reactive oxygen species and hence bacterial killing. The key finding reported in this thesis is that hypoxia substantially increases the release of all neutrophil granule subsets, as measured by the release of (active) hallmark proteins (elastase, myeloperoxidase, lactoferrin and matrix metalloproteinase-9). In consequence, supernatants from hypoxic neutrophils induced substantially more damage to lung epithelial cell layers than supernatants from neutrophils cultured under normoxic conditions; this damage was protein- and protease-dependent. This pattern of damage was seen consistently across lung adenocarcinoma-derived epithelial cells, primary immortalised lung epithelial cells, and primary human bronchial epithelial cells grown in physiological air-liquid interface culture. Surprisingly, the mechanism of hypoxia-augmented degranulation was found to be independent of protein synthesis and specifically, of the transcription factor HIF-1α (the ‘master-regulator’ of hypoxic responses); thus, hypoxia did not affect mRNA transcript or protein abundance of the major granule components, and hypoxia mimetics failed to recapitulate the phenotype. Inhibition of the key pathways known to be involved in neutrophil degranulation, including, phosphatidylinositol 3-kinase and phospholipase C, but not calcium flux prevented augmented granule release under hypoxia In conclusion, hypoxia induces a destructive neutrophil phenotype, with increased release of multiple histotoxic proteases. This may contribute to tissue injury and disease pathogenesis in a range of clinically important conditions.

This study sought to determine the independent influence of hypoxia on thermoregulatory responses to exercise in compensable and uncompensable hot conditions. Eight participants completed three experimental trials of cycling in either normoxia (21% O2) or hypoxia (13% O2) in order to manipulate relative exercise intensity (%VO2peak), since VO2peak was reduced by ~30% in hypoxia. When trials were matched for %VO2peak, changes in core temperature and local sweat rates (LSR) were significantly lower in the hypoxic trial as a result of a lower rate of metabolic heat production (Hprod) in order to maintain a similar %VO2peak compared to normoxia. However, when Hprod was fixed between normoxic and hypoxic trials the systematic differences in core temperature and LSR were eliminated. Conversely, at a fixed Hprod skin blood flow (SkBF) was greater in hypoxia compared to normoxia by ~40%. Despite improvements in SkBF, the potential for maximum heat loss was unaffected during an incremental humidity ramp protocol, resulting in no difference between normoxia and hypoxia in the critical ambient vapour pressures at which core temperature inflected upwards. These data further demonstrate, using a within-subjects design, that metabolic heat production, irrespective of large differences in %VO2peak, determines thermoregulatory responses during exercise. Furthermore, this study suggests that the influence of large differences in skin blood flow on heat dissipation may be lesser than previously thought.

Serotonin (5-HT) containing neuroepithelial cells (NECs) are O2 sensitive chemoreceptors found throughout the skin of larval zebrafish (Danio rerio). Zebrafish larvae are sensitive to changes in ambient PO2 as early as 2 days post fertilisation (dpf) and hyperventilate in response to hypoxia beginning at 3 dpf. Tryptophan hydroxylase (tph) is the rate-limiting enzyme in 5-HT synthesis; three tph paralogs are present in zebrafish (tph1a, tph1b and tph2). Although 5-HT has been implicated as a key neurotransmitter mediating hypoxic hyperventilation, it has not been possible to discern the role of 5-HT specifically contained within the NECs in promoting hypoxic hyperventilation. The purpose of this study was to determine the role of NEC 5-HT in regulating the hypoxic ventilatory response in larval zebrafish. It was hypothesised that 5-HT is a key neurotransmitter released from NECs which contributes to hypoxic hyperventilation. Immunohistochemistry was used to determine the distribution of tph paralogs and their role in 5-HT production in NECs. Tph1a was present in NECs and nerves innervating NECs. Exposure to the non-selective tph inhibitor, para-chlorophenylalanine (pCPA), or translational gene knockdown of tph1a, diminished 5-HT expression within NECs. Exposure to acute hypoxia (PO2 = 30 mmHg) revealed a blunted hypoxic ventilatory response (reduced breathing frequency) in fish exhibiting depleted 5-HT in NECs. The hypoxic hyperventilatory response was rescued with application of 5-HT. The results of these experiments demonstrate that tph1a is responsible for 5-HT production in NECs of larval zebrafish, and that 5-HT released from NECs is involved in establishing their hypoxic hyperventilatory response.

Identifying exploitable differences between cancer cells and normal cells has been ongoing since the dawn of cancer therapeutics. This task has proven difficult due to the complex genetic makeup of cancers. Tumours, however, share a low oxygen (hypoxic) microenvironment that selects for malignant cancer cells. It has recently been shown that cells switch from eIF4E to eIF4E2-mediated protein synthesis during periods of hypoxia, similar to those found in tumour cores. We hypothesize that this hypoxic translation complex is required for cell survival in hypoxia and can be targeted by inhibiting the eIF4E2 cap-binding protein. Here, we show that genetically diverse cancer cells require the cap-binding protein eIF4E2 for their growth, proliferation, and resistance to apoptosis in hypoxia, but not in normoxia. Furthermore, in vitro and in vivo eIF4E2-depleted tumour models cannot grow or sustain hypoxic regions without the reintroduction of exogenous eIF4E2. Thus, tumour cells could be targeted over somatic cells by selectively inhibiting their protein synthesis machinery, much like the function of antibiotics that revolutionized medicine.

For organisms relying on an aerobic metabolism, a constant oxygen (O₂) supply must be available to energy demanding tissues. In this thesis. the effects of hypoxia exposure and altered ionoregulatory demands on O₂ uptake of the larval zebrafish (Danio rerio) were evaluated. In Chapter 2, it was hypothesized that a pre-exposure to hypoxia would alter the O₂ uptake capacity of 4- and 7-days post-fertilisation (dpf) larvae through a modified vasculature system. Additionally, using a genetic knockout line, the role of Hif-1α in regulating cutaneous O₂ flux (JO₂) was tested. It was predicted that hypoxia-exposed larvae would display a higher JO₂ across the body due to a hypoxic, acclimatory response, explained by an increased vascularity and supported by an increased whole-body O₂ consumption (ṀO₂) and decreased critical O₂ tension (Pcrit). Consequently, this response was expected to be negated in the Hif1aa⁻/⁻ab⁻/⁻ larvae. Ultimately, JO₂ measured using the scanning micro-optrode technique (SMOT) remained unchanged between WT and Hif1aa⁻/⁻ab⁻/⁻ and normoxia- and hypoxia-exposed larvae, a finding which was supported by an unchanged vascularity across all treatments. The results from this chapter suggest that changes in hypoxia performance mediated by Hif-1α are unrelated to cutaneous JO₂ and vascularity. In Chapter 3, the aerobic costs of ion transport in 4 dpf larval zebrafish was assessed. We hypothesized that changes in rates of Na⁺ uptake evoked by acidic or low Na⁺ rearing would result in changes in ṀO₂ and/or JO₂, measured at the ionocyte-expressing yolk sac epithelium using SMOT. Ultimately, it was found that the measured JO₂ and ṀO₂ did not correlate with the corresponding Na⁺ uptake rate triggered by the acidic and low Na⁺ rearing environment. Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na⁺, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence for a low aerobic cost for ion regulation in zebrafish larvae.

Le diabète est associé à une augmentation de risque de maladie cardiovasculaire et une dérégulation du métabolisme. Il a été suggéré que la variabilité glycémique (VG) pouvait avoir un rôle dans le développement des complications du diabète. Afin d’étudier et de caractériser les dysfonctions induites par la VG, nous avons mis au point un modèle in vitro mimant la VG sur la lignée de cardiomyocytes HL-1. Nous avons ainsi développé un traitement de 12 heures, mimant hypoglycémie, normoglycémie, hyperglycémie et VG. L’étude de la signalisation cellulaire ne nous a pas permis de montrer un rôle délétère de la VG. Nous avons toutefois mis en évidence que la VG participait à des dysfonctions mitochondriales. En effet en situation de fluctuations en glucose, les mitochondries des cellules HL-1 présentent une augmentation de leur potentiel de membrane, ainsi qu’une augmentation de la production d’anions superoxydes. Bien que nous n’ayons pas réussi à montrer de perturbation de la chaîne respiratoire après 12 heures d’exposition, nous avons pu montrer que 72 heures d’exposition provoquaient une baisse de la respiration mitochondriale. Nous avons enfin étudié l’impact des fluctuations en glucose sur la susceptibilité au développement de lésions d’hypoxie, et avons montré que les lésions sont majorées après 36 heures d’hypoxie en cas d’exposition à des fluctuations en glucose. Nos résultats montrent un rôle délétère de la VG, néanmoins des expériences complémentaires sont nécessaires afin de caractériser de manière plus précise les mécanismes impliqués. / Diabetes mellitus is associated with higher risk of cardiovascular disease and metabolism dysregulation. Glycemic variability (GV) has been suggested as a risk factor in diabetic complication. In order to characterize dysfunctions induced by GV, we developed an in vitro model that transpose GV on the cardiac cell line HL-1. We exposed our cells to a treatment of 12 hours miming hypoglycemia, normoglycemia, hyperglycemia and GV. The exploration of signaling pathways didn’t allow us to show a deleterious effect of glucose fluctuation. However we were able to point mitochondrial alteration under glucose fluctuation. HL-1 cells mitochondria exhibit a higher membrane potential and an increase of superoxide anion production. Although we didn’t show any alteration in mitochondrial respiration after 12 hours of exposition, we showed that after 72 hours of glucose fluctuation, HL-1 cells showed a decrease in mitochondrial respiration. We finally studied the impact of glucose fluctuation on the susceptibility to develop hypoxic injuries. We showed that after 36 hours of hypoxia, injuries were higher for cells exposed to glucose fluctuation. Our results indicate a deleterious effect of GV, but additional experiments are needed to better characterize the mechanisms.