Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and is a risk factor for lung cancer development. COPD encompasses both emphysema and chronic bronchitis, the pathogenesis of which are unclear. In this dissertation, I leveraged genome-wide gene-expression studies of emphysema and lung cancer to investigate pathogenesis and carcinogenesis in COPD. Tobacco smoke is the primary cause of emphysema. The most severe form is also associated with alpha1-antitrypsin deficiency (AATD) resulting from a mutation. In this study, I leveraged multiple lung samples from patients with emphysema, with or without AATD. While genes involved in tissue repair decreased with emphysema severity, the unfolded protein response (UPR) was uniquely changed in AATD lungs. AATD may play multiple roles in emphysema and UPR activation suggests AAT replacement therapy may be insufficient to treat this form of emphysema. Emphysema is a progressive disease, and the mean linear intercept (Lm) can serve as a surrogate of progression. I evaluated whether Lm increases in non-diseased lungs may represent similar processes to those occurring in emphysema, and could offer insight into early stages of disease or homeostasis. Genes involved in tissue repair increased with Lm in controls but decreased in disease. Tissue repair processes may be active in even the non-insulted lung, suggesting their activity is necessary for lung homeostasis and their deficiency may drive emphysema progression. Finally, COPD patients are at increased lung cancer risk, and transcriptomic changes common to both diseases could explain this risk. In both COPD and lung cancer, I discovered that H3K27Me3 regulated genes are repressed, and that the methyltransferase responsible for H3K27me3, EZH2, is induced. H3K27Me3, an oncogenic histone modification, may drive carcinogenesis and pathogenesis in COPD. Though usual and AATD emphysema share transcriptomic signatures associated with tissue repair, which may be active in the normal homeostatic lung, the UPR changes in AATD emphysema only; successful therapeutic strategies in emphysema will need to account for this difference. In COPD, H3K27Me3 may play a role in both pathogenesis and carcinogenesis, making it an attractive target for therapeutic interventions, but one that would need further augmentation in AATD. / 2019-11-01T00:00:00Z

Die in den letzten Jahren erhobenen genetischen Befunden untermauern das Konzept, daß ein Ungleichgewicht von Proteasen und ihren Inhibitoren wesentlich an der Pathogenese der chronischen Pankreatitis beteiligt ist. Die Identifizierung von Mutationen im kationischen Trypsinogen bei Patienten mit hereditärer Pankreatitis hat das Verständnis der Erkrankung entscheidend beeinflußt. Der Nachweis von SPINK1-, CFTR- und PRSS1-Mutationen bei Patienten ohne Familienanamnese für eine Pankreatitis deutet darauf hin, daß auch die idiopathische Pankreatitis genetisch determiniert ist. Die bisher durchgeführten Studien legen nahe, daß die erblich bedingte chronische Pankreatitis eine genetisch heterogene Erkrankung ist, die in Abhängigkeit von den defekten Genen bzw. den zugrundeliegenden Mutationen einem autosomal dominanten, einem autosomal rezessiven oder einem komplexen Erbgang folgt. Das gehäufte Auftreten von SPINK1-Mutationen bei alkoholischer chronischer Pankreatitis ist ein Hinweis darauf, daß genetische Faktoren auch zur Suszeptibilität von primär nicht erblichen Formen der chronischen Pankreatitis beitragen. Im weiteren konnte gezeigt werden, daß genetische Dispositionsfaktoren auch bei der Pathogenese der tropischen Pankreatitis einen wesentlichen Stellenwert besitzen. Diese Daten stellen das Konzept der tropischen Pankreatitis als eigene, tropenspezifische Krankheitsentität in Frage. In der vorliegenden Arbeit wurde die Bedeutung genetischer Faktoren bei der Entstehung der hereditären und idiopathischen wie der alkoholischen Pankreatitis untersucht. Die vollständige Aufklärung der genetischen Ursachen wird vermutlich die Unterscheidung zwischen hereditärer und idiopathischer bzw. tropischer chronischer Pankreatitis obsolet werden lassen. Nach Ausschluß sekundärer Ursachen sollte auch bei Patienten ohne Familienanamnese eine Genanalyse auf Mutationen in den obengenannten Genen veranlaßt werden. / The recent discoveries of trypsinogen (PRSS1) and trypsin inhibitor (SPINK1) mutations in patients with hereditary and idiopathic chronic pancreatitis support the hypothesis that an inappropriate activation of pancreatic zymogens to active enzymes within the pancreatic parenchyma initiates the inflammatory process. Thus, pancreatitis may be the result of an imbalance of proteases and their inhibitors within the pancreatic parenchyma. Since the first description of inherited pancreatitis reported an autosomal dominant trait, hereditary CP was defined as an rare dominant inherited disease. Subsequently, the fact of familial clustering in one generation only, which indicates other inheritance pattern such as recessive or complex trait, was blinded out in the disease concept of hereditary CP for a long time. The Identification of PRSS1, SPINK1 and CFTR mutations in patients with so-called idiopathic chronic pancreatitis, however, shows that inherited cases of CP are much more frequent and that different mutations in different genes might lead to different inheritance pattern. Evaluation of patients with CP without an obvious predisposing factor should include genetic testing for mutations in the above mentioned genes even in the absence of a family history of pancreatitis. The finding of SPINK1 mutations in alcohol-induced pancreatitis indicates that genetic factors genetic factors may increase disease susceptibility to primary non-hereditary CP types. This work summarises the significance of genetic factors in the pathogenesis of hereditary and idiopathic as well as alcoholic chronic pancreatitis. Thus, the identification of further genes involved into the pathogenesis of inherited CP probably will also enhance our knowledge about more common types of CP such as alcoholic or tropical CP.

Chronic pulmonary diseases include a wide range of illnesses that differ in etiology, prevalence, symptomatology and available therapy. A common link among these illnesses is their impact on patients’ vital function of breathing, high symptom burden and significantly impaired quality of life. This dissertation research evaluates disease severity, symptom burden and health behaviors of patients with three different chronic pulmonary conditions. First, alpha-1 antitrypsin deficiency (AATD) is an inherited condition that typically is associated with an increased risk of early onset pulmonary emphysema. This study examines differences in demographic, health, and behavioral characteristics and compares clinical outcomes and health related behaviors and attitudes between two severe genotypes of AATD - ZZ and SZ. The findings of the study suggest that patients with SZ genotype and less severe form of deficiency report higher number of exacerbations, comorbidities, as well as unhealthy behaviors such as lack of exercise and current smoking. In addition, individuals with the more severely deficient ZZ genotype are more adherent to disease management and prevention program recommendations and maintain a healthier lifestyle than individuals with SZ genotype. Second chronic lung disease examined in this research was chronic obstructive pulmonary disease (COPD), the fourth leading cause of death and second leading cause of disability in the United States. Prevalence and burden of cough and phlegm, two of the most common symptoms of the COPD, were assessed among participants of the COPD Foundation’s Patient-Powered Research Network (COPD PPRN). In addition, association between patient-reported levels of phlegm and cough, clinical outcomes and patients’ quality of life were evaluated. Participants’ quality of life was assessed using Patient Reported Outcome Measurement Information System instrument PROMIS-29. Association between changes in symptom severity over time and patient-reported quality of life were examined. Findings of this study indicated that severity of cough and phlegm were associated with higher number of exacerbations, greater dyspnea, and worsened patient-reported quality of life including physical and social functioning. Improvement in cough and phlegm severity over time was associated with better patient-reported quality of life. Third pulmonary illness described in this dissertation is non-cystic fibrosis bronchiectasis (NCFB), a rare and etiologically diverse condition characterized by dilated bronchi, poor mucus clearance and susceptibility to bacterial infection. Association between presence of Pseudomonas aeruginosa (PA), one of the most frequently isolated pathogens in patients with NCFFB, and disease severity was assessed utilizing enrollment data from the Bronchiectasis and NTM Research Registry (BRR). NCFB disease severity was evaluated using modified versions of validated in large international cohorts instruments, the Bronchiectasis Severity Index (BSI) and FACED. The findings of this study indicate that PA infection is common in NCFB patients, and presence of PA in patients’ sputum is associated with having moderate and high severity of bronchiectasis. In addition, the results of this study suggest that the two severity assessment instruments classify patients with NCFB differently which may be attributed to a greater number of severity markers utilized in the calculation of the BSI compared to FACED. In conclusion, the proposed dissertation aims to enhance understanding of differences in health outcomes between genotypes of AATD within AlphaNet registry, and to guide future health-promoting behaviors. It highlights the burden of common symptoms such as cough and phlegm in patients with COPD within COPD PPRN and their association with patients’ quality of life. In addition, it introduces modified indices of NCFB severity and emphasizes high burden of the disease in patients with presence of PA within the US BRR.

Die Erkrankung Huntington s Chorea ist eine autosomal dominant vererbte Erkrankung, die gewöhnlich im mittleren Lebensabschnitt beginnt und unausweichlich zum Tode führt. In unserem Bestreben, Proteine zu identifizieren, welche an Prozessen "Upstream" oder "Downstream" des krankheitsverursachenden Proteins Huntingtin beteiligt sind, wurde das Proteom eines sehr gut etablierten Mausmodells mit Hilfe der Großgel 2D-Elektrophorese untersucht. Es konnte zum ersten Mal auf Proteinebene nachweisen werden, dass die Expression von zwei Serinproteasehemmern, alpha1-Antitrypsin und Contraspin und darüber hinaus eines Chaperons, alphaB-Kristallin, im Verlauf der Erkrankung abnimmt. Reduzierte Expression von alpha1-Antitrypsin und Contraspin konnte in Gehirn, Leber, Herz und Testes nahe dem Endstadium der Erkrankung nachgewiesen werden. Hier ist es wichtig festzustellen, dass die Expressionsabnahme von alpha1-Antitrypsin im Gehirn der Abnahme in der Leber im Herzen und in den Testes vorangeht. Eine verminderte Expression des Chaperons alphaB-Kristallin wurde nur im Gehirn gefunden. Für ein weiteres Protein, das Major Urinary Protein, wurde eine verminderte Expression in der Leber und im Urin von betroffenen Mäusen festgestellt. Damit konnte demonstriert werden, dass die Erkrankung auf Proteinebene auch ein Protein, das im Gehirn von transgenen Mäusen nicht vorkommt, beeinflusst. Bei Untersuchungen am Menschen wurde in drei Gehirnregionen von Postmortem-Gehirnen von Huntington s Chorea Patienten eine veränderte Expression von alpha1-Antitrypsin festgestellt. Wenn gewährleistet werden kann, dass die Konzentration von alpha1-Antitrypsin und alphaB-Kristallin während Huntington s Chorea im Gewebe nicht absinkt, könnte dies vielleicht neuronalen Zelltod verhindern und somit bei der Verzögerung des Krankheitsverlaufs nutzbringend eingesetzt werden. / Huntington disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In an effort to identify proteins involved in processes upstream or downstream of the disease causing huntingtin, the proteome of a well-established mouse model was studied by large-gel 2D electrophoresis. It could be demonstrated for the first time at the protein level that two serin protease inhibitors, alpha1-antitrypsin and contraspin and the chaperone alphaB-crystallin decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver, heart and testes in mice. Reduced expression of alpha1-antitrypsin and contraspin could be detected in the brain, liver heart and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. Reduced expression of the liver specific major urinary proteins not found in the brain, was seen in affected mice, demonstrating that the disease exerts its influence on a protein not present in the brain of transgenic mice at the protein level. When investigating three human brain regions obtained post-mortem from Huntington s disease patients, alpha1-antitrypsin expression was also altered. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington s disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.

<div>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.</div>