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Subcloning, Expression and Purification of Functional E. coli Nucleotide Excision Repair Protein UvrA Using IMPACT-CN SystemLin, Cathy W, Mrs 01 May 2014 (has links)
DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. DNA repair is a cellular machinery that counters DNA damage and thus preserve genome integrity. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. As the first step of E. coli NER, DNA damage recognition is achieved through the UvrA2B complex. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA, a 115 kDa protein, has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step using the cloned expression construct. Furthermore, the purified protein was found to be fully functional in the UvrABC incision assay in which the DNA adduct of FABP [N-(20-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl] was efficiently cleaved in a time course-dependent manner.
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NUCLEOTIDE EXCISION REPAIR: IMPACTS OF ENVIRONMENTAL CARCINOGENS AND ITS ROLE IN CANCER SUSCEPTIBILITY IN APPALACHIAN KENTUCKYHolcomb, Nathaniel C. 01 January 2017 (has links)
Lung cancer is a particularly devastating disease, accounting for the most deaths among all cancer types in the United States. Despite a reduction in the country’s smoking rates, cigarette smoking remains the number one risk factor for lung cancer. Additionally arsenic exposure, which occurs primarily through contaminated drinking water in the U.S., is associated with increased lung cancer incidence. The nucleotide excision repair (NER) pathway is critical for maintenance of genomic fidelity, removing DNA lesions that could otherwise promote DNA mutations and drive carcinogenesis. Tobacco smoking introduces significant amounts of DNA damage and produces characteristic DNA mutations found in lung cancers of smokers, and arsenic increases lung cancer risk in smokers beyond the risk of smoking along. The contributions of these chemicals to DNA damage and cancer have been well documented, but few studies have examined their effects on DNA repair pathways, particularly the nucleotide excision repair (NER) pathway. Arsenic, while not directly mutagenic, promotes the carcinogenicity of other compounds including agents that produce DNA damage that is repaired by the NER pathway. In this dissertation I investigated the effects of cigarette smoke condensate (CSC, a whole-smoke tobacco surrogate) and arsenic on NER. I observed that CSC or arsenic treatment inhibited NER as measured by a slot-blot assay using UV-induced photolesions as model substrates to measure NER. The abundance of Xeroderma Pigmentosum complementation group C (XPC), a critical NER protein, was significantly reduced in all lines treated with either chemical, while XPA protein was unaffected. CSC and arsenic also affected RNA levels of certain NER genes. Finally, proteasome-regulated XPC turnover was affected by CSC and arsenic treatment, suggesting a potential mechanism for XPC protein inhibition. The observed impairment of NER by CSC is critically important in tobacco cancer etiology – CSC introduces DNA damage, some of which is repaired exclusively by NER, and CSC inhibits the NER pathway as well, providing a two-sided assault on cellular genetic fidelity. I then adapted the NER assay to measure repair in lymphocytes isolated from human subjects of a study investigating the high incidence of lung cancer in Appalachian Kentucky. I observed an age-dependent decline in NER efficiency that was modulated by subject smoking status and a reduced NER efficiency among current smokers in the lung cancer patient population compared to control subjects in the youngest age group, suggesting individual DNA repair capacity measured with this repair assay may be a biomarker for lung cancer susceptibility.
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Régulation transcriptionnelle UV-induite de gènes de la réparation de l'ADN par le facteur de transcription USF-1 (Upstream Stimulating Factor 1)Baron, Yorann 14 December 2009 (has links) (PDF)
La peau est la première barrière de protection de l'organisme face aux agressions de l'environnement incluant notamment les radiations UV solaires, responsables de nombreux dommages sur l'ADN. Afin de prévenir l'apparition de lésions et de maintenir l'intégrité du génome, la cellule a mis en place des systèmes de protection avec la réponse pigmentaire, et de réparation avec le mécanisme du NER (nucleotide excision repair). Le NER est un mécanisme complexe et versatile, conservé au cours de l'évolution, qui repose sur l'action coordonnée d'acteurs protéiques dont les mutations conduisent à l'apparition de syndromes d'hyper-sensibilité aux UV. Ainsi, parallèlement à la régulation transcriptionnelle UV induite dépendante du facteur USF-1 (Upstream Stimulating Factor 1) des gènes clés de la pigmentation, nous avons étudié l'induction transcriptionnelle potentielle de gènes codant pour les acteurs du NER. Par une approche combinant expériences in-vivo (RT-QPCR et ChIP) et in-vitro (EMSA, luciférase essais), nous impliquons le facteur USF-1 dans l'induction transcriptionnelle de gènes codant deux acteurs des étapes de reconnaissance des lésions UV-induites par le NER. L'utilisation de souris invalidées pour le gène USF-1 confirme le modèle observé. L'ensemble de ces données obtenues au cours de ma thèse vient compléter la liste des gènes cibles du facteur de transcription USF-1 et élargit son réseau d'implication en réponse aux UV. Ces résultats suggèrent un rôle potentiel du facteur USF-1 dans le processus tumoral des cancers cutanés UV-induits.
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Experimental Diagnostics and Therapeutics of Invasive Urinary Bladder CancerSherif, Amir January 2003 (has links)
<p>The two purposes of this thesis were to evaluate new diagnostic techniques of lymphnode staging in invasive bladder cancer and to evaluate the results of neoadjuvant chemotherapy in invasive bladder cancer.</p><p>Sentinel node detection was performed in 13 patients in preparation for radical cystectomy. The method showed to be feasible, and the results displayed the occurrence of metastatic nodes outside the traditional area of diagnostic dissection in a majority of patients. Four patients were metastasized, each one with one metastatic node detected with the help of the sentinel node procedure.</p><p>Four randomly selected sentinel nodes from four different unmetastasized patients were compared to the four metastatic sentinel nodes from the first series. After microdissection, p53 genomic structure, immunohistochemical expression and MVD (microvessel density) were assessed in the primary tumors and corresponding sentinel nodes. The results suggested that invasive bladder cancer mainly involved monoclonal proliferation with predominantly homogenous biomarker profile, but there were also signs of clonal evolution.</p><p>The Nordic Cystectomy Trial 2 (NCT2), is a randomized prospective trial investigating the possible benefit of neoadjuvant chemotherapy versus cystectomy only, in 311 eligible patients with urinary bladder cancer T2-T4aNXM0.Evaluation of overall survival did not show any statistically significant benefit in the experimental arm. This probably due to lack of statistical power.</p><p>To increase the statistical power we performed a combined analysis of randomized patients from both the Nordic Cystectomy Trial 1 (NCT1) and NCT2, n = 620. Eligible patients from NCT1 had T1G3, T2-T4a NXM0 urinary bladder cancer. Standard meta-analysis methods were used. The only end-point analysed was overall survival. Neoadjuvant platinum based combination therapy was associated with a 20 % reduction in the relative hazard in probability of death.</p>
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Rectal Cancer : Can the Results be Further Improved?Folkesson, Joakim January 2006 (has links)
<p>The treatment of rectal cancer is complex and comprises: diagnostic measures; different preoperative treatments; a multitude of surgical and technical choices; possibilities of postoperative treatments and postoperative care and follow up. In this thesis, some aspects of this complex paradigm have been further investigated. One of the most feared complications after rectal cancer surgery is anastomotic leakage. The risk of anastomotic leakage is affected by non-influenceable factors related to the tumour and the patient. In the first paper, the risk of anastomotic leakage in relation to a surgical instrument, the circular stapler, was investigated. The risk of leakage was 7% or 11%, depending on the choice of instrument. In the second paper, a long-term evaluation of survival and local recurrence rates in the Swedish Rectal Cancer Trial was made. Randomisation was to either preoperative radiotherapy followed by surgery or surgery alone. After 13 years median follow-up, survival was 38% in the radiotherapy group and 30% in the surgery alone group. Differences in local recurrence rates were seen in all stages. Most rectal cancer operations carry a high risk of morbidity and mortality. For early stage cancers, a local procedure may be sufficient and in the third paper, population-based results of local excision of rectal cancer were explored. In stage I, cancer specific survival was the same after local excision as after major resection, but the relative survival was lower. The risk of local recurrence was higher after local excision than after resections. In the fourth paper, differences in survival rates in the Nordic countries and Scotland were investigated. The relative excess risk of death was highest in Denmark, but only in the first 90 postoperative days. </p><p>Through applying already existing knowledge and successively introducing new treatments, the results for rectal cancer treatment will be further improved.</p>
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Saccharomyces Cerevisiae as a Model Organism to Delineate Initial Lesion Detection Events in Chromatin Repair: A Focus On Ddb2-Mediated GG-NERJones, Kristi L 07 June 2011 (has links)
DNA damage repair is an essential and complex cellular process. Although the basic mechanisms of nucleotide excision repair (NER) have been studied for decades, some mechanistic details remain elusive. The lesion detection step remains one of the most elusive in the process of NER in the contest of chromatin. The work described herein addresses the initial events in the lesion detection step of chromatin repair, also referred to as global genome repair (GG-NER). Both the role of post-translational modifications of lesion identification proteins, and the initial sequence of events in recruitment of repair and remodeling factors are investigated. First, the controversial role of ubiquitination of DDB2 (a human lesion detection protein) is investigated. Due to documented DDB2 function in alternative physiological processes, its direct role in GG-NER is hard to study in human cells. To overcome this obstacle, we established the budding yeast, Saccharomyces cerevisiae as an alternative, simplified model organism to study DDB2-mediated GG-NER. Using this system, we show that inconsistent with the widely accepted model, rapid degradation of DDB2 post-UV irradiation is not an absolute requirement for progression of GG-NER. However, interestingly, our data suggest a role for ubiquitination in the release of DDB2 from chromatin. In both UV and mock treated samples, ubiquitin deficient cells had significantly higher amounts of DDB2 remaining bound to the chromatin compared to the isogenic parent cells. The discussion focuses on the possible physiological relevance of these observations. Additionally, the recruitment of the SWI/SNF chromatin remodeling complex to the silent HML (Hidden MAT Left) locus was also investigated. SWI/SNF is known to require recruitment for its role in transcription; therefore we investigate this requirement in GG-NER. Based on previously published data that indicate an UV-stimulated association of SWI/SNF and Rad4 (a lesion detection protein), we hypothesized that Rad4 is involved in recruitment of SWI/SNF to damaged DNA. Interestingly, our data suggest that Rad4 is not an absolute requirement for recruitment of Snf6 to the HML locus following UV irradiation. However, Rad16 appears to be. These data present an interesting insight into the lesion detection step in GG-NER and this will be discussed.
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Developing biocontainment strategies to suppress transgene escape via pollen dispersal from transgenic plantsMoon, Hong Seok 01 August 2011 (has links)
Genetic engineering is important to enhance crop characteristics and certain traits. Genetically engineered crop cultivation brings environmental and ecological concerns with the potential of unwanted transgene escape and introgression. Transgene escape has been considered as a major environmental and regulatory concern. This concern could be alleviated by appropriate biocontainment strategies. Therefore, it is important to develop efficient and reliable biocontainment strategies.
Removing transgenes from pollen has been known to be the most environmentally friendly biocontainment strategy. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.
A novel approach for selective male sterility in pollen was developed and evaluated as a biocontainment strategy. Overexpression of the EcoRI restriction endonuclease caused pollen ablation and/or infertility in tobacco, but exhibited normal phenotypes when compared to non-transgenic tobacco. Three EcoRI contained 0% GFP positive pollen, while GFP control plants contained 64% GFP positive pollen based on 9,000 pollen grains analyzed by flow cytometry-based transgenic pollen screening method. However, seven EcoRI events appeared to have 100% efficiency on selective male sterility based on the test-crosses. The results suggested that this selective male sterility could be used as a highly efficient and reliable biocontainment strategy for genetically engineered crop cultivation.
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Experimental Diagnostics and Therapeutics of Invasive Urinary Bladder CancerSherif, Amir January 2003 (has links)
The two purposes of this thesis were to evaluate new diagnostic techniques of lymphnode staging in invasive bladder cancer and to evaluate the results of neoadjuvant chemotherapy in invasive bladder cancer. Sentinel node detection was performed in 13 patients in preparation for radical cystectomy. The method showed to be feasible, and the results displayed the occurrence of metastatic nodes outside the traditional area of diagnostic dissection in a majority of patients. Four patients were metastasized, each one with one metastatic node detected with the help of the sentinel node procedure. Four randomly selected sentinel nodes from four different unmetastasized patients were compared to the four metastatic sentinel nodes from the first series. After microdissection, p53 genomic structure, immunohistochemical expression and MVD (microvessel density) were assessed in the primary tumors and corresponding sentinel nodes. The results suggested that invasive bladder cancer mainly involved monoclonal proliferation with predominantly homogenous biomarker profile, but there were also signs of clonal evolution. The Nordic Cystectomy Trial 2 (NCT2), is a randomized prospective trial investigating the possible benefit of neoadjuvant chemotherapy versus cystectomy only, in 311 eligible patients with urinary bladder cancer T2-T4aNXM0.Evaluation of overall survival did not show any statistically significant benefit in the experimental arm. This probably due to lack of statistical power. To increase the statistical power we performed a combined analysis of randomized patients from both the Nordic Cystectomy Trial 1 (NCT1) and NCT2, n = 620. Eligible patients from NCT1 had T1G3, T2-T4a NXM0 urinary bladder cancer. Standard meta-analysis methods were used. The only end-point analysed was overall survival. Neoadjuvant platinum based combination therapy was associated with a 20 % reduction in the relative hazard in probability of death.
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Rectal Cancer : Can the Results be Further Improved?Folkesson, Joakim January 2006 (has links)
The treatment of rectal cancer is complex and comprises: diagnostic measures; different preoperative treatments; a multitude of surgical and technical choices; possibilities of postoperative treatments and postoperative care and follow up. In this thesis, some aspects of this complex paradigm have been further investigated. One of the most feared complications after rectal cancer surgery is anastomotic leakage. The risk of anastomotic leakage is affected by non-influenceable factors related to the tumour and the patient. In the first paper, the risk of anastomotic leakage in relation to a surgical instrument, the circular stapler, was investigated. The risk of leakage was 7% or 11%, depending on the choice of instrument. In the second paper, a long-term evaluation of survival and local recurrence rates in the Swedish Rectal Cancer Trial was made. Randomisation was to either preoperative radiotherapy followed by surgery or surgery alone. After 13 years median follow-up, survival was 38% in the radiotherapy group and 30% in the surgery alone group. Differences in local recurrence rates were seen in all stages. Most rectal cancer operations carry a high risk of morbidity and mortality. For early stage cancers, a local procedure may be sufficient and in the third paper, population-based results of local excision of rectal cancer were explored. In stage I, cancer specific survival was the same after local excision as after major resection, but the relative survival was lower. The risk of local recurrence was higher after local excision than after resections. In the fourth paper, differences in survival rates in the Nordic countries and Scotland were investigated. The relative excess risk of death was highest in Denmark, but only in the first 90 postoperative days. Through applying already existing knowledge and successively introducing new treatments, the results for rectal cancer treatment will be further improved.
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Test fonctionnel de mesure des activités enzymatiques de réparation de l'ADN par excision resynthèse sur support miniaturisé : mise au point et applicationsMillau, Jean-François 15 November 2006 (has links) (PDF)
La réparation de l'ADN est un processus cellulaire très important comme le montre son implication dans de nombreuses maladies génétiques et la carcinogenèse. Les mécanismes des différents systèmes de réparation présentent des interactions et des complémentarités. Les tests fonctionnels utilisés jusqu'alors pour étudier les activités de réparation ne prennent pas en compte toute cette complexité.<br />Nous avons développé un test in vitro offrant une mesure parallélisée, fonctionnelle et spécifique d'activités enzymatiques de réparation de l'ADN. Pour ce faire, nous avons adapté un test d'excision resynthèse au format biopuce.<br />Nous avons mis au point les différentes étapes du test : la fabrication de la biopuce, la normalisation et l'analyse des données, les conditions de réactions biologiques. Nous avons ensuite validé le test en démontrant que nous mesurions des activités enzymatiques de réparation. Enfin deux expériences applicatives de ce test ont été réalisées. Nous avons tout d'abord mis en évidence la similitude des profils de réparation de trois souches de fibroblastes humains issus de cultures primaires, mais aussi les différences des capacités de réparation qu'il existe entre les fibroblastes, kératinocytes, et cellules mononuclées du sang périphérique. Par la suite, nous avons démontré que la réparation de l'ADN est impliquée dans la réponse adaptative des cellules au rayonnement ionisant. <br />Les applications futures de ce test sont importantes, tant en recherche fondamentale qu'appliquée pour le criblage de molécules, ou encore dans le domaine diagnostic.
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