Thesis (Ph. D.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 45-52). Also available online in ProQuest, free to University of Oregon users.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 253-257).

Made available in DSpace on 2014-06-11T19:30:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-09Bitstream added on 2014-06-13T18:40:34Z : No. of bitstreams: 1 dantas_bf_dr_botfca.pdf: 285129 bytes, checksum: 63cec0eda142ffb4303d4c3cd029e95a (MD5) / Foram realizadas análises de qualidade fisiológica, bem como ensaios enzimáticos de a-amilase e amilases totais em sementes de milho, cv. Saracura BRS4154 e CATI AL34, submetidas a diferentes períodos de deficiência de oxigênio (hipoxia), com a finalidade de avaliar a resposta das sementes a essa condição. Para tanto, as sementes das cultivares AL34 e Saracura foram submersas em 50mL de água destilada e incubadas em germinadores a 27oC, no escuro, durante 0, 1, 2, 3, 4 e 5 dias. Após esse período foram realizados testes de germinação e vigor (primeira contagem do teste de germinação, condutividade elétrica, crescimento de plântulas, emergência das plântulas em campo, índice de velocidade de emergência e tempo médio de emergência). Para a avaliação da atividade de a-amilase e de amilases totais, as sementes submetidas aos períodos de submersão foram divididas em quatro grupos. Um grupo foi prontamente congelado e os outros três foram semeados em gerbox e mantidas a 27oC durante 2, 4 e 7 dias. Para a extração das enzimas amilolíticas, retirou-se os tecidos de reserva das sementes, que foram macerados em tampão TRIS-HCl 0,1mol.L-1, pH 7, contendo NaCl 0,1mol.L-1e CaCl2 10mmol.L-1 e centrifugadas a 12.000g, 4oC durante 10 minutos. O sobrenadante foi congelado até a realização dos ensaios. A atividade de amilases totais e de a-amilase foi medida em um sistema de reação, contendo Acetato de sódio 50mmol.L-1, pH 5,2 e CaCl2 10mmol.L-1 e amido solúvel de batata 2,5% como substrato, incubado a 35oC durante 15minutos. Para inativar outras amilases, permanecendo apenas a a-amilase, o extrato cru foi mantido a 70oC, durante 15 minutos. A atividade amilolítica foi expressa em mmol de açúcares redutores produzidos pela degradação de amido, por mg de proteína, por minuto... / Maize seeds cultivars Saracura BRS-4154 and CATI AL34, subjected to several flooding periods, were accessed for physiological quality and total amylase and a- amylase activity aiming to evalute seeds response to this condition. The seeds were submerged in 50mL of distilled water and incubated at 27oC, in the dark during 0, 1, 2, 3, 4 and 5 days. After that, germination and vigor (first counting, electrical conductivity, seedlings growth, seedlings field emergence, germination index and germination speed) tests were carried out. For the amylolitic activity, the seeds subjected to hypoxia were divided in four portions. One portion was readily frozen and the other three were germinated during 2, 4 and 7 days. The enzymes were extracted by the seeds reserve tissues homogenization in buffer 0.1mol.L-1 TRIS-HCl, pH 7, with 0.1mol.L-1 NaCl and 10mmol.L-1 CaCl2. After that, the solution with the homogenized tissues was centrifuged at 12000g at 4oC during 10 minutes. The pellet was discarded. The amylolitic activity was measured in a reaction buffer containing, sodium acetate 50mmol.L-1, pH 5.2 and CaCl2 10mmol.L-1 along with soluble potato starch 2.5% as substrate incubated at 35oC during 15minutes. To inactivate other amylases, and purify a- amylase, the crude extract was incubated at 70oC, during 15 minutes. The amylase activity was expressed in reducing sugars mmol produced by starch degradation per protein mg, per minute. After the hypoxia periods the seeds were germinated in germitest paper imbibed in 100 mg.L-1 GA3, 0.375% CaCl2 and 100 mg.L-1 GA3 + 0.375% CaCl2 solutions and assessed for germination and vigor tests and amylolitic activity. The results indicate both cultivars are tolerant to hypoxia but had their reserve degradation and mobilization inhibited by oxygen deficiency. Calcium and GA individually have no effect on submerged seeds, but...(Complete abstract, click electronic access below)

Made available in DSpace on 2015-04-09T12:28:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-07Bitstream added on 2015-04-09T12:48:25Z : No. of bitstreams: 1 000816830.pdf: 3606121 bytes, checksum: b72f959db176b160845ce86fd37d1575 (MD5) / Baixa pressão parcial de O2 durante o desenvolvimento embrionário e/ou fetal pode diminuir a taxa metabólica e a taxa de crescimento do embrião, podendo levar a alterações morfofisiológicas após o nascimento, além de atenuar a resposta ventilatória hipóxica no recém-eclodido de galinhas. No presente estudo, várias incubações foram realizadas para avaliar o efeito da hipóxia pre-natal sobre a taxa metabólica e a resposta à hipóxia aguda em diferentes linhagens e idades de Gallus gallus. Além disso, foi verificada a possibilidade de que a redução da taxa metabólica por si e não especificamente induzida por hipóxia prolongada durante desenvolvimento pré-natal seria um fator contribuinte sobre a diminuição da taxa metabólica em neonatos. Para isso embriões de galinha foram incubados a 35 ◦C (grupo de frio), que é conhecido por diminuir o consumo de oxigênio embrionário por ~30% ao longo de incubação, e aumenta o período de incubação em ~2 dias. Quanto a aves precoces, nenhum estudo havia abordado a influência da hipóxia em diferentes fases de incubação no desenvolvimento morfométrico do intestino, importante não somente para os processos digestivos e de absorção, mas também para a proteção contra patógenos; por isto avaliamos as características morfométricas e a quantidade de células caliciformes por vilo das regiões do intestino delgado em pintainhos recémeclodidos de frango de corte / Low O2 partial pressure during embryonic and fetal development can decrease the growth and metabolic rate of the embryo, leading to morphophysiological changes after birth, and blunt the hypoxic ventilatory response in newly hatched chicken. In the present study, different conditions during incubation (cold and hypoxia) were performed to evaluate the effect of prenatal hypoxia on metabolic rate, and the response to acute hypoxia in different strains and ages of Gallus gallus. We also considered the possibility that prolonged metabolic depression per se, but not specifically induced by hypoxia, during incubation would be a contributing factor to decreased metabolic rate in neonate. Thus chicken embryos were incubated at 35◦C (cold group), which is known to decrease the embryonic oxygen consumption (VO2) by ~ 30% throughout the incubation period and increases the incubation time in ~2 days. Finally, in precocial birds, no study has approached the influence of hypoxia at different stages of incubation in the morphometric development of the intestine, not only important for the digestive processes and absorption, but also for protection against pathogens, so we evaluated the morphometric characteristics and quantity of goblet cells per villi of the small intestine regions in newly hatched broiler chicks

The JmjC-histone lysine demethylases (JmjC-KDMs) are epigenetic regulators responsible for the demethylation of methylated lysine residues on the N-terminal histone tails. As Fe²⁺ and 2-oxoglutarate dependent oxygenases (2OG oxygenases), the JmjC-KDMs possess an absolute requirement for molecular oxygen and are related to the cellular oxygen sensing HIF hydroxylases, PHD2 and FIH. Several JmjC-KDMs are known HIF target genes, hence are upregulated in hypoxia. Moreover, a number of JmjC-KDMs have been shown to have differential oxygen dependences, while aberrant histone methylation has been observed in both hypoxic cells and disease states such as cancer and cardiovascular disease. The work described in this thesis aimed to investigate the impact of hypoxia on the JmjC-KDM, KDM4A. In vitro kinetic analyses revealed a K_m^app(O₂) for recombinant KDM4A of 173 ± 23 μM, which is higher than reported values for the 2OG oxygenases C-P4H, mPAHX and even FIH, and approaching those evaluated for the key oxygen sensor PHD2 (230-1746 μM). These results indicate that KDM4A activity is highly sensitive to oxygen availability, and has the biochemical potential to act as an oxygen sensor in the context of epigenetic regulation. Subsequent investigation of the cellular oxygen dependence of KDM4A, and found that the activity of ectopically expressed KDM4A in U2OS cells demonstrates a graded response to oxygen. Importantly, this trend correlates with the in vitro results, providing further evidence that hypoxia may impact upon epigenetic regulation by the JmjC-KDMs. The various factors that may contribute to the hypoxic inhibition of KDM4A were investigated both in vitro and in cells. The results of these studies suggested that altered concentrations of TCA cycle intermediates, comprising reduced levels of the 2OG oxygenase co-substrate 2OG and increased concentrations of the reported inhibitor 2HG, are likely to only minimally affect the activity of KDM4A in hypoxia. Interestingly, the 2OG oxygenase inhibitor IOX1 possessed increased inhibitory potency against KDM4A under conditions of low oxygen, implying that the use of mixed-mode inhibitors against KDM4A may be of therapeutic benefit in hypoxic disease states. This may be of particular pertinence to cardiac hypertrophy (CH), in which KDM4A activity is reported to have pathophysiological consequences. In a collaboration with Dr Tim McKinsey (University of Colorado, Denver), the KDM4 inhibitor CCT1 was tested in a phenotypic screen of cardiomyocyte hypertrophy, the results of which further support a role for KDM4A in this disease, and suggest that the use of small-molecule inhibitors of KDM4A may be a viable therapeutic strategy in CH. Finally, the effect of reactive oxygen species, levels of which may be increased in hypoxia, on KDM4A activity was explored. Recombinant KDM4A was found to be acutely sensitive to inhibition by hydrogen peroxide (H₂O₂) when compared to the HIF hydroxylases PHD2 and FIH. These results imply that KDM4A may act as a sensor of oxidative stress at the chromatin level, and further investigation in a more biologically relevant context is proposed. Overall, the work described herein demonstrates that the activity of KDM4A is sensitive to oxygen availability, a phenomenon that is likely to have significant implications for epigenetic regulation in hypoxia and the expression of KDM4A-regulated genes in ischaemic disease states.

Hypoxia regulates many hundreds of genes that play important roles in numerous physiological and pathophysiological processes. The hypoxia inducible transcription factors (HIFs) are central to the transcriptional activation of these hypoxia-regulated genes. However, to date, little is known about the determinants of HIF-1 and HIF-2 binding site selection. Both HIF-1 and HIF-2 appear to bind as HIF-α/HIF-1β heterodimers, and recognise the same core consensus DNA motif, the hypoxia response element (HRE). However, each has its own distinct but partially overlapping set of binding sites that accounts for only a subset of the total accessible HRE motifs. Here, I have utilised ChIP-seq to systematically compare pan-genomic HIF-1α, HIF-2α and HIF-1β DNA binding in multiple cell lines and have related this to RNA-seq analyses and other publically-accessible next-generation sequencing datasets. I show that endogenous HIF-Iα subunits exhibit a high-degree of binding site concordance with HIF-1β, consistent with largely canonical binding of intact heterodimers. Despite cell-type specific differences in HIF-1 and HIF-2 binding site occupancy, each isoform exhibits a remarkable rigidity in its preference to bind either promoter-proximal or promoter-distal sites, respectively. These specific distribution patterns are unaffected by the absence of the other HIF-Iα subunit, suggesting that they do not result from exclusion of one isoform by competition with the other, but rather are discrete properties of each. Furthermore, hypoxia regulated genes neighbouring sites that are shared by both HIF-1 and HIF-2 are more likely to be regulated by HIF-1 when the site is closer to the gene and by HIF-2 when further away, indicating that post-binding mechanisms of transcriptional regulation also follow a similar pattern. Comparison of sites preferentially bound by HIF-1 and HIF-2, respectively, revealed associations with distinct histone environments, distinct accessory transcription factor binding motifs and distinct patterns of transcription factor binding site occupancy, suggesting that each may be influencing specific HIF-1 and HIF-2 binding site selection. In particular, both the AP-1 motif and AP-1 binding site occupancy were enriched within HIF-2 binding sites compared to HIF-1 sites. Intervention on AP-1 DNA-binding using the dominant-negative protein, AFos, attenuated HIF binding, specifically at sites co-occupied by AP-1 and HIF. This indicates that a cooperative relationship exists between the two transcription factors. However, binding of both HIF-1α and HIF-2α were affected suggesting that while AP-1 binding may account for the ability of HIF to bind some HRE motifs but not others, it is not a determinant of differential binding between the two isoforms. Overall, this work reveals remarkably distinct and functionally relevant patterns of HIF-1 and HIF-2 binding across the genome, and provides insight into underlying mechanisms of binding.

It has been shown that tumour cells are capable of switching to glycolytic metabolism for the production of ATP even in the presence of oxygen, this is known as aerobic glycolysis or the 'Warburg effect'. The glycolytic phenotype has been associated with tumour aggressiveness and poor outcome in several cancer types. This makes the area of cancer metabolism an attractive area for the potential identification of new therapeutic targets. One key component, required for cells to maintain the glycolytic phenotype, is the presence of monocarboxylate transporters that are capable of exporting lactate. These transporters are vital for the maintenance of the intracellular pH of cells under these conditions. This study was centred around the hypothesis that altering expression of MCTs would impact on the metabolism of tumour cells and, therefore, other key characteristics of cells relating to metastatic capabilities and survival following treatment. For the purpose of this work, prostate cancer cell lines were transfected with lentiviral particles targeting overexpression of MCT1 or MCT4, or knockdown of MCT4. Following transfection, cellular metabolic profiles were assessed under normoxic and hypoxic conditions and the metastatic phenotype of each cell line was investigated. Additionally, the effect of MCT expression on response to chemotherapy and radiation therapy was explored, and a siRNA metabolome screen was performed to identify combinations of targets that may produce synthetic lethality in prostate cancer cell lines. It was shown that changes in the expression of MCT1 or MCT4 did not cause significant changes in the metastatic phenotypes of the prostate cancer cell lines investigated. Some differences were observed in the metabolic pathways used by these prostate cancer cells following alterations in MCT expression. For example, overexpression of MCT1 in DU145 cells resulted in an increase in intracellular lactate. Additionally, MCT4 knockdown in PC3 cells was able to reduce OXPHOS under reduced oxygen. MCT1 overexpression was able to sensitise androgen-independent prostate cancer cells to treatment with chemotherapy and radiation therapy. Furthermore, combinations of siRNA treatments were identified that may be capable of producing synthetic lethality. In summary, findings in this study indicated that targeting MCT1 and MCT4 expression could offer therapeutic benefit in prostate cancer. However, it was also highlighted that the roles of these transporters are specific to cancer type, and even cell line.

Hypoxia and ocean acidification (OA) are amongst the major environmental threats to marine ecosystems worldwide. Biofilms, the signpost to guide larval settlement of many benthic invertebrates, are known to be responsive to environmental changes and thus can become the crucial factor for the response of benthic invertebrate communities. This study aimed at investigating the individual and interactive effects of hypoxia and OA on biofilms and the subsequent effects on larval settlement. Biofilms collected from two sites (clean, hypoxic) were treated with a factorial design of low dissolved oxygen and/or low pH conditions in microcosms and the bacterial cell density and viability (by LIVE/DEAD® cell viability assays) were analyzed. Larval settlement preference was tested with the marine invertebrate, Crepidula onyx. The total bacterial cell densities of biofilms of the hypoxia and hypoxia and OA combination treatment were lower than that of the control biofilms for both sites. There was generally no significant difference in cell viability among control and different treatments for both sites. While the larval settlement rate on hypoxia and hypoxia and OA combination treated biofilms was significantly lower. In conclusion, this study revealed that hypoxia and OA are likely to affect larval settlement by alteration of biofilms, and this may lead to alterations in future coastal communities.

Background: Current studies in radiotherapy aim to develop better methods for curing patients fromcancer. Since different types of radiation interact with biological matter in various ways, the resultsof their interaction and their effectiveness with respect to the biological damage to cells have ageneral investigation interest. Aim: The work in this project aims to use a mathematical model to fit a pre-existing data onclonogenic survival of cells irradiated by different types of radiation and report the fittingparameters. Various radiobiological concepts were investigated and compared between differentradiation qualities used in this work. Materials and Methods: The repairable-conditionally repairable (RCR) damage model parametrisedwith respect to the linear energy transfer (LET) of the cell oxygenation was used for fittingexperimental cell survival data for human salivary gland cells irradiated in oxic and hypoxicconditions with protons, 12C-, 20Ne- and 3He-ions. Results: Good consistency with the entire cell survival data was achieved. RCR-model was robustenough to achieve agreement with cell survival data for LET values excluded from fitting procedure.Slope of cell survival curves for the three ions increased up to optimal LET value reaching maximumthere and it decreased at higher LETs. RBE of 3He-ions showed the most rapid increase in low-LETregion and reached a higher maximum as compared with other ions. RBE of the three ions increasedapproximately in the same LET region as a and c parameters of RCR-model, but no underlyingradiobiological mechanism could explain any of curve shape similarities. The RBE of 12C-ions reachedmaximum approximately at 126 keV/μm, which is the optimal LET that could possibly correspond tothe steepest cell survival curve. It was observed how the cell oxygenation became less important forcell irradiation with very high LET values. Conclusion: The results showed that it is feasible to use the RCR model to fit the broad range of cellsurvival curves corresponding to different radiation qualities and the assessment of their relativebiological effectiveness in oxic and hypoxic irradiation conditions. RCR-model may have a possible application in cell irradiation with other ion beams than those used in this work.