<p>Microbeam radiation therapy (MRT) is an experimental radiotherapy concept that has been primarily developed for the treatment of malignant brain tumours. MRT uses high flux synchrotron x-rays delivered as an array of parallel microbeams in high doses of irradiation in fractions of seconds. The aims of this study were to 1) investigate the induction of bystander effects after normal and tumour-bearing rat brains were exposed to MRT and homogenous radiation; 2) validate a brain bystander proteome by detecting protein expression throughout immunohistochemistry: and 3) to investigate whether communication of bystander signals can be produced between animals.</p> <p>Healthy and tumour-bearing Wistar rats were exposed to 17.5, 35, 70 or 350 Gy of MRT or homogenous field of synchrotron radiation to the right brain hemisphere. To study the communication of bystander effects between animals, irradiated rats shared the same cage with non-irradiated rats over a period of 48 hours. After euthanasia of the animals, brains and bladders were dissected, and samples for immunohistochemistry and bystander clonogenic assays were set up.</p> <p>Clonogenic survival of the reporter HPVG cells showed that bystander effects occurred in both the non-irradiated hemisphere and bladder of normal and tumour-bearing rats, while the irradiated hemisphere showed the direct effects of radiation. Moreover, communication of bystander signals was confirmed in the non-irradiated rats.</p> <p>In conclusion, the results suggest that the MRT and homogenous radiation of unilateral normal and tumour-bearing rat brains produce bystander signals that affect the whole organism and that those signals also can be transmitted to non-irradiated animals.</p> / Master of Science (MSc)

<p>Ionizing radiation is without a doubt an invaluable tool in diagnostic imaging as well as radiation therapy. With the growing number of medical and occupational exposures, together with challenges against the LNT model, low dose exposures and non-targeted effects have been subject to intensive research. Additionally, with the advances in the field of radiation therapy and longer life expectancy after the treatment, the risks associated with second malignancies following radiation therapy for various cancers has received a tremendous amount of attention. On the other hand, nicotine, as the addictive component of tobacco has been known for its adverse health effects and its relation to various types of cancers, accounting for one in 10 adult deaths worldwide. Both nicotine and low doses of radiation are amongst the stressors that widely affect the public. Surprisingly, the interactions between low-dose effects and nicotine exposure have not received the proper scientific attention. Our group has been involved in investigation of the non-targeted effects of radiation with a variety of endpoints. Different natural compounds and signalling molecules have also been studied in our lab for their possible role or contribution to bystander signalling. This research involves the study of the impact of nicotine on radiation-induced bystander effects and also radioadaptive responses. Different concentrations of nicotine were used to study the kinetics of the drug as well as any detrimental or modifying effects when used together with radiation. It was shown that nicotine has a protective effect on survival of the cells in certain concentrations that follows a biphasic model. Similar bimodal behaviour was observed with bystander effect. No adaptation to a challenge dose of radiation occurred as a result of incubation with varying concentrations of nicotine, nor was such an effect shown with a priming dose of radiation. The results of the present study suggest that nicotine has a complicated effect on the cells which can vary significantly depending on the concentrations used and also the duration of exposure. nAChRs may have an important role in the response of the bystander cells when nicotine is involved as the results showed a shift in the response of the receptors to nicotine. This thesis is aimed to shed light on the impact of nicotine and initiate more detailed investigations on pathways through which these effects are mediated.</p> / Master of Science (MSc)

<p>The endoplasmic reticulum (ER) is a specialized organelle that facilitates correct protein folding and maturation. Disruptions in ER homeostasis lead to ER stress and activation of a series of signal transduction cascades known as the unfolded protein response (UPR), which acts to restore ER homeostasis. In recent years, ER stress and UPR dysfunction have been linked to obesity, fatty liver and insulin resistance. Lipid-laden adipocytes, the main cellular component of white adipose tissue (WAT), play a critical role in whole body energy homeostasis as well as lipid and carbohydrate metabolism. Mature adipocytes, which are metabolically active endocrine cells, differentiate from precursor fibroblast-like preadipocytes, through a process called adipogenesis, leading to formation of cells capable of secreting numerous proteins, cytokines and hormones. ER homeostasis and UPR activation are essential to the function/differentiation of highly secretory cells, however, the role of ER stress/UPR activation in adipogenesis had previously not been examined. We hypothesized that<em> adipogenesis may rely on physiological UPR activation to accommodate the demand on the ER for increased folding and secretion of proteins.</em></p> <p>Initial experiments examining UPR activation during 3T3-L1 adipogenesis identified that expression of ER stress/UPR markers was modulated during adipocyte differentiation. Furthermore, inhibition of ER stress/UPR activation by the chemical chaperone, 4-phenyl butyric acid (4-PBA), inhibited adipogenesis and blunted high fat-diet induced weight gain in 4-PBA supplemented mice. These findings suggested that UPR activation modulates adipogenesis and adipose tissue metabolism.</p> <p>Subsequently, we sought to identify novel candidate ER stress/UPR responsive genes that may be involved in adipogenesis and WAT metabolism. The expression of a recently recognized ER stress-responsive gene, T-cell death associated gene 51 (TDAG51) was identified to be differentially regulated during adipogenesis. However, the function of TDAG51 in adipogenesis or energy regulation was not known. Studies from this thesis showed that TDAG51 protein expression is attenuated by ER stress/UPR activation in preadipocytes and declines during adipogenesis. Based on these results, and given the importance of adipogenesis in WAT function and whole body energy metabolism, it was<em> </em>hypothesized that<em> TDAG51 may be a novel regulator of adipogenesis and energy homeostasis.</em> Indeed, as reported here, knock-down or absence of TDAG51 (<em>TDAG51^-/-</em>) in pre-adipocytes increased lipogenesis and lead to earlier and more potent expression of adipogenic markers.</p> <p>Finally, we investigated whether absence of TDAG51 in mice affected adiposity and metabolic outcomes. Consistent with the <em>in vitro </em>results, we found that <em>TDAG51^-/-</em>mice fed a standard chow diet, exhibited an age-associated increase in WAT, developed fatty liver, and exhibited insulin resistance as compared to wild-type mice.</p> <p>Taken together, the findings in this thesis indicate that physiological UPR activation and the UPR-responsive gene TDAG51 play important roles in regulating adipogenesis, lipogenesis and whole-body energy metabolism. Thus, therapeutic approaches aimed at modulating ER folding capacity, UPR activation and/or TDAG51 expression may have great potential in the treatment of obesity and its co-morbidities.</p> / Doctor of Philosophy (PhD)

Malignant cutaneous melanoma is the deadliest form of skin cancer, and a majority of melanoma diagnoses are a result of exposure to ultraviolet (UV) radiation. UV radiation causes DNA damage, which if not repaired correctly via nucleotide excision repair (NER) can result in mutations and melanomagenesis. The melanocortin 1 receptor (MC1R) is a Gs protein coupled receptor located on melanocyte plasma membranes and is involved in protecting the skin from UV induced damage. MC1R signaling results in the activation of two protective pathways: 1) induction of eumelanin synthesis downstream of micropthalmia-associated transcription factor (MITF) and 2) acceleration of NER downstream of ataxia telangiectaseia mutated and Rad3 related (ATR). MC1R signaling, however, also promotes melanocyte proliferation, therefore, the activation of the MC1R pathway must be regulated. The overall hypothesis of this dissertation is that the pathways downstream of MC1R can be manipulated to protect against UV induced damage. Chapter 2 investigates the regulation of the MC1R neutral antagonist human β-defensin 3 (βD3). UV damage did not induce βD3 mRNA expression in ex vivo human skin explants. The induction of βD3 expression instead correlated with inflammatory cytokines including TNF. Chapter 3 investigates the interdependence and cross talk between the two protective pathways downstream of MC1R. We directly tested the effect of MITF on the acceleration of NER and the effect of ATR on the induction of eumelanin synthesis following MC1R activation. MITF was not required for the acceleration of NER as mediated by ATR, however, the induction of transcription of enzymes involved in eumelanin synthesis was dependent upon ATR kinase activity. Finally, Chapter 4 investigates the mechanism by which MC1R promoted proliferation and whether the two UV protective pathways downstream of MC1R could be selectively activated without the risk of melanocyte proliferation. MC1R signaling resulted in activation of the mechanistic target of rapamycin complex 1 (mTORC1), a major regulator of cell growth and proliferation. Inhibition of mTORC1 signaling via rapamycin prevented MC1R induced proliferation in vitro. Rapamycin, however, did not prevent MC1R induced eumelanin synthesis or the acceleration of NER in vitro or in vivo suggesting it is possible to selectively activate the beneficial signaling pathways without the risk of melanocyte proliferation. The results of this dissertation suggest that MC1R signaling could be augmented in individuals to prevent UV induced damage.

Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.

Huntington’s disease is an incurable, progressive neurological disorder characterized by loss of motor control, psychiatric dysfunction, and eventual dystonia leading to death. Despite the fact that this disorder is caused by a mutation in one single gene, there is no cure. The mutant Huntingtin (mHtt) protein is expressed ubiquitously throughout the brain but frank cell death is limited to the striatum. Recent work has suggested that Rhes, Ras homolog enriched in striatum, which is selectively expressed in the striatum, may play a role in Huntington’s disease neuropathology. In vitro studies have shown Rhes to be an E3 ligase for the post-translational modification protein SUMO. Rhes increases binding of SUMO to mHtt which competes for the same binding site as Ubiquitin. SUMOylation of mHtt leads to disaggregation and cellular death, whereas ubiquitination leads to aggregation and cellular protection. In a previous study we showed that deletion of Rhes caused a decrease in the Huntington’s disease phenotype in mice. We hypothesized that mice lacking Rhes would also show increased aggregation in the striatum and this increased aggregation would correlate in a rescue of behavioral symptoms. Despite the prior in vitro and in vivo evidence, deletion of Rhes in vivo did not alter the aggregation of mHtt in the striatum of mice however deletion of Rhes still showed a rescue from the diseased phenotype. This result would indicate that deletion of Rhes alters the neurobehavioral phenotype of Huntington’s disease through a different pathway than promoting aggregation in striatal cells.

Clinical radiotherapy is used to treat a variety of brain tumors within the central nervous system. While effective, it can result in progressive and debilitating cognitive impairment that can diminish quality of life. These impairments have been linked to hippocampal dysfunction and corresponding deficits in spatial learning and memory. Mossy cells are a major population of excitatory neurons located within the dentate hilus and highly involved in hippocampal circuitry. They play critical roles in spatial navigation, neurogenesis, memory, and are particularly vulnerable to a variety of neurotoxic insults. However, their sensitivity to ionizing radiation has yet to be investigated in detail. I hypothesize that mossy cells are critical targets for ionizing radiation, whereby damage to these targets contributes to the mechanisms associated with radiation-induced hippocampal dysfunction. To test this idea, wild-type mice were exposed to clinically relevant doses of cranial x-ray irradiation and their hippocampi were examined 1 month and 3 months post treatment. A significant decline in both the number of mossy cells and their activity were observed. In addition, dentate granular cells demonstrated reduced levels of activity, as well as reduced proliferation within the subgranular zone. A second cohort of mice was introduced to a novel environment in order to induce the expression of immediate early genes. Analysis of c-Fos mRNA yielded a significant increase in control but not irradiated animals, suggesting that radiotherapy impaired immediate early gene expression and resultant functional behavioral outcomes. These findings support the proposition that radiation-induced damage to mossy cells contributes to hippocampal deficiencies which result in cognitive dysfunction.

The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.

During embryonic development, adipocytes emerge from microvasculature. Lineage-­‐tracing studies in mice have shown that adipocyte progenitors reside in the adipose tissue capillaries. However, the direct evidence of an association between adipocyte progenitors and vasculature in humans is lacking. A specific class of adipocytes (brown and beige/brite) expresses the uncoupling protein 1 (UCP1), which consumes glucose and fatty acids to generate heat. The abundance of UCP1- containing adipocytes correlates with a lean metabolically healthy phenotype in human. However, a causal relationship between the presence of these cells and metabolic benefits in human is not clear. In this thesis, I report human adipocyte progenitors proliferate in response to pro-angiogenic factors in association with adipose capillary networks in-vitro. The capillary-derived adipocytes transform from being UCP1-negative to positive upon adenylate cyclase activation, a defining feature of the brite/beige phenotype. Activated cells have denser, round mitochondria with UCP1 protein, and display uncoupled respiration. When implanted into NOD-scid IL2rgnull (NSG) mice, the adipocytes can form a vascularized fat pad that induces vascularization and becomes integrated into mouse circulatory system. In normal or high fat diet-fed NSG mice, activated brite/beige adipocytes enhance systemic glucose tolerance and improved hepatic steatosis, thus providing evidence for their potential therapeutic use. The adipocytes also express neuroendocrine and secretory factors such as Interleukin-33, proprotein convertase PCSK1 and proenkephalin PENK, which are correlated with human obesity. Finally, analyses on single-cell clones of capillary-sprout cells reveal the existence of diverse adipogenic progenitor populations. Further characterization of the clones will define the identifying features of the diverse adipocyte progenitor types that exist in human adipose tissue.