Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Petra Blažková Supervisor: Doc. PharmDr. Martin Beránek Ph.D. Title of diploma thesis: Alpha-1-antitrypsin deficiency analysis using real-time PCR This diploma thesis focuses on the validation of the method PCR in real-time (real-time PCR) to investigate the Z and S mutations in the gene SERPINA1, located at the long arm of chromosome 14 (14q32.13) and provides instructions for making a protein named alpha-1- antitrypsin (A1AT). A1AT is a serine protease inhibitor that protects tissues from degradation by neutrophil elastase. Deficiency, which is most commonly caused by these mutations, can cause children and adult's liver and lung disease. A method of real-time PCR was applied after successful validation to a set of 46 clinical samples of DNA extracted from blood samples and 30 samples of the DNA from cells of the buccal mucosa. DNA isolation was performed by kit extraction QIAamp ® DNA Mini Kit by QIAGEN. I used a set of primers and hybridization probes according to Snyder (2006) for genotyping. Melting curve analysis was carried out in the thermocycler LightCycler 1.2. Results of DNA samples obtained from blood were compared with the results obtained through an accredited method...

The purpose of the project was to increase awareness about alpha-1 antitrypsin deficiency (AATD) in chronic obstructive pulmonary disease (COPD), particularly among those with a familial history of genetic factor AATD; an additional goal was to understand its relationship to COPD. COPD is the third leading cause of death in the United States, with more than half of COPD patients experiencing significant disabilities. Major causes for COPD include smoking, air pollution, secondary smoke, upper respiratory infections, hereditary factors, occupational factors, environmental factors, and socioeconomic factors. Genetic factors, however, also play a significant role in early onset COPD and in those who smoke and have the genetic factor related to COPD (AATD), symptoms are more severe and exacerbations more frequent. Undiagnosed AATD can result in under treatment and lack of planning for preventing COPD onset and exacerbation in these patients. COPD clients of a local pharmacy (n =31) were invited to complete a Likert survey and given materials on COPD exacerbation prevention and information on AATD. Results indicated that 38.7% of respondents had early onset symptoms, positive family history, and no improvement in symptoms with smoking cessation. The results support that targeting those family members with COPD and providing information on genetic factors for this condition could decrease the frequency and severity of exacerbations. This is in keeping with the health belief model that guided this study in that a perceived risk for harm has the potential to improve the use of preventative health measures in individuals.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.

Walden University College of Health Sciences This is to certify that the doctoral study by Maureen Barta has been found to be complete and satisfactory in all respects, and that any and all revisions required by the review committee have been made. Review Committee Dr. Cheryl Holly, Committee Chairperson, Health Services Faculty Dr. Eric Anderson, Committee Member, Health Services Faculty Dr. Vincent Hall, University Reviewer, Health Services Faculty Chief Academic Officer Eric Riedel, Ph.D. Walden University 2015 â?? Increasing Knowledge About Alpha-1 Antitrypsin Deficiency in the Chronic Obstructive Pulmonary Disease Population by Maureen Ann Wentink Barta MSN, Pacific Lutheran University, 1996 BSN, Pacific Lutheran University, 1992 Project Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Nursing Practice Walden University November 2015 â?? The purpose of the project was to increase awareness about alpha-1 antitrypsin deficiency (AATD) in chronic obstructive pulmonary disease (COPD), particularly among those with a familial history of genetic factor AATD; an additional goal was to understand its relationship to COPD. COPD is the third leading cause of death in the United States, with more than half of COPD patients experiencing significant disabilities. Major causes for COPD include smoking, air pollution, secondary smoke, upper respiratory infections, hereditary factors, occupational factors, environmental factors, and socioeconomic factors. Genetic factors, however, also play a significant role in early onset COPD and in those who smoke and have the genetic factor related to COPD (AATD), symptoms are more severe and exacerbations more frequent. Undiagnosed AATD can result in under treatment and lack of planning for preventing COPD onset and exacerbation in these patients. COPD clients of a local pharmacy (n =31) were invited to complete a Likert survey and given materials on COPD exacerbation prevention and information on AATD. Results indicated that 38.7% of respondents had early onset symptoms, positive family history, and no improvement in symptoms with smoking cessation. The results support that targeting those family members with COPD and providing information on genetic factors for this condition could decrease the frequency and severity of exacerbations. This is in keeping with the health belief model that guided this study in that a perceived risk for harm has the potential to improve the use of preventative health measures in individuals.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 127 pages. Includes Vita. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19402号 / 医博第4053号 / 新制||医||1012(附属図書館) / 32427 / 京都大学大学院医学研究科医学専攻 / (主査)教授 佐藤 俊哉, 教授 小川 誠司, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / マギル大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第24203号 / 医博JD第1号 / 新制||医||JD1(附属図書館) / 京都大学大学院医学研究科京都大学マギル大学ゲノム医学国際連携専攻 / (主査)教授 稲垣 暢也, 教授 YOUSSEFIAN Shohab, 准教授 Majewski Jacek (マギル大学), 准教授 Gravel Simon (マギル大学), 教授 Gagneur Julien (ミュンヘン工科大学) / 学位規則第4条第1項該当 / Doctor of Philosophy in Human Genetics / Kyoto University / McGill University / DFAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) show high prevalence and are economically important in equine athletes. RAO is considered a multifactorial disease due to genetic and environmental components in their patophysiology. The aim of this study was to determine the presence of polymorphism at exons 2, 3, 4 and 5 of the SPi2 gene and a possible association between them and ORA or IAD on 51 thoroughbred horses through single-strand conformational polymorphism (SSCP). Exons 2, 3 and 4 of the Spi2 gene showed no polymorphism. On exon 5, 3 alleles and 6 genotypes were identified. Frequency of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO but no association was found between any polymorphism and horses with RAO or IAD. / A obstrução recorrente das vias aéreas (ORA) e a doença inflamatória das vias aéreas (DIVA) são doenças de alta prevalência e economicamente importantes em cavalos atletas. A ORA é considerada uma enfermidade multifatorial por apresentar componentes ambientais e genéticos em sua fiosiopatologia. O presente trabalho teve por objetivo determinar a presença de polimorfismos nos éxons 2, 3, 4 e 5 do gene SPi2 e verificar uma possível associação destes com a ORA e/ou DIVA em 51 cavalos Puro Sangue de Corrida através da técnica de polimorfismo conformacional de fita simples (SSCP). Os éxons 2, 3 e 4 não apresentaram polimorfismo. No éxon 5 do gene Spi2 foram identificados três alelos e seis genótipos. Apesar do alelo A e o genótipo AA apresentarem freqüência (0,6388 e 0,3888, respectivamente) mais elevada nos animais com ORA, não houve associação entre os polimorfismos observados e ORA ou DIVA.

Indiana University-Purdue University Indianapolis (IUPUI) / Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt% and 36 wt%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.