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.

ABSTRACT Rifampicin is a major chemotherapeutic agent used against mycobacterial and nocardial infections. High level resistance is primarily due to mutational alterations in the rpoB gene encoding the β subunit of RNA polymerase. When challenged, these bacteria may inactivate rifampicin by one of four mechanisms: decomposition, ADP-ribosylation, glucosylation and phosphorylation. ADPribosylation occurs in many mycobacterial pathogens but nothing is known about the properties of the enzyme responsible. Consequently mutational analysis may be used to explore structure-function relationships in this protein. Three mutants with changes in the open reading frame were selected and studied. The altered arr gene in pMG1 was obtained by in vivo selection whilst in pMG2 and pMG4 by in vitro mutagenesis. The mutated arr gene in pMG1 and pMG2 conferred resistance to 50 μg/ml of rifampicin while in pMG4 to 200 μg/ml. This suggested that alterations near the N-terminus resulted in lowered activity because of closer proximity to the active site. This is the first successful report of induced arr gene expression. This over-expression of the Arr ADP-ribosyltransferase and its mutants assisted in their later purification by metal affinity chromatography.

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

Examensarbetet ”ADP Generator” går ut på att skapa ett datorprogram och en databas somskall effektivisera hanteringen av information från alla stadier ur rymdproduktionen vidJirotex Furudahlsgruppen AB.Informationen i databasen skall hämtas med programmet på ett lätthanterligt sätt.Slutligen skall programmet skapa en sammanställning från produktionen i form av ettdokument, ett s.k. ADP, Acceptance Data Package.Programmet har skapats i Java och databasen i MySQL. Dokumenten är i formatet RTF, RichText Format.I rapporten står en systembeskrivning och förklaringar för valda lösningar. / Uppsatsnivå: C

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.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

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

Antiplatelet therapy with clopidogrel is widely used in patients with coronary artery disease, but the recent development of the new P2Y12 receptor antagonists prasugrel and ticagrelor will increase treatment options. An overview of systematic reviews was performed to summarize available evidence on clopidogrel. Current data on prasugrel and ticagrelor were identified by a systematic review and used for an indirect treatment comparison (ITC) of the drugs against each other and versus placebo in the absence of head-to-head clinical trials. Adjusted indirect comparison according to Bucher, Bayesian methods for mixed treatment comparisons using Winbugs, and generalized linear mixed models using SAS were employed for ITC, yielding almost identical results: prasugrel was favored regarding stent thrombosis and ticagrelor regarding major bleeding. However, substantial differences in trial design were identified, demanding caution when interpreting these results. On the basis of the obtained results, a randomized controlled trial was designed within the gap of current evidence.

Antiplatelet therapy with clopidogrel is widely used in patients with coronary artery disease, but the recent development of the new P2Y12 receptor antagonists prasugrel and ticagrelor will increase treatment options. An overview of systematic reviews was performed to summarize available evidence on clopidogrel. Current data on prasugrel and ticagrelor were identified by a systematic review and used for an indirect treatment comparison (ITC) of the drugs against each other and versus placebo in the absence of head-to-head clinical trials. Adjusted indirect comparison according to Bucher, Bayesian methods for mixed treatment comparisons using Winbugs, and generalized linear mixed models using SAS were employed for ITC, yielding almost identical results: prasugrel was favored regarding stent thrombosis and ticagrelor regarding major bleeding. However, substantial differences in trial design were identified, demanding caution when interpreting these results. On the basis of the obtained results, a randomized controlled trial was designed within the gap of current evidence.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 49-53).