PurA is an abundant and an evolutionary conserved protein that is known to bind to single stranded DNA or RNA and regulate both transcription and translation. PurA has been shown to be implicated in many neurological and neurodevelopmental deficits due to its multifunctional roles. In this study, we have studied the cells’ stress response in the presence and absence of PurA by introducing oxidative stress to the cells derived from wildtype (PurA+/+) mouse embryo fibroblasts (MEFs) and (PurA -/-) knockout mice. Our observations indicated that the presence of PurA is making cells more sensitive to toxicity compared to those cells lacking PurA, emphasizing on the importance of PurA in cell survival and cell cycle regulation. Our MTT results and western blot analysis were in agreement with each other confirming PurA ‘s importance in normal cell response against stress, in particular oxidative stress induced by Paraquat. PurA+/+ wildtype MEFs showed more sensitivity and a decrease in relative cell viability after 24 Hrs. exposure to 1.5 mM paraquat while less sensitivity was observed in PurA -/- knockout MEFs under same conditions. Western blot analysis showed a significantly increased expression of the apoptotic marker cleaved caspase3 in PurA+/+ wildtype MEFs under oxidative stress induction compared to PurA -/- knockout MEFs. Also, autophagy activation was measured by checking the expression of LC3-I and LC3-II in both MEFs under control and stressed conditions and the results were confirming that autophagy induction under oxidative stress is greater in the presence of PurA. PurA appears to be very critical for cell survival, and any alteration in its expression may lead to undesired consequences. / Biology

Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia

Bone is a metabolically active tissue that is continuously remodeled throughout life. Osteocytes, the most abundant cells in bone, regulate bone homeostasis in response to hormonal and mechanical cues. Parathyroid hormone (PTH), a calciotropic hormone secreted from the parathyroid glands, has been widely used in the clinic to treat age-related osteoporosis. PTH acts on cells of the osteoblast lineage, including osteocytes, by signaling via the PTH receptor (PPR) to promote bone formation. However, the role of PPR signaling in osteocytes during aging has not been investigated. The hypothesis of this study is that PPR signaling in osteocytes plays a key role in maintaining skeletal health in aging mice. To address this hypothesis, mice in which the PPR was ablated in mature osteoblasts/osteocytes (Dmp1-Cre+;PPRfl/fl or Dmp1-PPRKO) were used to study their skeletal phenotype at 4 and 13 months of age. Compared to control littermates (Dmp1-Cre–;PPRfl/fl), Dmp1-PPRKO animals displayed age-dependent osteopenia due to reduced osteoblast activity and increased osteoclast numbers and activity. These changes were associated with a significant decrease in osteoprogenitors and an increase in marrow adiposity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes was accompanied by a marked increase in serum sclerostin, RANKL-expressing marrow adipocytes, and early onset of oxidative stress in osteocytes. In vitro studies demonstrated that PTH protected osteocytes from oxidative stress-induced cell death by suppressing the intracellular accumulation of reactive oxygen species. Mechanical forces are also important regulators of bone mass and quality. For instance, immobilization and reduced mechanical loading, such as prolonged bed rest or long-duration spaceflight, lead to bone loss or osteopenia due to reduced bone formation and increased bone resorption. Osteocytes are known to sense and transduce mechanical forces applied to the skeleton into biochemical signals. However, the exact molecular mechanism remains unclear. To unravel the mechanism by which osteocytes sense and respond to mechanical unloading, an osteocytic cell line, Ocy454, was exposed to microgravity (µG) conditions for 2, 4, or 6 days onboard the SpaceX Dragon-6 and the International Space Station. Global transcriptomic analysis demonstrated that µG leads to downregulation of key osteocytic marker genes compared to ground controls (1G), suggesting the impaired differentiation of osteocytes. Importantly, glycolysis was the most activated signaling pathway in osteocytes subjected to µG compared to 1G. Gene comparison analysis further identified a set of mechano-sensitive genes that are consistently regulated in multiple types of cells exposed to µG, suggesting a common, yet to be fully elucidated, genome-wide response to µG. In summary, these studies demonstrated that osteocytes are highly regulated by PTH and mechanical forces. We found that PPR signaling in osteocytes is important for protecting the skeleton from age-induced osteopenia by promoting osteoblast’s bone-forming activity and mitigating osteoclast’s bone resorption. We also demonstrated that PTH protects osteocytes from oxidative stress. Finally, we showed that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

Hippocampal neurogenesis is linked to the onset, progression and remission of major mood disorder such as anxiety and depression. Neurogenesis is the process by which new neurons are formed in the brain. Mitochondria mediate cellular adaption and provide energy to support growth of new neurons. Chronic stress and mood disorders have been associated with impairments in mitochondrial function and neuronal growth. Individuals experiencing stress and mood disorders reportedly use cannabis as a means to self-medicate. The impacts of cannabis on stress-related effects on hippocampal neurogenesis and mitochondria are vastly unexplored. To investigate these effects we generated an in vitro model of hippocampal neuron stress by treating HT22 cells with corticosterone, the major effector molecule of stress in rodents. We first characterized the impacts of corticosterone on markers of neurogenesis and mitochondrial function in HT22 hippocampal cells. We found that corticosterone decreased gene markers of neurogenesis, mitochondrial biogenesis, content, dynamics and decreased mitochondrial membrane potential. Corticosterone also decreased levels of antioxidant enzymes but did not alter levels of reactive oxygen species (ROS) or elicit lipid peroxidation. We then investigated with potential impacts of cannabis components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on corticosterone-induced stress. Individually, THC and CBD decreased markers of neurogenesis, dysregulated mitochondrial dynamics and decreased mitochondrial membrane potential. Interestingly, both THC and CBD increased a marker of mitochondrial biogenesis. Finally, we co-treated HT22 cells with corticosterone and THC or CBD to interrogate the impacts of THC and CBD on corticosterone-induced alterations. Our results indicated THC and CBD had no effect on corticosterone-related reductions in neurogenesis markers or mitochondrial membrane potential. However, THC demonstrated a rescuing effect on a marker of mitochondrial biogenesis and CBD normalized a marker of mitochondrial fission; both of which were decreased with individual corticosterone treatments. This thesis ultimately identifies some of the pathways THC and CBD may impact stress response in relation to neurogenesis and mitochondria. / Thesis / Master of Science (MSc) / Neurogenesis is a process that describes the production of new nerve cells in the brain. It mainly occurs during early life, but persists in a central brain structure responsible for learning and memory, known as the hippocampus, throughout our lives. This active brain structure relies on the function of certain organelles called mitochondria, which are the primary cellular energy producers and promote nerve cell production. Mood disorders, such as anxiety and depression, may result as a consequence of impaired hippocampal neurogenesis. Evidently, people suffering from anxiety and depression turn to cannabis use for management and treatment of their mood disorders. Considering cannabis has been shown to affect neurogenesis and mitochondrial function, our primary objective was to explore its effects on hippocampal neurogenesis by focusing on mitochondrial function, in the context of stress. We demonstrate that components found in cannabis, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), alter the stress-induced changes in mitochondrial functions related to neurogenesis, suggesting that cannabis may play a role in protecting nerve cells.

Inhibiting inflammatory processes or eliminating inflammation represents a logical role in the suppression and treatment strategy of cancer. Several studies have shown that anti-inflammatory drugs (NSAIDs) have promise as anticancer agents while reducing metastases and mortality. NSAIDs are seriously limited by side effects and their toxicity, which can become cumulative with their long-term administration for chemoprevention. The huge development in nanotechnology allows the drugs to exhibit novel and significantly improved properties compared to the large particles of the respective bulk compound, leading to more targeted therapy and reduced dosage. The overall aim of this thesis is to add to our understanding of cancer prevention and treatment through studying the genotoxicity mechanisms of NSAIDs agents in lymphocytes. In this study, the genotoxicity mechanisms of NSAID in bulk and nanoparticles forms a strategy to prevent and minimise the damage in human lymphocytes. Aspirin nano (ASP N) caused a significant decrease in deoxyribonucleic acid (DNA) damage compared to aspirin bulk (ASP B). Also, ibuprofen nano (IBU N) showed a significant reduction in DNA damage compared to ibuprofen bulk (IBU B). Micronuclei (MNi) decreased after ASP N, ASP B and IBU N in prostate cancer patients and healthy individuals, and the ibuprofen bulk showed a significant increase of MNi formation in lymphocytes from healthy and prostate cancer patients when compared to untreated lymphocytes from prostate cancer patients. In order to study the geno-protective properties of these drugs, the protective effect of NSAIDs and the quantification of the DNA repair capacity in lymphocytes was studied. ASP N was found to increase the DNA repair capacity and reduced the reactive oxygen species (ROS) formation significantly more than ASP B. Finally, the role of NSAIDs on some key regulatory signal transduction pathways in isolated lymphocyte cells was investigated by studying their effect on ataxia-telangiectasia-mutated kinase (ATM) and ataxia-telangiectasia and Rad3-related kinase (ATR) mRNA. ATM mRNA significantly increased after treatment with ASP B, ASP N and IBU N. ATR expression also increased after treatment with IBU B and IBU N, but was only significant with IBU N. These findings indicate that a reduction in particle size had an impact on the reactivity of the drug, further emphasising the potential of nanoparticles as improvement to current treatment options.

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