Das Lungenkarzinom ist weltweit die häufigste Krebstodesursache. Ein möglicherweise erfolgsversprechender Angriffspunkt in der Therapie stellt die Tumorangiogenese dar, welche sich immunhistochemisch quantifizieren lässt. In der vorliegenden Arbeit wurde CD31, welcher als Marker für vorhandene Neoangiogenese fungiert, sowie PEDF als Antiangiogensesfaktor beschrieben und deren Einfluss auf die Gefäßbildung untersucht. Die Zielsetzung der Arbeit war es zu untersuchen, ob Unterschiede im Expressionsmuster bei einem Plattenepithel- und Adenokarzinom der Lunge zu sehen sind, ob eine Korrelation in der Expression von CD31 und PEDF zu erkennen ist und ob eine Aussage zur Prognose anhand der Expression möglich ist. Das Kollektiv umfasste klinisch anotierte Proben von 42 Patienten mit einem Plattenenpithelkarzinom der Lunge und 27 Patienten mit einem Adenokarzinom der Stadien Ia-IIIB. Sowohl die CD31 als auch die PEDF-Expression waren zwischen den Tumorentitäten nicht signifikant unterschiedlich. Im Expressionsvergleich von CD31 und PEDF zeigte sich beim Kollektiv der Plattenepithelkarzinome eine signifikante negative Korrelation bezogen auf CD31 und PEDF, gemessen anhand der Intensität. Beim Kollektiv der Adenokarzinompatienten zeigt sich eine positive Korrelation bezogen auf das CD31-Signal und der PEDF-Fläche. Für die Untersuchung zur Überlebensanalyse wurde das Kollektiv der Patienten, welche an einem Plattenepithelkarzinom erkrankt waren betrachtet. Die in der Literatur beschriebenen klassischen Einflussgrößen wie Tumorgröße, Lymphknotenstatus und Tumorstadieneinteilung auf die Überlebensraten konnten in dieser Arbeit bestätigt werden, allerdings erbrachte lediglich die Untersuchung der Tumorgröße eine statistisch signifikante Aussage. Hier konnte gezeigt werden, dass Patienten mit einem pT1-Tumor eine deutlich bessere Überlebensprognose haben als Patienten mit einem Tumor der Größe T2 oder größer (p=0.029). In der vorliegenden Studie ergab eine hohe CD31-Expression ein statistisch signifikant besseres Überleben der Patienten mit einem Plattenepithelkarzinom (p=0.038). Die 5JÜR der Patienten mit hoher CD31-Expression beträgt 67%, die mit niedriger 37%. Die Untersuchungen zur PEDF-Fläche als auch zur PEDF-Intensität ergaben keine Korrelation auf das Überleben und somit auf die Prognose.

Cigarette smoke (CS) exposure is the leading cause of preventable death in the United States contributing to over 480,000 deaths a year with over 300 billion dollars in CS related costs spent per year. While the dangers of CS exposure have been studied and characterized for decades being largely attributed to reactive oxygen species and oxidative stress, increasing evidence suggests that reactive aldehydes in CS, specifically the α,β-unsaturated aldehyde acrolein, are responsible for many of the negative pathologies associated CS exposure. Previous work has shown that acrolein can bind directly to a number of cellular proteins containing redox sensitive cysteine residues. The non-receptor tyrosine kinase Src contains nine cysteine residues and is known to be activated in response to CS and oxidative stress. Despite being the first characterized and one of the most widely studied oncogenes, the exact mechanism for Src activation remains unclear. In the current studies we examined the effects of acrolein on Src activation and the resulting outcomes on the lung epithelium in an effort to better understand how reactive electrophiles in CS contribute to the development of lung disease. To determine the effects of acrolein on Src activation, we first exposed NCI-H292 cells to acrolein and measured activity by western blot. We observed an increase in Src activity detected by an increase in Src phosphorylation at Y416 and an increase in phosphorylation of Src target proteins Caveolin1 and p120. Interestingly the increase in activation occurred without dephosphorylation of the inhibitory phosphorylated tyrosine Y527. Using biochemical-labeling strategies we identified Src as a direct target of acrolein adduction in vitro and in vivo, and we used mass spectrometry to confirm acrolein adduction to cysteine residues C245, C277 and C487, all which have been implicated in a redox dependent Src activation mechanism. Furthermore, increased Src activity following acrolein exposure was confirmed using an in vitro kinase activity assay and recombinant Src in a cell free system. To study the effects of acute acrolein exposure on lung epithelial function we exposed cultured mouse tracheal epithelial cells (MTECs) to acrolein and show impaired epithelial barrier function, measured by a decrease in trans epithelial resistance (TER) and increased epithelial permeability to FITC-dextran, which could be prevented using the Src inhibitor PP2. Src inhibition also attenuated acrolein-induced loss of E-cadherin and ZO-1. Acute exposure of C57BL/6 mice to acrolein (5 ppm for 4 hrs) led to increased epithelial permeability, measured by enhanced leakage of i.v. injected FITC-dextran into the airspaces, and induction of HO-1 in the lung while chronic acrolein exposure resulted features of epithelial to mesenchymal transition including a reduction of E-cadherin, increased vimentin, increased expression of MMP9 and increased collagen deposition. Chronic acrolein exposure in vitro resulted in a reduction of E-cadherin that could be prevented using the Src inhibitor AZD0530. Together our studies demonstrate that Src is a direct target for acrolein and plays an important role in epithelial alterations due to acrolein exposure. This work provides further insight into a potential mechanism involved in the development of cigarette smoke related disease and could provide a potential target for novel therapeutics.

Prognosis for lung cancer patients remains poor. For those receiving radiation therapy, local control and survival have been shown to improve with increased doses; however, deliverable dose is often limited by associated toxicity. Therefore, methods that reduce dose to normal tissues and allow isotoxic escalation are desirable. Adaptive radiation therapy seeks to improve treatment by modifying the initial plan throughout delivery, and has been shown to decrease normal tissue dose. Studies to date suggest a trend of increasing benefit with increases in replanning frequency; however, replanning is costly in terms of workload and past studies implement at most weekly adaptation. The purpose of this thesis is to quantify the benefit associated with daily replanning and characterize the tradeoff between replanning frequency and adaptive benefit. A software tool is developed to facilitate planning studies and to introduce complimentary methods for evaluating adaptive treatments. Synthetic images and contours are xii generated for each fraction of a typical fractionation schedule using principal component analysis and a novel method of sampling coefficients that preserves temporal trends in the data (e.g. tumor regression). Using the synthetic datasets, a series of adaptive schedules ranging from no adaption to daily replanning are simulated and compared to quantify adaptive benefits and characterize tradeoffs with frequency. Daily replanning resulted in significant reductions in all normal tissue planning metrics when compared to no adaptation, and incremental reductions were observed with each increase in replanning frequency while the magnitude of average reductions decreased with each step. Modest correlation between absolute change in planning target volume over the course of treatment and reductions in both mean lung dose and mean esophageal dose were observed.

Les kallicréines tissulaires humaines (KLK) ont récemment émergé comme une famille de protéases à serine pouvant jouer un rôle important dans la tumorigenèse. Le but de cette étude a été de mieux comprendre comment KLK6 et KLK12 pourraient intervenir dans la physiopathologie pulmonaire. Nous avons démontré qu’une expression ectopique de KLK6 induisait la prolifération des cellules A549 d’une manière dépendante de son activité enzymatique, une résistance à l’apoptose, ainsi qu’une translocation nucléaire de la β-caténine. Nous avons également montré une voie de signalisation impliquée dans la prolifération induite par KLK6 impliquant PAR2, EGFR et ERK. Nous avons identifié de nouveaux subtrats pour KLK12 : les CCN. Nous avons démontré que le clivage de CCN1 et CCN5 par KLK12 limitait leurs fixations avec le VEGF, BMP2 et le TGF-β1. / Recently, human tissue kallikreins (KLK) emerged as a new family of serine proteases which might play a major role in the tumorigenesis. The project aims at determining the contribution of KLK6 and 12 in lung pathophysiology. We showed that ectopic KLK6 promoted A549 cell proliferation in a protease activity-dependant manner, inhibited cell apoptosis and induced β-catenin nuclear translocation. Furthermore, this study uncovered a signaling pathway mediated by KLK6 in promoting A549 cell proliferation trough activation of the PAR2-EGFR-ERK pathway.We have also identified novel substrates of KLK12, the CCN family. We reported that KLK12-mediated proteolysis of CCN1 and CCN5 can reduce or abolish the binding of VEGF, BMP2, and TGF-β1.

Cystic Fibrosis (CF) is one of the most common heriditary diseases. This congenital condition, caused by CFTR gene mutation, affects gastrointestinal and respiratory system especially. The affection of respiratory system is considered the most serious life-threatening symptom. Pacients suffer from reccurent infections proceeding to the development of chronic inflammation and progressive pulmonary tissue destruction. A typical specific microoganism colonizating pulmonary tissue of thouse suffering from CF is recognised as Pseudomonas aeruginosa. Pulmonary infections caused by this microorganism are the most often cause of death in patients suffering from CF. Antiobiotics are the first-line therapy of this condition currently. Nevertheless, the need to find alternatives occurs due to antibiotics resistance development. Passive immunization by specific hen-egg-yolk antibodies against P. aeruginosa is a possible alternative. An observation of IgY influence on a bacterial adhesion to pulmonary epithelial cells required an appropriate model. In this context an appropriate adhesion testing method based on P. aeruginosa and pulmonary epithelial cells visualisation was searched. At first bacterial cells labelling was tested by CellTracker, resazurin, FITC and consequently PKH 26. P. aeruginosa fluorescent...

This study determines effects of oxygen levels on morphology and VEGF expression of developing chicken lungs following incubation in normoxia (21% O2), hypoxia (15% O2) or hyperoxia (30% O2), until developmental days 16 or 18. Lung morphology was assessed using light microscopy, while VEGF expression was determined with ELISA. In hypoxia, the proportion of parabronchial tissue and parabronchi including lumina increased from day 16 to 18 (61 to 68% and 74.2 to 82.2%, respectively). Non-parabronchial tissue was higher in hypoxia than in hyperoxia on day 16 (26 to 20%). However, by day 18, there were no differences between groups. VEGF expression was 33% higher in hypoxia than in hyperoxia on day 16 (736 vs. 492 pg/ml). On day 18, VEGF expression was 43% higher in hyperoxia than in normoxia (673 to 381pg/ml), and remained elevated by 40% in hypoxia over normoxia (631 pg/ml). VEGF may be a mechanism by which parabronchial tissue is stimulated from day 16 to 18 following exposure to chronic hypoxia.

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as an E3 ubiquitin ligase due to the presence of a RING finger domain and also assumed to have a regulatory role of p53 due to the presence p53BD through MdM2, although no substrate has been identified up to now. RBBP6 gene mutants are reported to be resistant to apoptosis inducers, which led to a belief that mutation of this gene might result in the development of lung cancer. Earlier localization and expression studies have shown that RBBP6 expression and apoptosis levels are indirectly proportional. The purpose of this study is to establish the expressional pattern of the RBBP6 gene in lung cancer at both mRNA and protein levels. The objective is also to characterize the role of this gene and apoptosis in diverse lung diseases. An understanding of the role of RBBP6 in the development of lung diseases may lead to insights into developing new therapeutic measures for those lung diseases in which apoptosis plays a prominent part. This thesis elucidate the possible role of RBBP6 in lung cancer and apoptosis, to establish tissue distribution and expression levels of RBBP6 at protein and mRNA levels in lung cancer by immunocytochemistry (ICC), in situ hybridization (ISH) and confirm findings by quantitative RT-PCR. RBBP6 mRNA transcripts were expressed in the cytoplasm of normal and tumour lung epithelium. In general, expression was highest in the cytoplasm of welldifferentiated carcinoma and invasive carcinoma that showed signs of cells undergoing mitosis. Immunolabelling results further showed high level of expression in all lung cancer types except in Small and large cell carcinomas. The immunolabeling were confirmed by ISH experiments and RT-PCR. In relation to p53, RBBP6 mRNA expression was higher in lung cancer cell lines that had p53 silenced and apoptosis induced by TRAIL and camptothecin. There was no notable change in the levels of p53 expression following RBBP6 silencing and apoptosis induction. However, there was a little correlation between RBBP6 expression and apoptosis levels in both lung cancer tissues by TUNEL and lung cancer cell line following apoptosis induction by TRAIL. The ratio of Bax/Bcl-2 was seen to be upregulated following p53 and RBBP6 silencing after apoptosis induction. The most common mutation notable after RBBP6 DNA sequencing was point mutations where only single nucleotide was mutated and mostly they were observed in lung cancer tissues. This was the first demonstration that RBBP6 is expressed in lung cancers. Because of the ubiquitin-like nature of the protein and its localized up-regulation and corresponding proapoptotic activity in lung cancer cells, it is possible that further characterization of this gene could lead to its manipulation as a diagnostic marker and a potential therapeutic target for cancer treatment.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg for the degree of Master of Science in Medicine. / A study was undertaken to ascertain whether a low intensity, long term home walking exercise programme could produce physiological changes in patients with chronic obstructive pulmonary disease (COPD). Subjective psychological effects of such a programme were also evaluated. (Abbreviation abstract) / AC2017

PhD, Faculty of health Sciences, University of the Witwatersrand, 2000

Stereotactic Body Radiation Therapy (SBRT) is a modern precision radiation therapy to deliver the dose in 1 to 5 fractions with high target dose conformity, and steep dose gradient towards healthy tissues. The dose delivered is influenced by the leaf width of the MLC, especially in case of SBRT. Treatment plans with high definition (HD) MLC having leaf-width 2.5 mm and normal MLC having leaf-width 5 mm, were compared to quantify dosimetric and radiobiological parameters. Dosimetric parameters conformity indices (CI), gradient indices (GI) and heterogeneity indices (HI) were compared. The radiobiological parameters were evaluated by normal tissue complication probability (NTCP) and tumor control probability (TCP) based on the equivalent uniform dose (EUD). The results show that there is dosimetric and radiobiological merit of the HD MLC over the normal MLC. However, the improvement is not consistent with all the plans and thus further research is required prior to conclusion. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection