This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.

Adenosine diphosphate (ADP) ribosylation is a post-translational modification dependent on the transfer of ADPr units from nicotinamide adenine dinucleotide (NAD+) on to a plethora of biomolecules (i.e., proteins, DNA, RNA, etc.) in response to physiological stressors (i.e., nutrient deprivation, oxidative stress, DNA strand breaks). Poly-ADP-ribosylation (PARylation) is primarily mediated by the family of poly(ADP-ribose) polymerases (PARPs) and enzymatically degraded (dePARylation) by hydrolases such as poly(ADP-ribose) glycohydrolase (PARG). This thesis characterizes the role of poly(ADP-ribose) polymerase 1 (PARP1) and PARG in the skeletal muscle of healthy mature mice under normal physiological conditions. Specifically, we validate the deletion of Parp1 and Parg in inducible skeletal muscle-specific KO mouse models followed by performing general phenotyping of both male and female mice. The thesis concludes that under normal physiological conditions the activity of Parp1 or Parg in (de)PARylation is dispensable for maintaining skeletal muscle mass, function, and homeostasis in healthy mature mice.

Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 — the enzyme responsible for synthesis of 90% of cellular PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension. Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry. Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.

Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.

Changes in myocardial energetics have been implicated in the pathophysiology of heart failure (HF). However, the precise contribution of creatine (Cr) / phosphocreatine (PCr) / creatine kinase (CK) energy buffer and transfer remains unclear. The aim of this thesis was to study the effects on murine cardiac function of both impairment and enhancement of creatine metabolism. In order to longitudinally follow the cause and effect relationship of myocardial creatine concentration, a non-invasive method of quantification was required. Cardiac Cr levels measured in vivo by 1H-MRS were therefore compared with gold-standard invasive HPLC and found to correlate over a wide-range (r2=0.91). 1H-MRS was reproducible for measuring Cr levels in the heart, brain, and skeletal muscle. The cardiac phenotype of a novel model of creatine depletion, the AGAT-/- mouse, was characterized using in vivo MRI, 1H-MRS and LV catheterisation, under conditions of gradually reducing Cr concentrations; zero Cr; and attempted phenotype rescue with dietary Cr. For the first time in the heart, the rate of Cr turnover was quantified (~3 % per day) and demonstrated that cardiac function was preserved even when creatine levels reduced by ~70-90%. Total absence of myocardial Cr induced impairment of inotropic and lusitropic cardiac function and reduced inotropic reserve. Cardiac dysfunction was only partially rescued by replenishment of the Cr pool, suggesting this to be a consequence of long-term adaptations to chronic low Cr. Finally, we tested the hypothesis that combined elevation of myocardial creatine and ribose would be beneficial in a mouse model of chronic HF by increasing cardiac energy availability. Despite an increase in myocardial ribose concentration, this did not prevent loss of total adenine nucleotides (TAN), and there was no improvement in post-infarct LV remodeling or function. Future studies are needed to explore alternative approaches for maintaining TAN in combination with total creatine.

Assim como o número de casos de câncer vem aumentando em nível global, a busca por abordagens terapêuticas visando uma maior eficácia com um menor poder de causar efeitos prejudiciais aos pacientes também vem crescendo. As antraciclinas e antracenodionas, as quais tem como exemplos, doxorrubicina (DOX) e mitoxantrona (MTX), respectivamente, são fármacos utilizados na quimioterapia em diversas neoplasias incluindo tumores sólidos e não sólidos tais como de mama, leucemias, linfomas, sarcomas etc. Embora sejam eficazes ao que se propõem, o tratamento com estas moléculas pode acarretar em efeitos secundários, tais como arritmias e insuficiência cardíaca. Estas drogas além de interagirem com o ferro e apresentarem capacidade de gerar espécies reativas de oxigénio (ROS), apresentam como principal mecanismo a inibição da enzima topoisomerase 2 (Top2). Os inibidores de PARP-1 emergiram como uma nova alternativa para tratar determinados tipos de neoplasias em que a letalidade sintética possa ser explorada. Além disto, já foi relatado que a toxicidade cardíaca induzida por DOX seja influenciada pela atividade de PARP-1. O objetivo desta tese foi, portanto, avaliar a influência da inibição de PARP-1 na toxicidade cardíaca de DOX e MTX em células cardíacas. Células foram incubadas durante 24h com DOX ou MTX na presença ou na ausência de inibidor de PARP-1. Ensaios de viabilidade, apoptose e genotoxicidade e foram realizados. Além disso, a fosforilação de proteínas envolvidas na resposta a danos no DNA (ATM, MRE-11 e H2AX) foram avaliadas por western blot e imunofluorescência. Os resultados demonstraram que a inibição de PARP-1, apesar de diminuir a concentração de ROS, diminui a viabilidade de células H9c2 tratadas com DOX ou MTX por aumentar a geração de quebras duplas no DNA induzida por estes fármacos. / As the number of people with cancer are globally increasing, the search for therapeutic approaches that increases efficiency decreasing harmful effects to patients is also growing, giving rise to cardio-oncology. Anthracyclines, e.g., doxorubicin (DOX), and anthracenediones, e.g., mitoxantrone (MTX), are drugs used in the chemotherapy of several cancer types, including solid and non-solid malignancies such as breast cancer, leukemia, lymphomas, and sarcomas. Although they are effective in tumor therapy, treatment with these two drugs may lead to side effects such as arrhythmia and heart failure. These drugs interact with iron to generate reactive oxygen species (ROS), target topoisomerase 2 (Top2), and impair mitochondria. PARP-1 inhibitors have emerged as a new alternative for treating certain types of malignancies in which the synthetic lethality can be exploited. Furthermore, it has been reported that DOX-induced cardiac cardiotoxicity is influenced by PARP-1 activity. The main goal of this thesis was, therefore, to evaluate PARP-1 inhibition influence in cardiac toxicity of DOX and MTX in cardiac cells. Cells were incubated for 24h with MTX or DOX in presence or absence of PARP-1 inhibitor. Viability, oxidative stress and genotoxicity assays have been conducted. Furthermore, phosphorylation of proteins involved in response to DNA damage (ATM, H2AX and MRE-11) were evaluated by western blot and immunofluorescence. Results demonstrated that inhibition of PARP-1, although decreasing ROS generation, decreases H9c2 cells viability after DOX or MTX by increasing DNA double strand break generation induced by these drugs.

Thèse (M.Sc.)--Université Laval, 2005. / Bibliogr.: f. 75-90. Publié aussi en version électronique.

No description available.

During periods of intense exercise the adenine nucleotide pool in skeletal muscle becomes partially depleted. Ribose has been shown to increase rates of both purine salvage and adenine nucleotide de novo synthesis in rat skeletal muscle. However, to date no research has been conducted on the effects of ribose on adenine nucleotide levels in human skeletal muscle. Therefore, the purpose of this investigation was to determine the effects of ribose supplementation on adenine nucleotide levels in human skeletal muscle during high-intensity exercise. To do this, an 11-day supplementation of either ribose (20 g.d-1) or placebo (glucose 20 g•d-1) was given to 16 healthy male subjects. After 72 hours of supplementation, the subjects performed five-days of high-intensity exercise designed to elicit significant reductions in adenine nucleotides. A 65 hour recovery period was completed following the exercise protocol. Muscle biopsies were performed at four different time points during the supplementation/exercise period.The exercise protocol elicited significant decreases in skeletal muscle adenine nucleotide levels of both the ribose and placebo groups (p<0.05). However, ribose supplementation was shown to partially attenuated the adenine nucleotide decrease. The placebo group demonstrated a 39% decrease, while the ribose group dropped 23% in TAN levels (p<0.05). The largest decreases were observed in ATP for both groups; however, the decreases were significantly greater in the placebo group (p<0.05). Both groups displayed a similar amount of replenishment in adenine nucleotides 65 hours following the 5-day exercise period. No differences were demonstrated in ADP or AMP for either the ribose and placebo groups throughout the investigation. Both groups displayed an increase in E MP post-exercise, but the increase was only significant in the ribose group (p<0.05). No differences in mean power, peak power, and fatigue were observed between the ribose and placebo group. However, the ribose group consistently displayed a non-significantly greater percent change (3.1 %) in mean power. / School of Physical Education

Thèse (M.Sc.) -- Université Laval, 1997. / Bibliogr.: f. 128-151. Publié aussi en version électronique.