As a classic neurotransmitter in the brain, norepinephrine (NE) also is an important modulator to other neuronal systems. Using primary cultures from rat ventral mesencephalon (VM) and dopaminergic cell line MN9D, the present study examined the neuroprotective effects of NE and its effects on the expression of tyrosine hydroxylase (TH). The results showed that NE protected both VM cultures and MN9D cells against 6-hydroxydopamine-caused apoptosis, with possible involvement of adrenal receptors. In addition, treatment with NE upregulated TH protein levels in dose- and time-dependent manner. Further experiments to investigate the potential mechanisms underlying this NE-induced upregulation of TH demonstrated a marked increase in protein levels of the brain-derived neurotrophic factor (BDNF) and the phosphorylated extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) in VM cultures treated with NE. In MN9D cells, a significantly increase of TH and pERK1/2 protein levels were observed after their transfection with BDNF cDNA or exposure to BDNF peptides. Treatment of VM cultures with K252a, an antagonist of the tropomyosin-related kinase B, blocked the upregulatory effects of NE on TH, BDNF and pERK1/2. Administration of MEK1 & MEK2 inhibitors also reversed NE-induced upregulation of TH and pERK1/2. Moreover, ChIP assay showed that treatment with NE or BDNF increased H4 acetylation in the TH promoter. These results suggest that the neuroprotection and modulation of NE on dopaminergic neurons are mediated via BDNF and MAPK/ERK pathways, as well as through epigenetic histone modification, which may have implications for the improvement of therapeutic strategies for Parkinson's disease.

Sepsis-induced myeloid-derived suppressor cells (MDSCs) increase mortality risk. We previously identified that long non-coding RNA Hotairm1 supports myeloid precursor shifts to Gr1+CD11b+ MDSCs during mouse sepsis. A major unanswered question is what molecular processes control Hotairm1 expression. In this study, we found by a genetic deletion that a specific PU.1-binding site is indispensable in controlling Hotairm1 transcription. We then identified H3K4me3 and H3K27me3 at the PU.1 site on the Hotairm1 promoter. Controlling an epigenetic switch of Hotairm1 transcription by PU.1 was histone KDM6A demethylase for H3K27me3 that derepressed its transcription with possible contributions from Ezh2 methyltransferase for H3K27me3. KDM6A knockdown in MDSCs increased H3K27me3, decreased H3K4me3, and inhibited Hotairm1 transcription activation by PU.1. These results enlighten clinical translation research of PU.1 epigenetic regulation as a potential sepsis immune-checkpoint treatment site.

In the aftermath of cancer surgery, Natural killer (NK) cells are severely suppressed. NK cells are critical for anti-tumour surveillance and their postoperative dysfunction creates an opportunity for metastases. I hypothesized that NK cell suppression is mediated by multiple suppressive mechanisms of surgery-induced Myeloid Derived Suppressor Cells (Sx-MDSCs). In this thesis, I first show that NK cell dysfunction is far worse than previously described. In a cohort of colorectal cancer (CRC) surgery patients (n=42), the ability of NK cells to secrete IFN-gamma in response to stimulation was suppressed for up to 2 months after surgery. Secondly, since Sx-MDSCs have been poorly characterized in humans, I thoroughly phenotyped Sx-MDSCs from cancer surgery patients using flow cytometry (n=32 patient samples) and single-cell RNA sequencing (n=6 patient samples). Additionally, upon screening a library of 150 compounds, I showed that Sx-MDSC rely on PI3K signaling for their suppression of NK cells in ex vivo NK cell suppression assays. The third part of this thesis explores the contribution of Sx-MDSCs to the rapid reduction in postoperative arginine, the perioperative importance of arginine for NK cells, and the therapeutic effects of a perioperative arginine enriched supplement (AES) on metastases in murine models of surgical stress. Here, I showed that perioperative AES attenuates postoperative metastases by accelerating NK cell recovery after surgery. These promising preclinical data combined with evidence from the scientific literature led us to initiate a Phase II randomized-controlled clinical trial assessing the ability of perioperative AES to improve NK cell function after surgery in CRC patients (n=12/arm). In the last part of this thesis, I present the results from our clinical trial, which showed only a transient and, at best, modest improvement in NK cell function. Importantly, this may have been heavily influenced by poor postoperative patient compliance in taking the AES. In conclusion, this body of work describes the multifactorial role that Sx-MDSCs play in mediating postoperative NK cell suppression, and that safe, effective, and targeted perioperative interventions should be further investigated as a strategy to attenuate metastatic disease recurrence after surgery.

Nearshore bathymetry in the Canadian Arctic is poorly surveyed, but is vital knowledge for coastal communities that rely on marine transportation for resources and development. Nautical charts currently available are often outdated and surveying by traditional methods is both time consuming and expensive. Satellite-derived bathymetry (SDB) offers a significantly cheaper and faster option to provide information on nearshore bathymetry. The two most common approaches to SDB are empirical and physics-based. The empirical approach is simple and typically does well when calibrated with high-quality in-situ data, whereas the physics-based approach is more difficult to implement and requires precise atmospheric correction. This project tests the practical use of five methods within the empirical and physics-based approaches to SDB, using Landsat 8 and Sentinel-2 satellite imagery, at seven sites across Nunavut. Methods tested include: the Ratio-Transform, Multiband, and Random Forest Regression methods (empirical) and radiative transfer modeling (physics-based) using two atmospheric correction models: ACOLITE and Deep Water Correction. All methods typically use geolocated water depth data for validation, as well as calibration for the empirical methods. Spectral reflectance for model inputs were collected in Cambridge Bay, NU. Water depth data were acquired from the Canadian Hydrographic Service. All processing was conducted within the framework of plugins developed for the open-source GIS software, QGIS. Results from the empirical methods were typically poor due to poor calibration data, though Random Forest Regression performed well when good calibration data were available. Due to poor quality validation data, error for the physics-based results cannot be adequately quantified in most places. Additionally, atmospheric correction remains a challenge for the physics-based methods. Overall, results indicate that where large, high-quality calibration datasets are available, Random Forest Regression performs best of all methods tested, with little bias and low mean absolute error in water less than 10 m deep. As such datasets are rare in the Arctic, the physics-based method is often the only option for SDB and is an excellent qualitative tool for informing communities of shallow bathymetry features and assessing navigation risk.

Bone is an important organ influenced by mechanical load, hormones, nutrition and disease. During bone remodelling, osteoclasts resorb bone and osteoblasts form new bone. Osteoblasts are derived from mesenchymal stem cells (MSCs) such as adipose-derived stromal cells (ASCs). The mitogen-activated protein kinase (MAPK) pathway has been shown to interfere with osteoblast differentiation from an early stage. Runt related transcription factor 2 (RUNX2) exerts an effect downstream from p38 MAPK. RUNX2 phosphorylation by p38 MAPK may increase osteoblast differentiation markers such as alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL). Palmitoleic acid (PLA), an omega-7 monounsaturated fatty acid (MUFA), promotes anti-osteoclastogenic effects, however, the effects of PLA on osteoblasts has not been reported. Osteoporosis is a condition which has debilitating effects in the elderly. Unsaturated fatty acids (UFA) have been studied for their beneficial effects on human health for a number of years. Polyunsaturated fatty acids (PUFA) have been studied as a potential therapeutic agent to prevent and assist in managing the condition. Few studies have been conducted on the effects of MUFA on bone therefore this study aimed to investigate the effects of PLA on osteoblast differentiation using ASCs and MG-63 osteosarcoma cells as an osteoblast model. ASCs and MG-63 osteosarcoma cell lines were exposed to PLA (20-100 μM) in osteogenic media (OM). The effects of PLA on cell viability was evaluated on undifferentiated cells. Thereafter, cells were exposed to PLA for 7, 14 or 21 days. Subsequently ALP activity, calcium mineralisation, gene expression, protein expression and adipogenesis were assessed. In this study, PLA had no significant effects on cell viability in undifferentiated cells. Furthermore, PLA had no significant effects on ALP activity, calcium mineralisation or phosphorylated extracellular signal-regulated kinase (pERK)/extracellular signal-regulated kinase (ERK) expression in differentiating cells, however, ALP activity increased at 7 day in ASCs and 21 days in MG-63 cells. Alizarin Red S staining increased at 21 days in both cell lines with a significant increase in the ASCs, however, calcium nodules were not visible. In the ASCs, PLA significantly increased the gene expression of ALP at 7 and 14 days compared to control (p<0.01 and p<0.05) while RANKL was significantly decreased at 7 days compared to the control (p<0.05). In the MG-63 cells, RUNX2 and OCN were significantly reduced at 7 days compared to control (p<0.05) and ALP, RUNX2, Osx and RANKL were significantly reduced at 14 days compared to control (p<0.001 and p<0.05). In the ASCs, lipid accumulation was not present after 21 days while in MG-63 cells, there was a significant increase in lipid accumulation at a high concentration of PLA after 21 days compared to control (p<0.05). This is the first study to explore the effects of PLA on osteoblast formation using ASCs and MG-63 osteosarcoma cells. Results suggest that PLA exerted changes in the ASCs and MG-63 cells during osteoblast differentiation, however, these changes were not significant. To conclude, PLA showed some significant effects on osteoblast-specific gene expression, however, most of the osteoblast-specific gene expression was downregulated, particularly in the MG-63 cells, after PLA treatment. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

Exercise alone is an efficacious intervention for depression, but few studies have identified the benefits of using exercise to augment other psychosocial treatments. The purpose of the current series of studies was to examine the feasibility, acceptability, efficacy, and potential mechanism of the augmentation of behavioral activation (BA) with exercise. The starting point for this series was a meta-analysis of the strength and reliability of brain-derived neurotrophic factor (BDNF) as a putative mechanism of the mood and cognitive effects of exercise. Evaluating 29 studies, I found: (1) a moderate effect for BDNF increases following acute exercise, (2) a moderate effect for the intensification of this effect following a program of exercise, and (3) a small effect on resting BDNF following a program of exercise. Given these effects, I hypothesized that exercise added to BA would improve mood beyond that for BA combined with a control condition and that changes in BDNF would mediate these effects. In a clinical trial, 32 sedentary, depressed patients received 9 sessions of BA over 12 weeks and were randomized to receive an exercise or control (stretching) augmentation. Assessments of depression, quality of life, distress intolerance, perceived stress, cognition (memory, attention), and amount of exercise were conducted across the treatment period. Results demonstrated strong credibility ratings and completion rates comparable to other exercise interventions. The randomized treatment failed to lead to differential exercise between groups; all participants exercised more over time. Similarly, participants, regardless of condition, significantly improved on all outcome measures over time. BDNF significantly increased following acute exercise. However, the amount of exercise completed over time was not significantly related to changes in BDNF across acute episodes, nor did resting BDNF significantly improve over time. Nonetheless, effect sizes for these changes were in the moderate range, reflecting values for the literature as a whole. Finally, contrary to my hypothesis, BDNF changes were not associated with subsequent improvement in depression symptoms. Results from this trial raise questions whether BA may be a powerful enough intervention to increase exercise, thus explicit exercise prescriptions may not be necessary for patients receiving this intervention.

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor with a 5% survival rate over 5 years. Though SCLC comprises 13% of all cases of lung cancer the median survival time of 14.5 months has seen little improvement over the last four decades. Standard treatment relies on DNA damaging agents such as Cisplatin/Etoposide (EP) which induce a high response rate of 60-70%. Despite this initial response, nearly all patients will relapse rendering first-line therapies ineffective. Furthermore, SCLC has been shown to develop chemotherapy cross-resistance in which resistance to first-line chemotherapies will confer resistance to additional DNA damaging agents thereby reducing treatment efficacy and duration of response. Cross-Resistance constitutes a major clinical issue whose underlying mechanisms remain a mystery. The modest improvements in SCLC patient outcomes over the decades may be partially explained by the existing systems of study. Current methodologies of SCLC study rely on cell lines, patient samples, and Genetically Engineered Mouse Models which have little functional correlation to clinical outcomes. While few sources have proposed Patient Derived Xenograft (PDX) systems as an improved alternative, significant data remains sparse. Without a robust model system which accurately recapitulates patient outcomes, molecular pathways driving resistance cannot be uncovered. Here we present the generation of 34 SCLC PDX models which maintain both genomic and functional fidelity. Furthermore, treatment of a 30-model subset with first-line chemotherapy EP and a novel chemotherapy Olaparib/Temozolomide (OT) allowed for functional and molecular comparison between groups. Our findings demonstrate incomplete independent resistance mechanisms between EP and OT treatment with a small overlap of 31 genes involved in glycolysis and xenobiotic metabolism.

Species distribution models are frequently used in ecology to predict the spatial and temporal occurrence of organisms. Direct interpretation of these models assumes that the relationships between the organisms and their environment are manifestations of causal mechanisms. However, in general, the mechanisms producing these associations have not been experimentally validated, which questions our confidence in their interpretation and application. Temperature is one of the most important factors influencing the fitness and distribution of aquatic organisms, and studying the thermal physiology of aquatic invertebrates could provide a useful approach for validating predictions of the species distribution models. Experimental thermal tolerance studies, which assess the physiological limits to temperature, should be useful in interpreting the causal basis for species distribution model predictions. Critical Thermal Maxima experiments are frequently used to measure the thermal tolerance of ectothermic organisms. They represent the temperature at which organisms exhibit disorganized locomotor activity to the point that they lose their ability to escape conditions that will promptly lead to death. Critical Thermal Maxima experiments could, therefore, provide a useful test of the inferred mechanisms of species distribution models. The objective of my study was to determine if Critical Thermal Maxima experiments are associated with the thermal limits inferred from species distribution models. If the models accurately describe causal relationships between predicted distributions of organisms and environmental temperatures, and if the thermal maxima are associated with the limits to organism fitness, I expected to see a strong correspondence between model-derived and experimentally-derived thermal limits. A strong correspondence between model predictions and experimentally obtained thermal maxima would both validate a physiological interpretation of the species distribution models and justify the use of Critical Thermal Maxima experiments alone in predicting species distributions and responses to climate change.

Children's language and world knowledge grows explosively in the preschool years. One critical contributor to this growth is their developing ability to infer relations beyond those that have been directly taught or modeled. Categorization is one type of skill commonly taught in preschool in which inference is an important aspect. This study explored the development of specific types of inferences within a categorization relation: those among naming items and categories, selecting items based on their names and categories, and answering questions that relate names and categories. Children learned names and categories for a set of unfamiliar cartoon characters through one of two training protocols: (a) Listener training involved selecting a picture upon hearing an item name or category; (b) Expressive training involved saying an item name or category upon seeing a picture. Following training, we tested whether children derived several kinds of untrained responses. Those children who received Expressive training (saying names) completed tests of listener responses (selecting pictures); similarly, those children who received Listener training (selecting pictures) completed tests of expressive responses (saying names). Next, children answered oral questions in the absence of pictures. Results show that children receiving Expressive and Listener training produce naming and question answering responses at levels above chance. However, many children failed to answer all questions correctly. The Expressive group produced naming and question answering responses at significantly higher levels than the Listener group. This suggests that Listener training is a weaker form of instruction when the goal of instruction is the production of untrained responses. However, these results are tentative because unequal proportions of children completed each type of training. Finally, we examined the relationship between naming and question answering. Few children answered questions at a higher level than they produced names. This study shows that children learn to infer responses from both Listener and Expressive trainings. This study also suggests that naming and question answering responses are related responses. The current study highlights the need for later research on teaching inference skills such as naming and question answering to those who do not develop them in the absence of specific instruction.

This applied dissertation compared traditional peer feedback activities, where students comment and edit a peer’s work using red pen on the document, with learner derived feedback critiques based on the task requirements provided by the instructor. The feedback rubrics were created using a Web 2.0 application wiki. Framed by the experiential learning theory in an anagogical learning setting, this task-based approach focused on instructing students how to self-correct and edit their work in English language classes at the university level in Japan. The objective of the investigation was to also identify learner preference for the approach they attempted, to how they conducted peer feedback, and whether or not student preferences were demonstrated in their efforts to meet the required outcomes. No specific preferences for the type of feedback could be clearly identified based in a quantitative survey of the participants. The results for the assigned task did show significance in learner performance: vocabulary, F(1, 85) = 4.055, p = .047; grammar, F(1, 85) = 7.720, p = .009. For content, the p = .143 was not significant; however, scores for the treatment group were stronger than those shown by the control group. The flipped assessment or peer derived feedback/critique approach to peer feedback activities, based on this research, is recommended in second language English writing classes and is a suggested activity in other educational disciplines.