Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Alzheimers disease (AD) is pathologically characterized by amyloid plaques, neurofibrillary tangles, inflammation, and neurodegeneration. According to the amyloid hypothesis of AD, the central mediating event of the disease is the deposition of amyloid. The inflammation hypothesis of AD states that it is the inflammatory response to plaques and tangles, rather than the actual lesions, which causes the disease. Studies described here combine the two approaches into a single model. Four studies are presented using a basic protocol of intrahippocampal lipopolysaccharide (LPS) injection to stimulate inflammation in transgenic mice. The first study looked at alpha7 nicotinic receptors during the glial response to Abeta deposits and LPS. Reactive astrocytes which immunolabeled for alpha7 were co-localized with Congophilic deposits in APP and APP+PS1 mice, and increased after LPS injection. Unfortunately, LPS injection into alpha7 knock out mice revealed the alpha7 labeling to be nonspecific. The second study evaluated the time course of protein and gene expression after LPS injection into nontransgenic mice. This experiment identified both a transient and chronic microglial inflammatory response, with changes in cell morphology. The third study evaluated a similar time course in APP mice. Concurrent with the inflammatory response, transient reductions in Abeta burden were seen, though compact plaque load was unaffected. The fourth and final study used dexamethasone to inhibit LPS-induced inflammation in APP mice. LPS injection reduced Abeta burden, but was completely blocked by dexamethasone co-treatment. Though dexamethasone inhibited LPS-induced CD45 and complement receptor 3 levels (markers of general microglial activation), dexamethasone had no effect on scavenger receptor A or Fc gamma receptor II/III levels. An overall hypothesis regarding LPS mediated reductions in Abeta can be proposed: It is not the presence of the LPS molecule, nor the upregulation of receptors involved in phagocytosis, but rather general glial cell activation that mediates Abeta removal. Thus, a phagocytic cell must not only bind Abeta (by various receptors) but must also be capable of engulfing the material (via general cell activation). Taken together, these studies suggest that some level of inflammation in AD is beneficial and responsible for maintaining a balance between amyloid deposition and removal.

Spinal cord injury (SCI) often results in irreversible paralysis and widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate that inhibition of astroglial-NF-κB activation reduces white matter damage and improves functional recovery in a mouse model of SCI. Here we provide novel evidence demonstrating that astrocytes directly regulate oligodendrocyte fate after trauma by a glutamate-mediated AMPA receptor dependent mechanism. Following trauma, elevated expression of the SLC39a10 zinc transporter correlated with an increase in zinc uptake by astrocytes, thereby reducing extracellular zinc concentrations required for AMPA receptor inhibition. Stimulation of AMPA receptors on oligodendrocytes by glutamate induced oligodendrocyte toxicity through the activation of the NADPH oxidase enzyme within oligodendrocytes. Genetic and pharmacological inhibition of active NADPH oxidase was sufficient to attenuate oligodendrocyte death in vitro. Following SCI, NADPH oxidase inhibition reduced oligodendrocyte death by ~75%, suggesting that glutamate-mediated oligodendrocyte death is dependent on the activation of the NADPH oxidase enzyme within oligodendrocytes. Combined treatment of the NADPH oxidase inhibitor apocynin and the AMPA receptor inhibitor NBQX significantly improved hind limb locomotor behavior, reduced white matter damage and lesion volume, and significantly spared descending serotonergic fibers. These studies provide a novel mechanism of oligodendrocyte death and may lead to clinically relevant therapeutics after SCI.

This thesis concerns the investigation of the neuroprotective effects of the peptides relaxin-2 and relaxin-3. Previous studies have shown that intracerebral relaxin-2 reduces brain lesion size in an in vivo model of stroke, thereby providing evidence of a neuroprotective action of relaxin-2. This thesis set out to extend this work to determine whether or not relaxin-2 and relaxin-3 protected neural tissues from stroke in vivo and to determine the mechanisms by which relaxin-2 and relaxin-3 may protect astrocytes from injury by affecting migration, resistance to hypoxia and prevention of apoptosis. The first set of experiments show that relaxin-2 and relaxin-3 pre- and post-treatments following stroke induction protect neural tissues from cerebral damage in vivo. The next experiments show that relaxin-2 and relaxin-3 increase astrocyte migration in vitro through nitric oxide, phosphoinositide 3-kinase and matrix metalloproteinase-mediated pathways. A third set of experiments show that relaxin-2 and relaxin-3 treated astrocytes exhibited a higher viability compared to untreated astrocytes when exposed to oxygen glucose deprivation for 24 hours. Astrocytes that were cultured with relaxin-2 or relaxin-3 also showed a lower production of reactive oxygen species compared to astrocytes that were exposed to oxygen glucose deprivation alone. Finally, relaxin-2 and relaxin-3 protected astrocytes from 24-hour apoptosis injury that was induced by tumor necrosis factor alpha and hydrogen peroxide. Taken together these experiments provide evidence that relaxin-2 and relaxin-3 peptides protect neural tissues from the deleterious effects of cerebral ischemia in vivo and help elucidate some of the cellular mechanisms by which relaxin peptides might protect the brain. Furthermore, these data show that relaxin-2 and relaxin-3 act directly on astrocytes, the most numerous cell type in the brain, to increase astrocyte migration and to protect these cells from some of the deleterious effects of stroke, namely hypoxia and apoptosis.

Several research groups have applied elevated hydrostatic pressure to ONH astrocytes cultured on a rigid substrate as an in vitro model for glaucoma. These studies have shown significant biological effects and this hydrostatic pressure model is now becoming generally accepted in the ophthalmic community. However, since the applied pressures were modest the finding of significant biological effects due to pressure alone is surprising. We hypothesized that the application of hydrostatic pressure as described in these studies also altered gas tensions in the culture media. Our goal was to design equipment and carry out experiments to separate the biologic effects of pressure from those of hypoxia on cultured astrocytes. We designed equipment and carried out experiments to subject cultures of DITNC1 astrocytes to the four combinations of two levels of each parameter. We explored the morphology and migration rates of astrocytes, but observed no significant change in any of these properties.

Molecular, cellular, and physiological processes within an organism are set to occur at specific times throughout the day. The timing of these processes is under control of a biological clock. Nearly all organisms on Earth have biological clocks, ranging from unicellular bacteria and fungi to multicellular plants, insects, reptiles, fish, birds, and mammals. The biological clock is an endogenous time-keeping mechanism that generates the onset of many processes and coordinates the phases of processes over 24 hours. While the biological clock allows these organisms to maintain roughly 24-hour, or circadian, timing in daily processes, many organisms have the ability to set their clocks, or entrain them, to changes in light. In mammals, the suprachiasmatic nucleus (SCN) is the master biological clock that entrains daily physiological and behavioral rhythms to the appropriate times of day and night. The SCN is located in the hypothalamus and contains thousands of neurons and glia that function in coordinating system-level physiological rhythms that are entrained to environmental light cues. Many of these neurons and glia are individual circadian oscillators, and the cellular mechanisms that couple them into ensemble oscillations are emerging. Adenosine triphosphate (ATP) is a transmitter involved in local communication among astrocytes and between astrocytes and neurons. ATP released from astrocytes may play a role in SCN cellular communication and synchrony. Extracellular ATP accumulated rhythmically in the rat SCN in vivo, and ATP released from rat SCN astrocytes in vitro was rhythmic, with a periodicity near 24 hours. ATP released from mouse SCN astrocytes was circadian, and disruption of the molecular clock abolished rhythmic extracellular ATP accumulation. SCN astrocyte cultures with disrupted molecular clocks also had marked reductions in total ATP accumulation compared to SCN astrocyte cultures with functional biological clocks. Furthermore, ATP-induced calcium transients were rhythmic, and this rhythmic purinergic sensitivity was abolished in clock mutant astrocytes. Pharmacological blockade of purinergic signaling, with antagonists of both the P2X7 and P2Y1 receptors, led to a gradual reduction in the amplitude of coordinated ATP accumulation over three days. These purinergic receptor antagonists, as expected, led to a reduction in calcium responses of SCN astrocytes to ATP and led to a dampening of clock gene expression rhythms as determined by PER2::LUC bioluminescence reporting in SCN astrocytes. These data demonstrate that astrocytes of the mammalian SCN rhythmically release ATP and are rhythmically sensitive to ATP in a manner dependent on their intrinsic molecular clock. Ensemble rhythmicity of SCN astrocytes is, in turn, dependent on that rhythmic purinergic signaling via both P2X and P2Y classes of ATP receptors. These results are indicative of a functional role for ATP accumulation within the SCN, with astrocytes releasing ATP every 24 hours for continual signaling onto astrocytes and neurons to maintain daily coordinated synchrony of the clocks in these cells.

abstract: Circular RNAs (circRNAs) are a class of endogenous, non-coding RNAs that are formed when exons back-splice to each other and represent a new area of transcriptomics research. Numerous RNA sequencing (RNAseq) studies since 2012 have revealed that circRNAs are pervasively expressed in eukaryotes, especially in the mammalian brain. While their functional role and impact remains to be clarified, circRNAs have been found to regulate micro-RNAs (miRNAs) as well as parental gene transcription and may thus have key roles in transcriptional regulation. Although circRNAs have continued to gain attention, our understanding of their expression in a cell-, tissue- , and brain region-specific context remains limited. Further, computational algorithms produce varied results in terms of what circRNAs are detected. This thesis aims to advance current knowledge of circRNA expression in a region specific context focusing on the human brain, as well as address computational challenges. The overarching goal of my research unfolds over three aims: (i) evaluating circRNAs and their predicted impact on transcriptional regulatory networks in cell-specific RNAseq data; (ii) developing a novel solution for de novo detection of full length circRNAs as well as in silico validation of selected circRNA junctions using assembly; and (iii) application of these assembly based detection and validation workflows, and integrating existing tools, to systematically identify and characterize circRNAs in functionally distinct human brain regions. To this end, I have developed novel bioinformatics workflows that are applicable to non-polyA selected RNAseq datasets and can be used to characterize circRNA expression across various sample types and diseases. Further, I establish a reference dataset of circRNA expression profiles and regulatory networks in a brain region-specific manner. This resource along with existing databases such as circBase will be invaluable in advancing circRNA research as well as improving our understanding of their role in transcriptional regulation and various neurological conditions. / Dissertation/Thesis / Appendix file containing list of enriched pathways and functions identified in Chapter 4 / Doctoral Dissertation Biomedical Informatics 2018

abstract: Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI. / Dissertation/Thesis / M.S. Bioengineering 2013

<p>Polybrominated diphenyl ether (PBDE) flame retardants are ubiquitous contaminants in the environment due to their heavy usage in plastics, foam, and textiles to comply with flammability standards from the 1970s through the late 2000s. Due to their toxicity and persistence in the environment, two of the three PBDE commercial mixtures (PentaBDE and OctaBDE) were banned by the Stockholm Convention on Persistent Organic Pollutants in 2009. The DecaBDE commercial mixture, which consists primarily of the fully brominated congener BDE-209, has been banned or phased out in the United States and Europe but is still in use in other parts of the world. Human exposure to PBDEs persists via environmental reservoirs of PBDEs and products produced before the bans/phase-outs. PBDEs disrupt thyroid hormone levels and neurodevelopment in fish and rodents and are associated with altered thyroid hormone levels and neurodevelopmental impairments in humans. However, the mechanism by which PBDEs alter neurodevelopment remains unclear. Knowledge of the mechanisms and molecular targets of PBDEs is necessary for a causal link to be established between PBDEs and neurodevelopmental impairments. The hypothesis of this thesis research is that PBDEs alter thyroid hormone levels in the brain by interfering with the activity of PBDE-metabolizing deiodinase enzymes in brain cells, which may result in decreased levels of thyroid hormones in the brain and impaired neurodevelopment. </p><p>In the first aim of this thesis research, the biotransformation of PBDEs was examined to determine whether hydroxylated PBDEs (OH-BDEs) are formed in the human brain. In biotransformation assays performed with human astrocytes, which are cells located at the blood brain barrier, no debrominated or OH-BDE metabolites were identified. The results indicate that the enzyme responsible for PBDE hydroxylation (CYP2B6) was not expressed in sufficient quantities to metabolize PBDEs in the astrocyte cells used in this study, but future studies should analyze the potential for PBDE hydroxylation in other brain cells. </p><p>In the second aim of this thesis research, the effects of PBDEs on the thyroid-activating enzyme Type 2 deiodinase (DIO2) were determined in human astrocyte cells. DIO2 converts thyroxine (T4) into triiodothyronine (T3), which is the primary ligand that binds to the thyroid nuclear receptors, and is a very important signaling molecule during neurodevelopment. Cultured primary astrocytes and a human glioma cell line (H4 cells) were exposed to PBDEs and OH-BDEs, and changes in DIO2 activity were measured using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Exposure to BDE-99, -153, and -209, 3-OH-BDE-47, and 5'-OH-BDE-99 all resulted in significant decreases in DIO2 activity in the H4 cells by up to 80% at doses of 500-1,000 nM. Further experiments deduced that the primary mechanism responsible for this decrease in activity was attributed to decreased DIO2 mRNA expression, increased post-translational degradation of DIO2, and competitive inhibition of DIO2. The reduction in DIO2 activity by PBDE and OH-BDE exposures could potentially reduce the concentration of T3 in the brain, which may be responsible for the neurodevelopmental impairments produced by exposure to this class of compounds and needs to be further explored. </p><p>In the third aim of this thesis research, the effects of PBDEs and OH-BDEs were examined in the H4 cells and in a mixed culture containing a human neuroblastoma cell line (SK-N-AS cells). The SK-N-AS cells express the thyroid hormone-inactivating enzyme Type 3 deiodinase (DIO3), which works in concert with DIO2 to buffer the concentration of T3 in the brain. Exposure to BDE-99 decreased the concentration of T3 and the inactive thyroid hormone rT3 in the cell culture medium of co-cultured cells by 59-76%. 3-OH-BDE-47 competitively inhibited DIO3 with an IC50 of 19 uM. 5'-OH-BDE-99 increased the rT3 concentrations in cell culture medium by 400%, increased DIO3 activity in exposed cells by 50%, and increased DIO3 catalytic activity in cellular homogenates by over 500%. Further effects on the mRNA expression of several thyroid-regulated genes (DIO3, TR-a, TR-b, MCT8, and ENPP2) and oxidative respiration were also assessed in the SK-N-AS cells. DIO3 mRNA expression increased by 9 fold in cells exposed to 400 nM BDE-99, and ENPP2 mRNA expression increased by 2 fold in cells exposed to 500 nM BDE-99 and a mixture of the three congeners, but no other significant effects on mRNA expression were observed. The basal respiration rates and other parameters of oxidative respiration were also not significantly altered by exposure to PBDEs or OH-BDEs, but proton leak was increased by over 400% in cells exposed to 2 uM 5'-OH-BDE-99. </p><p>This was the first study to examine the effects of an environmental contaminant on human DIO2 and DIO3 in cultured cells. The results indicated that BDE-99 and OH-BDEs decreased the activity of DIO2 and 5'-OH-BDE-99 increased the activity of DIO3, which combined would lead to decreased levels of T3 exported from the cells into the extracellular environment. These results provide more evidence that disruption of DIO2 and DIO3 by PBDEs during development may mediate the neurodevelopment effects associated with PBDEs.</p> / Dissertation