Browsing by Author "Buttigieg, Josef"

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Open Access
Alzheimer's Disease and Amyloid Beta: Effects of amyloid beta on hippocampal and dorsal root ganglion neurons' electrophyiology and morphology
(Faculty of Graduate Studies and Research, University of Regina, 2019-07) Castilla Bolanos, Maria Alejandra; Buttigieg, Josef; Chao, Tzu-Chiao; Mousseau, Darrell; Campanucci, Veronica
Alzheimer’s disease (AD) is the most common type of dementia, increasing by 10 new cases every day in Saskatchewan alone. AD results from pathological amyloid beta (Aβ) peptides that hamper neurons’ communication, cause progressive neuronal cell death in the central nervous system, and ultimately lead to memory loss in the individual. While disease symptoms are well known, the mechanism by which Aβ triggers the degeneration of the brain remains elusive. There is no cure for AD and present treatments only focus on slowing down the disease progression or mitigating symptoms. This study has used several techniques to investigate the effect of Aβ on the electrophysiology and morphology of isolated hippocampal and dorsal root ganglion (DRG) neurons in order to test whether the effects of Aβ exposure and clearance are exclusive to central nervous system neurons. Isolation and culture of hippocampal and DRG neurons were performed. By exposing neurons to combinations of Aβ42 and Aβ38, electrophysiological and morphological techniques were used to assess the effects of Aβ on neuronal cells. Using patch clamp electrophysiology, I demonstrated a significant decrease in the current density profile of neurons after 24-h of exposure to Aβ42 and Aβ38. Additionally, Aβ had a dose-dependent effect on hippocampal and DRG neurons’ morphology, reducing neuronal soma and nucleus size. The addition of Aβ38 negated the depressive effect of Aβ42, suggesting the cancellation of low current density profiles of neurons or the inhibition of their electrical activity. Thus, co-treatment of Aβ42 and Aβ38 neutralized the depressive Aβ effect on neuronal cells. This research has demonstrated the electrophysiology of hippocampal and DRG neurons after Aβ exposure to understand the fundamental biology of Aβ exposure at a cellular level, which is involved in the pathophysiology of AD and other types of dementia.
Open Access
Analysis of In Vitro Differentiation Cues in Adult Mouse Neural Stem and Progenitor Cells
(Faculty of Graduate Studies and Research, University of Regina, 2015-07) Ludlow, Katherine Alaine; Buttigieg, Josef; Manzon, Richard; Dahms, Tanya; Candow, Darren
Neural stem and progenitor cells (NSPCs) from the adult subventricular zone hold therapeutic potential for regenerative medicine. Specifically, differentiated oligodendrocytes can aid in remyelinating denuded axons in demyelinative disorders or spinal cord injury. The excitatory neurotransmitter, glutamate, has disputed roles in this multi-step process. The goal of my project was to determine if glutamate has an effect on myelination, with the primary focus on the differentiation aspect of NSPCs to oligodendrocytes. Changes to the gene expression profile of NSPC culture in response to oligodendrocyte driving media, glutamate, differentiation and attachment was investigated using reverse transcriptase quantitative polymerase chain reaction (RTQPCR) with cell marker gene targets. The combination of growth factors (FGF2, PDGF, NT3) used to drive, or increase the propensity of the NSPC to differentiate towards oligodendrocytes, had no effect on the proportions of cell types present in differentiated cultures. Therefore, data from driven or non-driven cultures could be combined for the rest of the treatments. Glutamic acid (200μM -1 mM) did not affect the survival of NSPC cultures when growing as floating neurospheres or attached to the extracellular matrix substrate, Matrigel. In addition, glutamate (200μM) did not have a significant effect on the gene expression profile of differentiated cultures. The differentiation media, containing fetal bovine serum (FBS) and no growth factors, significantly affected gene expression and promoted the development of neuronal lineage cells from preceding precursors, while decreasing the proportions of glial cells over time. It is suggested that the decrease in the majority of cell markers could also be the result of a large cell die off from proliferation and subsequent confluency issues during the differentiation period. Cultures in regular media, but also attached to Matrigel-coated dishes, saw significant increases in NSC and glial markers, with possible inhibition of the neuronal differentiation favoured by spontaneous attachment. The cause of spontaneous attachment was undetermined and promoted the differentiation of NSPCs towards neuronal and oligodendrocyte lineage cells. Necessary improvements to the current experimental design, such as an optimal driving, and ideally, cell sorting protocol, would allow for more conclusive evidence to be presented for glutamate’s role on NSPC differentiation. Attempts were made to investigate glutamate’s role on myelination by observing migration of oligodendrocyte precursor cells (OPCs) using co-cultures of differentiated NSPCs with dorsal root ganglion neurons and visualization by immunocytochemistry. There were antibody specificity and technique issues, most of which have been optimized, but these experiments did not produce substantive results. An apparent lack of mature oligodendrocytes in these co-cultures prompted the need for differentiation experiments, which demonstrated the presence of mature oligodendrocytes over the differentiation period required for co-culture experiments. Conversely, the lack of effect observed for the driving media may have also contributed to the apparent insufficient oligodendrocytes present for immunocytochemistry analysis. Therefore, improvements are suggested for the differentiation protocols, which would increase the ability to specifically investigate glutamate’s effect on myelination and migration.
Open Access
Analysis of the Differentiation of Adult Neural Stem and Progenitor Cells Post Treatment with the ASH1 Transcription Factor
(Faculty of Graduate Studies and Research, University of Regina, 2018-03) Teece, Shaneen Michelle; Buttigieg, Josef; Hart, Melanie; Campanucci, Veronica
Adult neural stem and progenitor cells hold the potential to regenerate lost tissue following injury or disease to the central nervous system. Before these cells can be used to regenerate tissue, they must be instructed to differentiate into the appropriate cell type. The objectives of this thesis were to investigate the differentiation induced from the proneural transcription factor ASH1 with an intracellular delivery mechanism in adult neural stem and progenitor cells cultured in vitro and to develop an in-house biosensor capable of detecting the neurotransmitter glutamate from non-myelinated axons. It was demonstrated that the addition of the cell permeable ASH1 promoted neurogenesis in neural stem and progenitor cells after two weeks of cell culture. Patch-clamp electrophysiology and immunocytochemistry were used to verify that the cells were differentiating into neurons. Although the cells did not develop into mature neurons capable of generating action potentials, they showed neuronal characteristics including the expression of neurofilament and the presence of voltage-gated Na+ channels. In addition, it was shown, using patch-clamp electrophysiology on HEK293T cells transduced to express the glutamate ionotropic receptor GRIK3, that glutamate can be detected in the bath solution. This work shows that HASH1 can be used to induce neuronal differentiation in adult neural stem and progenitor cells in vitro and the inhouse biosensor can be used to detect potential glutamate release from axons of neurons generated by ASH1.
Open Access
Applications of Synchrotron Radiation Techniques To The Study Of Taphonomic Alterations and Preservation in Fossils
(Faculty of Graduate Studies and Research, University of Regina, 2019-02) Popovoski Kolaceke, Anezka; Barbi, Mauricio; McKellar, Ryan; Buttigieg, Josef; Caicedo, Maria Velez; Huber, Garth; Bell, Phil
Fossils have traditionally been seen as sedimentary rocks that preserve little of the original composition of animals, except for their shapes, and perhaps some original material from recalcitant mineralized structures, such as bones, and teeth. However, recent studies have shown that not the case. Researchers have identified preserved organic molecules, such as collagen and melanosomes, as well as mineralized soft tissues, including feathers, muscle tissue and skin, tens of millions of years after the animal's death. These results have improved our understanding of extinct species, and have been obtained using a variety of characterization techniques, including the synchrotron-based approaches that are the focus of the research presented in this thesis. The main goal of the research discussed in this thesis was the application of synchrotron radiation techniques (X-ray uorescence and X-ray absorption spectroscopy, in particular) in order to determine the taphonomic alterations that fossils experience, and examine how different materials are preserved. In this thesis, I discuss the results of the chemical characterization on the remains of the Tyrannosaurus rex known as \Scotty", turtle shells, and a rare specimen of fossilized hadrosaur skin. I also examine the applicability of X-ray uorescence to determine the composition and elemental distribution of insect inclusions in amber. The results presented herein offer possible explanations on how some of these specimens were preserved and the extent of the chemical alterations they underwent during their taphonomic history. Beyond the specific results for each specimen, the overall research presented in this thesis shows that synchrotron radiation techniques have great potential to advance palaeontological research, as it becomes necessary to evaluate the chemistry of specimens in high resolution. These characterization techniques were able to con rm that more original material is preserved after fossilization than would have been believed possibly even a decade ago.
Open Access
Cardiac cycle timing intervals in acute COVID-19 and recovered COVID-19 with sustained symptoms
(Faculty of Graduate Studies and Research, University of Regina, 2022-06) Singh, Jyotpal; Neary, J. Patrick; Bardutz, Holly; Bhagaloo, Lanishen; Mang, Cameron; Buttigieg, Josef; Eves, Neil D.
Research is available to show that COVID-19 can result in both acute and sustained cardiac damage. Acute cardiac damage may be due to elevated inflammatory responses and can result in ischemia, which can lead to impairments in the cardiac cycle timing events. The purpose of this project was threefold: 1) to characterise cardiac cycle timing intervals in patients with COVID-19, 2) to understand the mechanisms that compromise cardiac function in post-acute COVID-19 syndrome (PACS)/long COVID, and 3) to identify unique cardiac dysfunction which can occur due to COVID-19 as compared to cardiac and respiratory disease. First, case studies showed that daily observation of cardiac function provided detailed information about the overall dynamic changes by which cardiac dysfunction occurs, and thus can be beneficial study on a case by-case basis, day-to-day during acute infection. The literature review supports the findings of altered cardiac mechanics and suggests that right ventricular dysfunction, along with global longitudinal strain and diastolic dysfunction are common findings in patients with PACS/long COVID, and a more severe acute myocardial injury during the index hospitalization appears to exacerbate cardiac function on follow-up. Finally, a Kruskal-Wallis ANOVA showed that participants with COVID-19 and sustained symptoms present with elevated systolic time and decreased IVCT in comparison to acute COVID-19, and those with respiratory and cardiac disease. These are reflected by decreases in heart, diastolic, and systolic performance indices (HPI, DPI and SPI, respectively), thereby showcasing a unique cardiac dysfunction in patients with sustained symptoms from COVID-19. Future research must consider the details of cardiac complications during the acute infection period and relate this to the cardiac function in patients with long COVID during a mid- and long-term follow-up.
Open Access
Community ecology of migratory bats in a northern sky island
(Faculty of Graduate Studies and Research, University of Regina, 2024-06) Green, Dana Maureen; Brigham, R. Mark; Baerwald, Erin; Somers, Chris; Buttigieg, Josef; Hurlbert, Margot; Fraser, Erin
Animal migrations are often motivated by the opportunity to exploit seasonal abundances of resources, a phenomenon particularly pronounced for species migrating to higher latitudes where seasonality is more extreme. Bats are the only mammalian order to have evolved flight, and three North American migratory bats are the long-distance migrating hoary (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans), and the regionally migrating little-brown Myotis bats (Myotis lucifugus). I describe a highly seasonal community of migratory bats in a northern “sky island” within the Cypress Hills of southern Saskatchewan, Canada, focusing on abundance trends, foraging behavior, niche ecology, and ecophysiology. By collecting data spanning 20 years, my first chapter describes that while long-distance migrating bats are experiencing population declines, the Cypress Hills currently has locally increasing abundances of hoary and silver-haired bats, likely driven by increased roosting habitat. Further in my second chapter, I found that three species of bat separate their ecological niches, allowing for current co-existence, but that silver-haired bats may experience competition with either the hoary or little-brown myotis. My final chapter describes the inter- and intra-specific differences of fur cortisol, a regulatory hormone often associated with stress. Notably, silver-haired bats exhibit elevated fur cortisol levels, but only in juveniles, suggesting it was transferred through their mothers milk while pups grew fur. Thus, female silver-haired bats likely have interactions within their environment causing increased cortisol circulation. Collectively based on my results, I postulate that long-distance migratory bats are disproportionally attracted to the Cypress Hills, and the local population of silver-haired bats may be experiencing increased inter- and intra- competition, resulting in heightened cortisol levels. Although each of the three bat species are seasonally abundant within the Cypress Hills, they are all currently experiencing population declines across their ranges. The environmental conditions hoary, silver-haired, and little-brown myotis bats face consist of highly seasonal resources in a relatively small area of land, while also experiencing habitat loss and increased risk during their annual migrations. My work highlights the importance of both long-term studies and datasets, and lays the foundation to continue to study the summer ecology of at risk species.
Open Access
Effects of Exercise Training on Heart Failure Measured Using Seismocardiography
(Faculty of Graduate Studies and Research, University of Regina, 2019-12) Silbernatel, Jonathan Eric; Neary, J. Patrick; Candow, Darren; Dorsch, Kim; Buttigieg, Josef
Heart failure has become a growing health concern across most of the Western world and will soon become a global health focus. The nature of the disease presents with a very high mortality rate. In Canada alone, the average mortality rate is approximately 50% within the first five years of diagnosis [1]. Exercise has now become a common treatment modality for many chronic disease conditions including heart failure. As heart failure advances, the myocardium goes through a remodeling phase that alters the contractility of the heart and its pumping efficiency. Exercise is known to lead to a positive remodeling of the myocardium in healthy populations, but the degree to which exercise reverses pathological remodeling in individuals with heart failure remains to be determined. The purpose of this study was to analyze the Systolic Timing Intervals (STI) of the myocardium, specifically left ventricular ejection time (LVET) and the pre-ejection period (PEP) using seismocardiography (SCG), as well as to investigate the functional health changes measured using the six-minute walk test (6MWT). Participants exercised three times per week for a period of 12 weeks involving a combination of aerobic and resistance type activities. The SCG screening and 6MWT were performed at the commencement, and at the 12 week point of their exercise program. Eleven individuals participated in this study with varying etiologies for heart failure (ischemic n=6 and nonischemic n=5). Among the individuals with ischemic heart failure, significant improvement were observed in 6MWT distance, (477.0±127.0m to 539.3±113.9m t(5)=- 3.01, p=0.030), but no significant improvements were noted in indices of myocardial function. However, in the non-ischemic group, significant changes were noted in indices iii of myocardial function including LVET (449.6±36.0ms to 438.4±30.5ms t(149)=4.28, p=<0.001), and PEP (128.0±23.5ms to 119.9±18.5ms t(149)=6.87, p=<0.001), but no statistically significant changes were observed in 6MWT distance (p=0.056). This study showed that SCG can be used to record the mechanical function of the heart in individuals with heart failure, that exercise training can produce positive mechanical changes to the heart for individuals with non-ischemic heart failure, and exercise capacity can increase in for individuals with ischemic based heart failure.
Open Access
Effects of Temperature and Thyroid Hormone on Oxidative and Lipid Metabolism in Juvenile Lake Whitefish (Coregonus Clupeaformis)
(Faculty of Graduate Studies and Research, University of Regina, 2018-01) Zak, Megan Alexandra; Manzon, Richard; Weger, Harold; Buttigieg, Josef; Anderson, W. Gary
Nearly every aspect of biological function is influenced by temperature, including metabolism. Factors such as seasonal change and climate change both contribute to environmental temperature variability. While many eurythermal fish species can acclimate to varying environmental conditions by adjusting metabolic processes, the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. I examined the interactions between elevated temperature exposure and hyperthyroidism on various metabolic markers in the cool-water teleost, lake whitefish (Coregonus clupeaformis). Juveniles were exposed to 13 (control), 17 or 21 ºC for 4, 8 or 24 days. Widespread changes in the abundance of mRNA transcripts were observed in enzymes associated with oxidative metabolism (citrate synthase, cytochrome c oxidase subunit 1), lipogenesis (acetyl-coA carboxylase , acetyl-coA carboxylase ) and peroxisomal -oxidation (acyl-coA oxidase 3) following exposure to elevated temperatures. However, the absence of effects on mitochondrial -oxidation enzyme isoforms, carnitine palmitoyltransferase  and carnitine palmitoyltransferase , combined with a decline in citrate synthase and cytochrome c oxidase activities, suggests that exposure to elevated temperature used in this study did not significantly increase overall metabolic demands in juvenile lake whitefish. To assess the role of hyperthyroidism in mediating thermal responses, thyroid status of fish was altered using thyroxine implants prior to the initiation of temperature change. Exogenous thyroxine treatment resulted in notable effects of on citrate synthase, acetyl-coA carboxylase , acetyl-coA carboxylase  and carnitine palmitoyltransferase  mRNA abundance, but only in fish exposed to 17 ºC or 21 ºC. Furthermore, these effects were transient, occurring primarily within the first 8 days of temperature change. Pronounced short-term decreases in carnitine palmitoyltransferase  mRNA abundance in the presence of exogenous thyroxine at all three temperatures suggests thyroid hormones may drive towards a less oxidative phenotype in liver, perhaps in an effort to promote/protect lipid stores. Overall, these results suggest temperature-dependent thyroid hormone action on the transcription of metabolic enzymes in juvenile lake whitefish which may be particularly influential in the early stages of temperature response.
Open Access
Effects of Velocity-Based Resistance Training Programs on Muscle Mass and Performance in Healthy, Older Adults
(Faculty of Graduate Studies and Research, University of Regina, 2020-08) Feasby, Jessica Lynn; Candow, Darren; Bruno, Paul; Neary, Patrick; Buttigieg, Josef
The purpose was to investigate the effects of different velocity-based resistance training programs on muscle mass and muscle performance in aging adults. Twenty-seven participants were randomized to one of three groups: slow-velocity (n = 9, 6 female, 3 male; 55.8 ± 6.8 yrs, 171 ± 9.9 cm, 75.0 ± 11.4 kg; taking 2 seconds to perform the concentric and eccentric phase of each muscle contraction), fast-velocity (n = 9, 7 female, 2 male; 55.3 ± 4.9 yrs, 167.2 ± 6.4 cm, 79.1 ± 15.9 kg; performing the concentric phase as fast as possible and taking 2 seconds to perform the eccentric phase), or mixed-velocity (n = 9, 7 female, 2 male; 57.1 ± 7.3 yrs, 167.1 ± 9.9 cm, 70.6 ± 16.1 kg; performing slow-velocity repetitions for sets 1 and 3 and fast-velocity repetitions for sets 2 and 4). The supervised resistance training program was performed 3x/week for 12 weeks and consisted of 4 sets of 10 repetitions to volitional fatigue for 9 whole-body machine-based exercises (leg press, chest press, latissimus-pull down, shoulder press, leg extension, leg curl, biceps curl, triceps extension, calf press). Prior to and following training, the primary dependent variables assessed were whole-body lean tissue mass (dual energy X-ray absorptiometry [DEXA]), muscle thickness (ultrasonography; elbow and knee flexors and extensors, ankle plantar- and dorsi-flexors), muscle strength (1 repetition max [1 RM]) leg press and chest press), and isokinetic peak torque (1.05 rad/s and 3.14 rad/s). There was a significant increase over time (p < 0.05) for muscle strength and isokinetic peak torque at both velocities and muscle thickness of the knee extensors and ankle plantar flexors, with no significant difference between groups. Therefore, resistance training, regardless of movement velocity, improves muscle strength, muscle torque, and some measures of lower-body muscle thickness in healthy aging adults.
Open Access
An Efficient Lentiviral-based Proteomics Approach Identifies Mitochondrial Complexes with Neurodegenerative Function
(Faculty of Graduate Studies and Research, University of Regina, 2015-04) Kassir, Sandy; Babu, Mohan; Suh, Dae-Yeon; Dahms, Tanya; Buttigieg, Josef
Perturbations in proteins involved in the mitochondrial processes have emerged as a causative factor in a wide range of life-threatening human diseases, such as neurodegenerative disorders. In fact, mitochondrial dysfunctions are argued to be the first step in triggering the onset of other deleterious events that collectively act causally in the pathogenesis of diseases. Accordingly, dysfunctions in mitochondrial proteins influence the proper functioning of basic processes, such as energy metabolism and reactive species production. Indeed, biochemical and genetic evidence has provided molecular insights into the role of mitochondrial proteins and complexes in isolation, yet our understanding of how mitochondrial proteins cause and/or contribute to the diverse array of human diseases, including neurodegeneration remains unclear. Like any other biological system, mitochondria are linked together by extensive networks of physical (protein-protein) interactions; therefore a detailed understanding of the systems properties is required to unravel their role in neurodegenerative diseases. To address this, I have developed and optimized an effective procedure to identify the physically interacting proteins for generating a mitochondrial protein-protein interaction network for those involved in various neurodegenerative diseases.
Open Access
Identification and Characterization of Developmentally Regulated Components of the Stress Axis in Petromyzon Marinus
(Faculty of Graduate Studies and Research, University of Regina, 2013-01) Endsin, Matthew Joel; Manzon, Richard; Buttigieg, Josef; Babu, Mohan
Genes resembling elements of the Corticotropin releasing hormone (CRH) receptor-ligand system (CRH system) have been identified in invertebrate species and suggest the CRH system has existed, in some form, for approximately a billion years. It is theorized that vertebrates inherited components of the CRH system from an invertebrate ancestor. The association of the CRH system with the stress response, however, is specific to vertebrate species and theorized to have accompanied the development of hypothalamic pituitary (HP) axes, specifically the HP interrenal (HPI) axis in fish. A functional HPI axis has recently been suggested in the lamprey species Petromyzon marinus, a member of the ancient vertebrate superclass agnatha, by identification of pituitary and inter-renal components corticotrophin (ACTH) and 11- deoxycortisol respectively. This study, however, is the first to identify the hypothalamic components, specifically the CRH system, of the HPI. In P. marinus the expression of six CRH system genes, including three hormones, CRH A, CRH B and UCN III-like; two receptors, CRH Rα and CRH Rβ; and a binding protein, CRH BP, are identified by PCR and in silico methodologies. Analysis of the P. marinus CRH system genes appear to support the occurance of the Agnathan superclass prior to a theorized second vertebrate whole genome duplication (WGD) event. This is supported by the P. marinus CRH hormones appearing to represent two of the four vertebrate CRH family paralogues; CRH A and B both being orthologous to vertebrate CRH, and UCN III-like being orthologous to vertebrate UCN III. Additionally, neither CRH Rα nor CRH Rβ, while identified as distinct from one another and related to other vertebrate receptors, were phylogenetically indistinguishable as either ii type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear to have arisen out of a lamprey specific duplication event they diverged separately from the formation of the type 1 and type 2 receptors. The P. marinus CRH BP deduced amino acid sequence was found to contain regions highly conserved and functionally significant in other vertebrates as well as invertebrate species, and occupies a unique phylogenetic branch. Expression of these genes in brain, gill, liver, kidney as measured by reverse transcription quantitative PCR (RT qPCR) over the life history of P. marinus (including pre-metamorphic larvae, each of the seven stages of metamorphosis, and juvenile parasites) indicated significant variation in gene expression both between tissues and through the life history. Differences in expression were observed for each P. marinus CRH system gene and correlate with significant physiological changes occurring in the developing P. marinus. Some of these include increases in Na+/K+ -ATPase activity in the gill, possibly relating to salt water tolerance, and lipogenic and lipolytic metabolic phases in the kidney and liver. Interestingly, comparatively high expression levels of CRH A, CRH B and CRH Rβ were observed in the JP gonad relative to other JP organs. This suggests these genes may have a paracrine role in this organ, possibly by local regulation of sex steroids, similar to that observed in mice and humans. Interestingly, CRH system mRNA expression did not vary in response to multiple successive acute stressors, including dewatering and salt water exposure, over a 24 hour period as measured by RT qPCR. This suggests that P. marinus CRH system genes may not respond to such stressors at the level of mRNA expression. Collectively, these data indicate that lamprey contain all necessary components of a complete HPI axis, and that the CRH system likely plays an important role in the normal development.
Open Access
Immune Modulating Peptide for the Suppression of Autoimmune Cells in Multiple Sclerosis
(Faculty of Graduate Studies and Research, University of Regina, 2021-08) Rustad, Karin Christen; Buttigieg, Josef; Hart, Mel; Tomesh, Trevor; Yong, Wee
Multiple Sclerosis is a chronic demyelinating inflammatory disease that causes symptoms ranging from fatigue to cerebellar ataxia. I propose a novel treatment for MS that targets the aberrant inflammatory cells directly in order to halt the progression of the disease. This protein complex will competitively bind to inflammatory cells that target the components of the central nervous system and subsequently eliminate them, thus eliminating the disease as it arises. Experimental autoimmune encephalomyelitis induced mice were treated with 0.2 mL of trimeric protein at peak disease. Gait score was used to measure disease progression. The cerebellum was fixed, sectioned and stained to measure lesion load. CD3+ and CD19+ cell populations from the spleens were counted. I found that administration of the trimeric protein at low doses significantly improved the motor function and significantly reduced the amount of cerebellar lesioned area of the EAE-induced mice.
Open Access
Physiological and Cellular Responses Used By Lake Whitfish (Coregonus Clupeaformis) in Response to Thermal and Hypoxic Stress at Different Points in Development
(Faculty of Graduate Studies and Research, University of Regina, 2018-06) Whitehouse, Lindy Marie; Manzon, Richard; Somers, Christopher; Buttigieg, Josef; Neary, Patrick; Bernier, Nicholas
Early life stages of fish are particularly susceptible to environmental change and even brief exposures to thermal or hypoxic stress can have detrimental effects. Fish may be able to respond to stress, using physiological and cellular mechanisms, to mitigate some of the damaging effects of stressor exposure and maintain homeostasis. I investigated the development of the hypothalamus-pituitary-interrenal axis (HPI-axis) and two key cellular stress responses, the heat shock response (HSR) and hypoxia response, in lake whitefish (Coregonus clupeaformis) at several life-stages, to understand when these responses could be activated in response to stress. The HSR and HR are mediated by heat shock proteins (Hsps) and hypoxia-inducible factor 1α (Hif-1α), respectively. The results from this thesis show that lake whitefish can respond to environmental fluctuations with the activation of the HPI-axis and both cellular stress responses. Increases in the mRNA levels of multiple hsps were observed in response to both thermal and hypoxic stress from the earliest ages studied, 15 and 21 days post fertilisation (dpf), respectively, and was the only response I observed at these ages. Further, increases in hsp70 were observed at all embryonic ages studied in response to both stressors, suggesting that the HSR plays a fundamental role in the response to stress in lake whitefish embryos. At 38 dpf, the HPI-axis could be activated in response to hypoxia, resulting in increased whole-embryo cortisol levels. This is the first time that this has been observed during early embryogenesis in a teleost fish and was not observed at any other embryonic ages studied. It was also at 38 dpf that the activation of the hypoxia-inducible factor pathway was first observed, with an increase in the mRNA levels of multiple Hif-1 responsive genes. In response to hypoxia, the ability to induce a cellular response persisted through to 2 weeks post hatch (wph), although this was age and gene specific. In contrast, by 3 and 4 wph, mRNA levels of several genes, including hsp70, and whole body cortisol levels were below those in controls, suggesting that at these ages larval lake whitefish were either unable to initiate a stress response or the response could not be sustained for the duration of the 6-hour treatment. Taken together, these data suggest that sensitive windows exist in lake whitefish development and that at these ages developing fish may be more vulnerable to stress exposure. Lastly, I show that young of the year (YOY) juvenile lake whitefish at 18 wph, can respond to acute thermal stress, but not hypoxia, with strong increases in hsp mRNA. Hypoxia exposure resulted in the up-regulation of multiple genes that are commonly induced by hypoxia in other organisms. Exposure to thermal and hypoxic stress simultaneously caused increases in hsps and hypoxia inducible genes. The more severe multi-stressor treatments resulted in some mortality which suggests that these stressors act synergistically or additively, highlighting the value in studying multi-stressor scenarios which are more indicative of what is occurring in natural systems. Overall, this thesis demonstrates that the physiological and cellular response of developing lake whitefish to temperature and hypoxia vary with age, severity and type of stressor and contributes to our understanding of the nature of the stress responses developing fish use when exposed to changes in their environment.
Open Access
Role of physiologically relevant hypoxia in neural stem and progenitor cell proliferation, migration and differentiation into oligodendrocytes
(Faculty of Graduate Studies and Research, University of Regina, 2023-04) Masood, Mahrukh; Buttigieg, Josef; Hart, Mel; Candow, Darren
Stem cells are undifferentiated cells, defined by their capability to self-renew and differentiate to give rise to different cells of the body. Neural stem and progenitor cells are a type of multipotent stem cell capable of giving rise to the cells of the mature central nervous system (CNS): neurons, astrocytes and oligodendrocytes. The mechanism by which various factors influence stem fate is of wide interest, as these cells play a key role in development, and have a potential role in repair of CNS injury. I investigated the role of physiologically relevant hypoxic levels as a driving force for the proliferation and differentiation of Neural Stem and Progenitor Cells (NSPCs) into Oligodendrocytes (OLs) as well as increased migration. In most research, cells are cultured at 21% O2, which is significantly higher than what these cells, and other cells of the CNS, are typically exposed to. Physioxia is what could be considered low concentrations of O2 in the external environment, but normal in the body. The O2 level in the human body is tightly regulated; particularly low levels of O2 may positively aid in NSPC differentiation through the regulation of certain genes. One mechanism that may aid in the differentiation process is the involvement of transcription factors that are sensitive to changes in O2 levels. Hypoxic Inducible Factor (HIF) is a transcription factor that plays an integral role in the detection of hypoxia and can induce changes in genes responsible for vascular growth (vascular endothelial growth factor (VEGF)), cell migration (matrix metalloproteinase 2 (MMP2)) or A disintegrin and metalloproteinase with thrombosponfin motifs 1 (ADAMTS-1)) and cell differentiation (platelet-derived growth factor (PDGF)). This study demonstrates that a low O2 environment can be confirmed through the upregulation of HIF-1a at low levels of physiologically relevant oxygen levels. Furthermore, the upregulation of VEGF at different O2 concentrations alludes to NSPC proliferation, especially at 5% O2. MMP2 upregulation showed that migration of the OL lineage cells is highest at 5% O2. Lastly, differentiation of NSPCs to OPCs seemed to increase when exposed to low levels of O2 and was the highest at 5% O2. Moderate levels of physiologically relevant oxygen levels such as 5% seem to have the optimal effect on NSPC proliferation, differentiation, and migration as gene expression for several of the gene is highest at that O2 concentration.
Open Access
Role of the Neurotransmitter Glutamate in Neural Stem and Progenitor Cells Differentiation into Oligodendrocytes
(Faculty of Graduate Studies and Research, University of Regina, 2017-12) Kisheev, Anastasye; Buttigieg, Josef; Chao, Tzu-Chiao; Dahms, Tanya
Various regions of the mammalian brain, including the subventricular zones (SVZs) of the hippocampus and the dentate gyrus (DG), contain niches of undifferentiated neural stem and progenitor cells (NSPCs) which persist from development, all throughout adulthood. These cells can differentiate into the three main cell types found within the brain: neurons, oligodendrocytes (OLs), and astrocytes. Adult NSPCs can be driven to specific cells of interest which can be used to replace lost myelin in some cases of neurodegenerative diseases such as multiple sclerosis (MS) or insults such as spinal cord injury. I investigated the requirements to drive immature NSPCs into oligodendrocyte precursor cells (OPCs) using various driving factors (PDGFα and NT-3) and the neurotransmitter (NT) glutamate. Glutamate is one of the main NTs in the central nervous system (CNS) hence, it may have a role in the differentiation of NSPCs into OPCs. My results demonstrated that among all the driving factors tested, glutamate treatment yielded the highest proportion of OPCs after six days. Flow cytometry was used to verify changes in cell lineage and proportions during the driving experiments. Enriched OPC cultures were purified using a magnetic bead sorting technique after which the cell lineage was confirmed using an immunocytochemistry (ICC) staining technique. Results demonstrated that cells differentiated alone with no external stimulation did not produce the myelin basic protein (MBP). In contrast, almost all OPCs stimulated with glutamate expressed MBP. Purified OPCs grown in a co-culture with DRG neurons for nine days also began expressing MBP which was found to be in close association with the neuronal axons. In order to verify the effect of glutamate in co-cultures of OPCs with DRG neurons, AMPA/kainate inhibitors such as CNQX and DNQX, were tested. Although MBP positive cells were still identified in cultures treated with AMPA inhibitors, in the case of CNQX, the myelin sheath organization appeared distorted compared to the nontreated samples, thus suggesting glutamate does play a key role in oligodendroglial development.
Open Access
Study of Neutron Reflector for the HALO-1kT Supernovae Neutrino Detector
(Faculty of Graduate Studies and Research, University of Regina, 2020-05) Patel, Divyaben Ashvinkumar; Barbi, Mauricio; Kolev, Nikolay; Grinyer, Gwen; Buttigieg, Josef
The Helium and Lead Observatory 1 Kilotonne (HALO-1kT) is a lead-based detector to study electron neutrinos emitted from core-collapse supernovae. It is proposed to follow the same purpose as the HALO detector located at SNOLAB but with higher detection efficiency. The sensitivity to electron neutrinos makes HALO-1kT (and also the current HALO detector) unique in the sense that all other detectors with capability to detect supernova neutrinos are sensitive to anti-electron neutrinos through charged- current inverse beta-decay such as the Super-Kamiokande, LVD, IceCube and KamLAND. HALO-1kT's sensitivity to supernova neutrinos is larger than that for HALO due to its proposed 12-fold target-mass increase relative to HALO and a more e cient neutron detection. The detector will consist of 1 kT of lead. Neutrinos from a supernova will interact with the lead via inverse beta-decay process producing bismuth or lead in highly-excited states (the excitation states depend on the incoming neutrino flavour). The daughter nuclei emit neutrons during de-excitation, which are then detected by 3He proportional counters. The layer of the detector immediately following the lead volume consists of a graphite reflector to recover neutrons that would otherwise escape the detector fiducial volume. The main goal of this thesis is to develop simulation studies for the design of a graphite layer which will serve as a neutron moderator and reflector, redirecting escaping neutrons back into the detector lead volume. The reflector layer will increase the detection efficiency by up to 50% relative to the 28% efficiency achieved in HALO. Geant4 simulations have been used to assess the optimal thickness and grade of the graphite to be used. It was found that a graphite material of 15 cm thickness is the favourable choice for a neutron reflector. As for the graphite grade, it should, ideally, be nuclear-reactor quality. However, costs involved in the acquisition of such high grade material should be considered. It was found that a < 1.0 ppm concentration level of boron in the graphite layer is an acceptable compromise between cost and effectiveness.
Open Access
Task-oriented exercise effects on walking and corticospinal excitability in multiple sclerosis: protocol for a randomized controlled trial
(BioMed Central, 2023-12-21) Moslemi, Zahra; Toledo-Aldana, Eduardo A.; Baldwin, Bruce; Donkers, Sarah J.; Eng, Janice J.; Mondal, Prosanta; Totosy de Zepetnek, Julia O.; Buttigieg, Josef; Levin. Michael C.; Mang, Cameron S.
Background Multiple sclerosis (MS) is a degenerative disease of the central nervous system (CNS) that disrupts walking function and results in other debilitating symptoms. This study compares the effects of ‘task-oriented exercise’ against ‘generalized resistance and aerobic exercise’ and a ‘stretching control’ on walking and CNS function in people with MS (PwMS). We hypothesize that task-oriented exercise will enhance walking speed and related neural changes to a greater extent than other exercise approaches. Methods This study is a single-blinded, three-arm randomized controlled trial conducted in Saskatchewan, Canada. Eligible participants are those older than 18 years of age with a diagnosis of MS and an expanded Patient-Determined Disease Steps (PDDS) score between 3 (‘gait disability’) and 6 (‘bilateral support’). Exercise interventions are delivered for 12 weeks (3 × 60-min per week) in-person under the supervision of a qualified exercise professional. Interventions differ in exercise approach, such that task-oriented exercise involves weight-bearing, walking-specific activities, while generalized resistance and aerobic exercise uses seated machine-based resistance training of major upper and lower body muscle groups and recumbent cycling, and the stretching control exercise involves seated flexibility and relaxation activities. Participants are allocated to interventions using blocked randomization that stratifies by PDDS (mild: 3–4; moderate: 5–6). Assessments are conducted at baseline, post-intervention, and at a six-week retention time point. The primary and secondary outcome measures are the Timed 25-Foot Walk Test and corticospinal excitability for the tibialis anterior muscles determined using transcranial magnetic stimulation (TMS), respectively. Tertiary outcomes include assessments of balance, additional TMS measures, blood biomarkers of neural health and inflammation, and measures of cardiorespiratory and musculoskeletal fitness. Discussion A paradigm shift in MS healthcare towards the use of “exercise as medicine” was recently proposed to improve outcomes and alleviate the economic burden of MS. Findings will support this shift by informing the development of specialized exercise programming that targets walking and changes in corticospinal excitability in PwMS. Trial registration ClinicalTrials.gov, NCT05496881, Registered August 11, 2022. https://classic.clinicaltrials.gov/ct2/show/NCT05496881. Protocol amendment number: 01; Issue date: August 1, 2023; Primary reason for amendment: Expand eligibility to include people with all forms of MS rather than progressive forms of MS only.
Open Access
Thermal Stress and the Heat Shock Response in Embryonic and Young of the Year Juvenile Lake Whitefish (Coregonus Clupeaformis)
(Faculty of Graduate Studies and Research, University of Regina, 2015-07) Stefanovic, Daniel Ivan; Manzon, Richard; Buttigieg, Josef; Babu, Mohan
I investigated the effects of thermal stress associated with changing environments and industrial thermal pollution by characterizing the kinetics of the heat shock response (HSR) throughout Lake Whitefish (Coregonus clupeaformis) development. Lake Whitefish are a cold water species that spawn in the late fall to early winter. Their embryos develop at 0.5 – 6 °C, usually under the cover of ice. The HSR is a universal response to thermal and other types of stressors that offer protection at the cellular level. This response is characterized by the synthesis of a group of highly conserved proteins called the heat shock proteins (Hsps). Here I isolated five different hsp cDNAs from Lake Whitefish and quantified changes in mRNA transcript levels in response to varying heat stress conditions in embryos and young of the year (YOY) juveniles. Lake Whitefish were subjected to three different heat shock temperatures (3 °C, 6 °C or 9 °C above control) and six different heat shock durations (0.25, 0.50, 1, 2, 3, and 4 hr) followed by a 2 hr recovery period prior to sampling. In addition, the duration of the HSR was examined by varying the post-heat shock recovery time period prior to sampling. In this recovery experiment, Lake Whitefish were permitted to recover for 1, 2, 4, 8, 12, 16, 24, 36, and 48 hr following a 2 hr heat shock. My data suggest that Lake Whitefish embryos may be resilient to short bouts of heat shock. In embryos, hsp70 mRNA levels were elevated following a 2 hr, 9 °C heat shock. However, levels of the hsp70 did not increase in response to any of the 3 °C or 6 °C heat shocks, irrespective of heat shock duration. It is also significant that embryos did not upregulate mRNA levels of the normally inducible hsp90α or hsp47, in response to any of the heat shock exposures. These embryo data are different from those in 60 days post-hatch YOY juveniles. In YOY juveniles, all three inducible hsps were upregulated in response to both a 6 °C and 9 °C heat shock, suggesting that YOY juveniles will more readily initiate a HSR and that this response is more robust in that it involves multiple hsps. In embryos, once triggered, the HSR was relatively long lasting with hsp70 mRNA levels remaining elevated 48 hr post-heat shock. In contrast, in YOY juveniles the HSR was shorter lived with hsp70 levels beginning to decrease as early as 4 - 8 hr post-heat shock. In summary, my data indicates that Lake Whitefish can initiate a HSR during embryogenesis. In comparison to YOY juveniles, embryos are more resistant to heat stress as they only initiated a HSR at relatively high heat shock temperatures and this response was limited to increases in hsp70. However, once initiated the HSR was relatively long lasting. Collectively, these data will help us better understand the potential impact of thermal stress associated with changing environments and industrial thermal pollution on development of this and other coldwater fish species.