No description available.

Microglia, the resident immune cells of the central nervous system (CNS), become activated in response to danger signals given out by other cells when homeostasis has been disturbed. Microglial activation is a multifaceted phenomenon that includes numerous distinct phenotypes. The type of activation often influences the survival of surrounding CNS tissue, and thus gaining a better understanding of how microglial activation is regulated has important therapeutic implications. Currently, it is known that the phenotype of activated microglia depends on both the type of CNS insult and the specific activating agent. The aim of this thesis was to investigate the potential involvement of other determining factors. Extrinsic regulators of microglial activation, including the severity of CNS insult and the stimulation strength of activating agents, were examined. Intrinsic differences among different microglial populations, namely differences in region of origin and age of origin, were also investigated. To study microglial behavior without interference from other cells, rat primary cultures were used as the system of study. With regard to extrinsic factors, it was found that different severities of hypoxic neuronal injury induced distinct microglial phenotypes. Among the activating agents released by injured neurons, adenosine 5-triphosphate (ATP) was studied in isolation and was found to induce trophic and toxic effectors in microglia depending on the strength of ATP stimulation. In regards to intrinsic factors, it was found that microglia derived from different regions of the brain had distinct responses to activators, with cortical and hippocampal microglia generating more toxic responses than brainstem, striatal, and thalamic microglia. Microglia derived from various ages of origin also responded differentially to activators, with neonatal and aged microglia being more reactive than microglia derived from other age groups. Together, the results here present several novel concepts, that the phenotype of activated microglia are dependent not only on the type of activating stimulus, but the strength of that stimulus, and that in addition to stimuli from other cells, the regional and age differences among microglia themselves are also crucial in determining their activation phenotype.

M. Phil. in Marine Environmental Science. Includes bibliographical references.

Connexin 43 (Cx43) is a gap junction protein that enables direct cell-to-cell communication in many tissues of the body and stressful environments such as injury and hypoxia affect its expression. In addition to affecting Cx43 expression, hypoxia also retards wound-healing rates. Using rat corneal organ cultures, this thesis aims to define hypoxia's role in the expression and localization of Cx43 in wounded corneas. Tissue samples were stained immunohistochemically at three different time points after injury, 2.5 hours, 5 hours, and 18 hours. It was determined that under hypoxic conditions, Cx43 is more highly expressed than under normoxic conditions after injury. While Cx43 expression follows the same spatial pattern throughout the healing process in the cornea under both normoxic and hypoxic conditions, the pattern is delayed under hypoxic conditions. These results provide a possible reason why wound closure is delayed in low oxygen environments.

Hypoxia-ischemia (HI) insults can have profound effects on the immature brain, impairing development and leaving survivors with lifelong physical and cognitive deficits. Improvements in neonatal care have resulted in more newborns surviving HI, but effective treatments for the long-term consequences of this disorder have yet to be established. Using the Rice-Vannucci model of hypoxia-ischemia at postnatal day (PND) 7, we investigated the effects of metformin and enriched rehabilitation on short and long-term motor and cognitive outcome in both male and female Sprague-Dawley rats. A battery of behavioural tests was used to assess early development and motor function from PND 8-21, while long-term motor and cognitive function was assessed from PND 49 onwards. Metformin, administered from PND 8-49, improved several aspects of early development that were compromised following HI (weight gain, neurological reflexes). However, it worsened motor impairments in the adhesive strip removal task and Montoya staircase. Enriched rehabilitation, beginning at PND 21, improved motor function in the adhesive strip removal task, open field and Montoya staircase. Additionally, it enhanced cognition in the Barnes maze and Morris water maze. Our results indicated that, despite early beneficial effects on development, metformin was not effective at improving long-term outcome. Enriched rehabilitation led to significant improvements in several aspects of motor and cognitive function, even when administered 2 weeks post-injury. This data suggests that enriched rehabilitation, but not metformin, may be a valuable intervention for treating behavioural impairments resulting from episodes of perinatal hypoxia-ischemia.

Hypoxia Inducible Factors (HIFs) are very important transcription factors that can respond to low oxygen concentrations in the cellular environment. Inhibition of HIF’s transcriptional activity represents a promising approach to new anticancer compounds. Herein, we describe the design and synthesis of a series of HIF-1 inhibitors. Evaluation of these inhibitors using a cell-based luciferase assay led to the discovery compounds with sub-micromolar potency.

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

It was hypothesized that chronic hypoxia will affect various biological processes including developmental trajectory and behavior. To test this hypothesis, embryos were raised to adulthood in severe hypoxic environments (0.5% O2 or 1% O2, 22°C) and analyzed for survival rate, developmental progression, and altered behaviors. Wildtype hermaphrodites survive chronic hypoxia yet developmental trajectory is slowed. The hermaphrodites raised in chronic hypoxia had different phenotypes in comparison to the normoxic controls. First, hermaphrodites exposed to chronic hypoxia produced a significantly lower number of embryos and had a slight increase in male progeny. This suggests that chronic hypoxia exposure during development affects the germline. Second, animals raised in chronic hypoxia from embryos to young adults have a slight increase in lifespan when re-exposed to a normoxic environment, indicating that chronic hypoxia does not negatively decrease lifespan. Finally, hermaphrodites that were raised in hypoxia will lay the majority of their eggs on the area of the agar plate where the bacterial lawn is not present. This is in contrast to animals in normoxia, which lay the majority of their eggs on the bacterial lawn. One hypothesis for this hypoxia-induced egg-laying behavior is that the animal can sense microenvironments in hypoxia. To examine if various pathways are involved with chronic-hypoxia responses RNAi and assayed genetic mutants were used. Specifically, genetic mutations affecting oxygen sensing (egl-9), aerotaxis (npr-1), TFG-ß signaling (dbl-1, daf-7) and predicted oxygen-binding proteins (globin-like genes) were phenotypically analyzed. Results indicate that mutations in several of these genes (npr-1, dbl-1) resulted in a decrease in hypoxia survival rate. A mutation in egl-9 also had a detrimental affect on the viability of an animal raised in chronic hypoxia. However, a similar phenotype was not observed in the vhl-1 mutation indicating that the phenotype may not be due to a mere increase in HIF-1 levels, per se. A mutation in the globin-like gene (glb-13(tm2825)) suppressed the hypoxia-induced egg-laying phenotype. That is, the glb-13(tm2825) animal raised in chronic hypoxia laid eggs on the bacterial lawn at a significantly higher rate in comparison to wildtype controls, thus suggesting that globin-like molecules may be involved with the sensing of microenvironments. Together, this research lays the foundation for understanding the implications of chronic hypoxia in developing organisms.

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

Ascending to high altitudes can cause severe threats for the human body. Hypoxia, the lack of oxygen due to a low atmospheric pressure is a major danger in great heights.Millions of people live or travel in elevated areas, means that mountain sickness due to hypoxia is a public health problem. The worst forms of mountain illness, known as high-altitude pulmonary edema and high-altitude cerebral edema, are potentially fatal. Due to a lack infrastructure and especially bad accessibility it is very challenging to support people with first aid or transport. The aim of this master thesis in Advanced Product Design was to explore the field of hypoxia in extreme altitudes in order to create a product solution that can contribute to overcome this threat and protect people from it