Multiple studies have implicated the POZ-ZF and methyl-DNA-binding transcription factor, Kaiso, in the regulation of genes and pathways that are important for development and tumorigenesis. In Xenopus embryos and mammalian cultured cells, Kaiso has been implicated as a negative regulator of the canonical Wnt signaling pathway. Paradoxically however, Kaiso depletion extends lifespan and delays polyp onset in the ApcMin/+ mouse model of intestinal tumorigenesis, where aberrant activation of Wnt signaling results in the development of neoplasias. These findings call into question Kaiso’s role as a negative regulator of canonical Wnt signaling and led us to hypothesize that Kaiso promotes intestinal tumorigenesis by a mechanism independent of its role in canonical Wnt signaling. To further delineate Kaiso’s role in intestinal tumorigenesis and to determine Kaiso’s role in regulating canonical Wnt signaling in the murine intestine, we generated a Kaiso transgenic mouse model expressing an intestine-specific murine Kaiso transgene. We then crossed our Kaiso transgenic mice with ApcMin/+ mice and analyzed the resultant progeny. Unexpectedly, Kaiso transgenic mice exhibited intestinal inflammation, increased expression of Wnt target genes and deregulated progenitor cell differentiation, although ectopic expression of Kaiso was not sufficient to drive tumorigenesis in the intestine. In agreement with previous studies, ectopic Kaiso expression in ApcMin/+ mice resulted in a significantly shortened lifespan and increased tumour burden. While we were unable to determine the precise mechanism by which Kaiso promotes intestinal tumour development, we found that Kaiso-induced inflammation is enhanced in the ApcMin/+ background and ectopic Kaiso expression further intensifies Wnt target gene expression in this model. Collectively, these studies have identified novel roles for Kaiso in regulating inflammation and cell-fate determination in the intestine. Furthermore, our findings suggest that Kaiso may contribute to intestinal tumorigenesis by promoting inflammation, which has been shown to be a predisposing factor for colorectal cancer development. Lastly, we have demonstrated distinct tissue and organism-specific roles for Kaiso in regulating canonical Wnt signaling. While, the aforementioned studies were the primary focus of this thesis, we also examined Kaiso’s role in DNA methylation-dependent repression of two tumour-associated genes, cyclinD1 and HIF1A. Our studies revealed that Kaiso binds and regulates the cyclinD1 locus via both sequence-specific and methylation-dependent DNA binding, suggesting that these alternate modes by which Kaiso binds to DNA may not be mutually exclusive. Furthermore, we identified a previously unexplored role for Kaiso in regulating the expression of the master regulator of hypoxia, HIF1A, which implicates Kaiso in modulating hypoxia-driven tumorigenic processes. / Thesis / Doctor of Philosophy (PhD)

Hypoxia as a stressor has physiological implications that have been a focal point for many physiological studies in recent years. In some studies, hypoxia had large effects on the organ tissue degeneration, which ultimately effects multiple ecological processes. These organ tissue studies played a part in the development of new fields like neurocardiology, a specialty that studied the relationship between the brain and the heart. This thesis focuses on how hypoxia-induced cardiac arrest alters the amounts of GLYT2, a glycine reuptake transporter, in the central nervous system of zebrafish, Danio rerio. At 7 days post-fertilization (dpf), zebrafish were exposed to acute, severe hypoxia until they lost equilibrium, and minutes later, subsequent cardiac arrest occurred. Zebrafish were then placed into recovery groups to measure the GLYT2 levels at multiple points in zebrafish recovery. Fish were then sacrificed, and their brains dissected. Using immunofluorescence, the outer left optic tectum of the zebrafish was imaged, and mean image pixel fluorescent intensity was taken. There were significant changes (one-way ANOVA) in the levels of GLYT2 compared to that of the control groups during the course of recovery. GLYT2 levels continued to rise through the 24-hour recovery mark but did not show significant difference after 3 hours of recovery. This suggest that GLYT2 levels increased rapidly in the first 3 hours of recovery and continued to increase through 24 hours at a slower rate. Changes in GLYT2 levels may affect motor and sensory information, movement, visualization, and audition in these zebrafish. Further research should be conducted to determine how long it takes for GLYT2 levels to return to baseline, as well as behavioral measurements through each recovery period as it relates to glycine function.

No description available.

Channel catfish (Ictalurus punctatus) are raised for aquaculture in ponds characterized by dramatic swings in dissolved oxygen concentration. If morning dissolved oxygen concentration falls below approximately 3.0 mg/L catfish consume less feed, leading to a reduction in growth and production. Although the effects of low oxygen on channel catfish appetite have been described, the underlying mechanisms responsible for those effects are unknown. Increased production of the neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) are implicated in other fishes as neuropeptides that reduce appetite once an environmental stressor is detected. This project characterizes the hematological responses and transcriptional response of the hypothalamus to acute, chronic, and cyclical (repeating periods of hypoxia and normoxia) hypoxia. During acute (12 hours) hypoxia, venous PO2 decreased within 6 hours, coupled with an increase in hematocrit and decreased blood osmolality. These changes reversed within 12 hours after returning to normoxia but were not coupled with a change in transcription of the genes for CRF and UI. If this pattern of hypoxia and normoxia is repeated cyclically for 5 days, the same physiological responses repeat continually. During chronic hypoxia up to 5 days in duration, channel catfish have a similar hematological response, but did not recover to normoxic control values over the duration of the challenge. Likewise, no significant change in gene expression of CRF or UI were detected during chronic hypoxia. The hypothalamic transcriptome was analyzed during a 12-hour exposure to hypoxia followed by a 12-hour normoxic recovery. Across all time points, 190 genes were differentially expressed, with the greatest numbers occurring during periods of hypoxia. Differentially expressed genes were grouped into Gene Ontology biological processes and were most overrepresented by the term “response to hypoxia,†which included genes involved with angiogenesis, red blood cell production, and negative feedback to hypoxia-inducible factor proteins. Although this study did not find a change in hypothalamic transcription of CRF and UI, it did identify multiple adaptive responses that work together to reduce the severity of hypoxia along with several gene candidates for future hypoxia studies.

Hypoxia is a characteristic pathophysiological feature of many solid tumors, which contributes significantly to resistance to chemotherapy and radiotherapy. It also induces numerous intracellular signaling pathways, which in turn trigger the upregulation of various key proteins promoting tumor cell survival, progression and metastasis. In this context, novel therapeutic approaches are urgently needed to facilitate the early detection and improve the treatment of hypoxic tumors. Focusing on the hypoxic tumor microenvironment, one can recognize that the membrane bound carbonic anhydrase IX (CA IX) isozyme represents a potential biomarker and a compelling therapeutic target for better diagnosis and management of hypoxic tumors. CA IX is significantly overexpressed under hypoxic conditions as compared to normal tissues and it assists tumor cell to maintain neutral intracellular pH values. Building on this hypothesis, we are focusing our efforts in this thesis towards the development and the optimization of drug delivery nanosystems capable of selectively targeting CA IX that is overexpressed in the hypoxic tumor niche, which in turn will enhance the early detection of hypoxic tumors as well as improve the accumulation of chemotherapeutic drugs in hypoxic cancer cells. This strategy is expected to overcome the chemoresistance associated with tumor hypoxia and minimize the systemic side effects associated with chemotherapeutic drugs administration. In chapter 2, we focused our efforts towards the development of the in vitro biological models for testing our nanoparticles. This process was achieved through screening a series of cancer cell lines for the expression of our target epitope under hypoxic conditions. We induced hypoxia either chemically, using cobalt chloride, or physicochemically, using a hypoxia chamber purged with hypoxia gas mixture containing 1% O2. Screening for CA IX overexpression under hypoxic conditions was done both in 2D monolayer cells and 3D tumor spheroids, which become naturally hypoxic due to their 3D growth. Western blot analysis was used to confirm the expression of our target protein and we have identified three cell lines with a high level of expression of CA IX under hypoxic conditions, namely HT-29 colorectal cancer, SKOV-3 ovarian cancer and MDA-MB-231 breast cancer cell lines. In chapter 3, we optimized a theranostic liposomal delivery system through the use of a combination of zwitterionic amphiphilies of different packing parameters to encapsulate a potent fluorescent carbonic anhydrase inhibitor (CAI), as a novel approach to facilitate the detection of colorectal cancer. Our main focus was to increase the aqueous concentration of poorly water-soluble CAI, to correlate its delivery efficiency with the lipid type and composition of the liposomal nanosystem, as well as to enhance the tissue permeability, allowing easy detection of small tumor polyps. Our optimized DMPC/DOPE liposomal formulation demonstrated an optimum size, high encapsulation efficiency of CAI, and a phase transition temperature below 37 ᴼC that allows efficient delivery of CAI and good tissue penetrability towards the hypoxic tumor cells overexpressing CA IX. In chapter 4, we optimized a CAI-targeted long circulating liposomal delivery system encapsulating doxorubicin. Our main focus was to enhance the accumulation of doxorubicin in hypoxic tumors through targeting CA IX protein overexpressed under hypoxic conditions. This strategy proved to enhance the internalization of the drug carrier into hypoxic cancer cells thus overcoming chemoresistance associated with hypoxia and also minimize the systemic side effects associated with the intravenous administration of non-targeted Doxil®-like formulations. In chapter 5, we optimized a pH sensitive gold nanoplatform functionalized with CAI based moieties to enhance the selective delivery of doxorubicin to hypoxic tumors in a controlled release manner. Our main focus was to combine the advantage of targeting CA IX overexpressed under hypoxic conditions with the intracellular triggered release of doxorubicin in the lysosomes inside the cell in order to enhance the delivery of doxorubicin inside the cancer cells and to overcome the chemoresistance associated with hypoxia. / Pharmaceutical Sciences

The purpose of this study was to investigate the effects of acute moderate hypoxia (14.5% inspired oxygen fraction) on the magnitude of fatigue development during the different phases (work, recovery, and both) of a repeated-sprint test (10 x 6-s sprints interspersed with 30-s recovery) in healthy, trained males. This study also sought to investigate the relationship between anaerobic power reserve and fatigue during the aforementioned repeated-sprint test. Fourteen exercise-trained males completed four trials of the repeated-sprint test under each of the following conditions: (a) normoxia (20.93% inspired oxygen fraction), (b) acute hypoxia during the work intervals only, (c) acute hypoxia during the recovery intervals only, and (d) acute hypoxia during both the work and recovery intervals. The order of the experimental conditions was systematically balanced. Fatigue scores were not different between experimental conditions, despite the fact that arterial oxygen saturation values from the acute hypoxia during recovery condition and the acute hypoxia during both work and recovery condition differed significantly from the normoxia and acute hypoxia during work intervals only conditions (p < .001). There was no relationship between anaerobic power reserve and fatigue in any experimental condition. The results of the present study indicate that although the participants experienced different levels of hypoxia during the experimental trials, the degree of hypoxia was insufficient to alter mechanical performance during a repeated-sprint test. The lack of an effect on mechanical performance does not appear to be influenced by an individual's anaerobic power reserve. It may be inferred that the degree of hypoxia employed was neither severe enough as to impair levels of muscle oxygenation beyond what was experienced in normoxia, nor as to induce further fatigue related to central mechanisms. / Kinesiology

The purpose of the present study was to determine whether or not the combination of normobaric hypoxia and exercise training would enhance adaptations in skeletal muscle over and above that which occurs with the same amount of training under normoxic conditions. Also investigated was the effect of such training conditions on performance as assessed by V0_2max and maximal aerobic capacity (MAC). Ten males performed unilateral cycle ergometry training 3 times per week for 8 weeks so that one leg was trained under normoxic conditions and the other while breathing an hypoxic gas mixture (FI0_2= 13.5%; equivalent to an altitude of 3,292 meters). Absolute power output was kept constant for both conditions and subjects performed both continuous (75% pre-training maximal power output) and interval (100% pre-training maximal power output) training. Needle biopsies were taken from the vastus lateralis of both legs to assess pre- and post-training differences in morphometric and biochemical data. Performance measures included V0_2max and MAC (time to fatigue at 95% pre-training maximal power output) for each leg. Significant increases in V0_2max (p <0.05) occurred in both legs with higher peak ventilation and blood lactate concentrations (p <0.05) post-training. Marked improvements (p <0.05) in MAC were also seen with an increase of 402% in the normoxically-trained leg and 513% in the hypoxically-trained leg. Citrate synthase (CS), succinate dehydrogenase, and phosphofructokinase activity was significantly (p <0.05) higher in both legs following training with a significantly greater (p <0.05) increase in CS in the hypoxically-trained leg. There were no differences in capillary/fiber ratio, capillary density, fiber area, fiber type, and mitochondrial volume density for either condition, pre- or post-training. It is concluded that hypoxia enhanced the muscle oxidative capacity (as marked by cs activity) but was unable to improve performance over and above that which occurs with the same training at sea level. / Thesis / Master of Science (MS)

The process of reversible growth arrest, otherwise known as cellular quiescence or the G₀ phase denoted by withdrawal from the cell cycle, is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to/dependent on the limiting factor or circumstance inducing growth arrest. Here I characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition and oxygen limitation (hypoxia). Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients is the limiting factor inducing growth arrest. Contact inhibition and hypoxia induced FABP4 expression, whereas cycling control CEF and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. The GAS gene p20k lipocalin has been shown to exhibit similar expression patterns to FABP4, with its regulation determined by the presence of the transcription factor C/EBP-β. CEF overexpressing C/EBP-β also showed strong FABP4 induction. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that C/EBP-β bound directly to the FABP4 promoter in both normoxic and hypoxic cells, although only the latter condition induced FABP4 protein expression. In summary, these results suggest that FABP4 is induced during growth arrest specifically when oxygen is the limiting factor, as induction was not seen during growth arrest mediated by starvation-induced endoplasmic reticulum (ER) stress, where nutrients was the limiting factor. The induction of these hypoxia-responsive genes suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes. Additionally, FABP4 was shown to be associated with growth arrest and the promotion of cell survival and proliferation, as depicted by proliferation assays. Lastly, C/EBP-β not only strongly induced FABP4 expression, but directly bound to the FABP4 promoter. This suggests that C/EBP-β is a regulator of FABP4, although there may be other interacting factors acting as activators or repressors as this FABP4-C/EBP-β interaction was observed in conditions permissive and non-permissive to FABP4 expression. / Thesis / Master of Science (MSc) / The process of reversible growth arrest is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to the limiting factor or circumstance inducing growth arrest. Here we characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition (CI) and hypoxia. Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients availability is the limiting factor. CI and hypoxia induced FABP4 expression, whereas control and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. When overexpressing C/EBP-β, CEF showed strong FABP4 induction. Additionally, a direct interaction with the FABP4 promoter was observed in both normoxic and hypoxic cells, although only the latter condition induced expression. In summary, the induction of this hypoxia-responsive gene suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes and that this induction may be regulated by C/EBP-β.

For animals at high altitude, low oxygen (hypoxia) is an unremitting stressor that has the potential to impair metabolism and performance. The hypoxic chemoreflex senses reductions in the partial pressure of O2 in the arterial blood and thus elicits many of the physiological responses to hypoxia, including increases in breathing and activation of the sympathetic nervous system. The hypoxic chemoreflex is vital to surviving acute exposure to severe hypoxia, but the advantage of this reflex during chronic hypoxia is less clear. The goals of my thesis were to examine how control of breathing by the hypoxic chemoreflex has evolved in high-altitude natives to maintain O2 transport in chronic hypoxia, and to elucidate the potential genetic mechanisms that were involved. This was accomplished using deer mice (Peromyscus maniculatus) native to high- and low-altitudes, in addition to a strictly low-altitude species (P. leucopus). I found that high-altitude deer mice breathe with higher total ventilation using preferentially deeper breaths, contributing to higher O2 saturation of arterial blood, but in contrast to lowland mice highlanders do not exhibit ventilatory plasticity in response to chronic hypoxia. These phenotypes appeared to be uniquely evolved in the highland population and arise during the onset of endothermy in early post-natal development. I then used second-generation inter-population hybrids to evaluate the effects of genetic variation (specifically, in the hypoxia-inducible factor 2a gene Epas1 and in haemoglobin genes) on an admixed genomic background. The high-altitude variant of α-globin could completely explain the deep breathing pattern of highland mice, whereas the high-altitude variant of Epas1 and possibly β-globin contributed to their apparent lack of ventilatory plasticity in response to chronic hypoxia. Together, the physiological changes elicited by these mutations contribute to maintaining O2 uptake and metabolism in the cold and hypoxic environment at high altitude. / Thesis / Doctor of Philosophy (PhD) / High-altitude environments are amongst the harshest on earth, with extremely low levels of oxygen, but some animals not only survive but thrive in these conditions. How these animals do so was previously not well understood. My thesis has uncovered how the evolution of respiratory physiology contributes to high-altitude adaptation in the deer mouse, the species with the broadest altitudinal distribution of any North American mammal, and has elucidated the genetic mechanisms involved. My work contributes to understanding nature’s solutions to oxygen deprivation – an all too common problem in many human and animal diseases.

Yes / Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells. / This work was primarily supported by a PhD studentship for SME (RAF) and partly by Yorkshire Cancer Research (PML, KP, RAF), a Prostate Cancer UK studentship for MS (KP) and a Wellcome Trust grant (RAF).