Monitoring and mapping the presence and/or intensity of an environmental hazard through space, is an essential part of public health surveillance. Radon, a naturally occurring radioactive carcinogenic gas, is an environmental hazard that is both the greatest source of natural radiation exposure in human populations and the second leading cause of lung cancer worldwide. Concentrations of radon can accumulate in an indoor setting, and, though there is no safe concentration, various guideline values from different countries, organizations and regions provide differing threshold concentrations that are often used to delineate geographic areas at higher risk. Radon maps demarcate geographic areas more prone to higher concentrations but can underestimate or overestimate indoor radon risk depending on the concentration threshold used. The goals of this thesis are to map indoor radon risk in the province of British Columbia, identify areas more prone to higher concentrations and their associations with different radon concentration thresholds and lung cancer mortality trends. The first analysis was concerned with developing a data-driven method to predict and map ordinal classes of indoor radon vulnerability at aggregated spatial units. Spatially referenced indoor radon concentration data were used to define low, medium and high classes of radon vulnerability, which were then linked to regional environmental and housing data derived from existing geospatial datasets. A balanced random forests algorithm was used to model environmental predictors of indoor radon vulnerability and predict values for un-sampled locations. A model was generated and evaluated using accuracy, precision, and kappa statistics. We investigated the influence of predictor variables through variable importance and partial dependence plots. The model performed 34% better than a random classifier. Increased probabilities of high vulnerability were found to be associated with cold and dry winters, close proximity to major river systems, and fluvioglacial and colluvial soil parent materials. The Kootenays and Columbia-Shuswap regions were most at risk. We built upon the first analysis by assessing the difference between temporal trends in lung cancer mortality associated with areas of differing predicted radon risk. We assessed multiple scenarios of risk by using eight different radon concentration thresholds, ranging from 50 to 600 Bq m-3, to define low and high radon vulnerability. We then examined how the following parameters changed with the use of a different concentration threshold: the classification accuracy of each radon vulnerability model, the geographic characterizations of high risk, the population within high risk areas and the differences in lung cancer mortality trends between high and low vulnerability stratified by sex and smoking prevalence. We found the classification accuracy of the model improved as the threshold concentrations decreased and the area classified as high vulnerability increased. The majority of the population were found to live in areas of lower vulnerability regardless of the threshold value. Thresholds as low as 50 Bq m-3 were associated with higher lung cancer mortality trends, even in areas with relatively low smoking prevalence. Lung cancer mortality trends were increasing through time for women, while decreasing for men. We suggest a reference level as low as 50 Bq m-3 is justified for the province. / Graduate

No description available.

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. While Radiation therapy is effective at killing tumor cells or inhibiting their growth initially, development of resistance to treatments and recurrence of tumors are major issues. One of the major goals of Dr. Gewirtz’s laboratory has been to develop strategies to overcome the resistance and attenuate disease recurrence. One of these attempts involve employing vitamin D and its analogs in combination with radiation therapy. Our proposed studies were based on a previous finding where vitamin D and vitamin D analogs such as EB 1089, were shown to enhance the response to radiation in breast cancer through the promotion of autophagy. We extended these studies to non-small cell lung cancer (NSCLC) and were able to validate that 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 does in fact sensitize A549 and H460 cells and prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack the vitamin D receptor or functional p53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when p53 was induced in otherwise p53 null H1299 NSCLC cells. Sensitization by 1,25-D3/ EB 1089 was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by the vitamin D receptor, p53 and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.

Overexpression of oncoprotein MDM2 and mutations of tumor suppressor p53 are frequently observed in human cancers. The NFκB pathway is one of the deregulated pathways in oncogenesis. The overall goal of the project was to study the regulation of NFκB pathway by MDM2 in lung cancer. Our first effort was to determine the frequency of MDM2 overexpression in human lung tumor samples and to identify co-occurring abnormal gene expression by studying the levels of MDM2 and members of NFκB pathway with respect to p53 status. Higher than normal levels of MDM2 were found in approximately 30% of the cancer samples harboring wild-type (WT) and mutant p53. Expression of NFκB2, a mutant p53 inducible gene showed significant statistical correlation with MDM2 in cancer samples that harbored WT p53. A downstream target gene of NFκB2, Bcl2, showed a significant correlation to MDM2 levels, independent of p53 status. Lung cancer samples harboring mutant p53 exhibited elevated levels of NFκB2 though not statistically significant. Our next step was to corroborate findings from lung tumor samples with data from lung cancer cell line harboring WT p53-H460. Consistent with lung tumor samples, ectopic overexpression of MDM2 in H460, showed elevated expression of NFκB2 and Bcl2 with promoter upregulation of NFB2. Silencing of MDM2 proportionally downregulated NFκB2 and Bcl2 in H460 cells. Domain analysis of MDM2 suggested that increase in the NFκB2 promoter activity was not confined to the p53 binding domain of MDM2 suggesting their interaction via p53-dependent and p53-independent mechanisms. A functional cell growth assay showed retarded cell proliferation with downregulation of MDM2. Data from human lung tumor samples and lung cancer cell line suggest that overexpression of MDM2 mediates NFB2 upregulation to confer growth advantage, thus favoring oncogenesis.

Appendices data can be found at: A: http://dx.doi.org/10.15142/T35P49 B: http://dx.doi.org/10.15142/T3201B C: http://dx.doi.org/10.15142/T3X59V D: http://dx.doi.org/10.15142/T3SG6K F: http://dx.doi.org/10.15142/T3NP4N

<p>The small GTPases <italic>HRAS, <italic>NRAS and <italic>KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in <italic>RAS, with 98% of those occurring in the <italic>KRAS isoform. While there have been many advances in the understanding of <italic>KRAS&ndash;driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of <italic>Kras&ndash;driven tumorigenesis in the lung:</p><p>(<italic>I) Despite nearly identical protein sequences, the three <italic>RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, <italic>KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to <italic>HRAS and <italic>NRAS. To determine the consequences of rare codon bias during <italic>de <italic>novo tumorigenesis, we created a knock-in <italic>Kras<super>ex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the <italic>Kras<super>ex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non&ndash;oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in <italic>Kras tumorigenesis.</p><p>(<italic>II) Lung cancer patients exhale higher levels of NO and <italic>iNOS<super>-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes <italic>Kras&ndash;driven lung adenocarcinoma, and NOS inhibition may decrease <italic>Kras&ndash;driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L&ndash;NAME in a genetically engineered mouse model of <italic>Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic <italic>Kras<super>G12D and dominant-negative <italic>Trp53<super>R172H in lung epithelial cells. L&ndash;NAME treatment was provided in the water and continued until survival endpoints. In this model, L&ndash;NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.</p> / Dissertation

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the Master of Science degree. 2 November 2017. / Screening methodologies have identified specific targets that could serve as potential therapeutic markers in cancer drug design, and the Retinoblastoma binding protein 6 (RBBP6) which is predominately expressed in lung and breast cancers is one critical protein identified. This study seeks to understand the 3D structure of RBBP6 domains, with emphasis on cancer. Three of these domains have been studied in this project, i.e. the Domain With No Name (DWNN), RING Finger, and the p53-binding domain. The ubiquitin-like structure of the DWNN has implicated this domain as a ubiquitin-like modifier of other proteins such as p53, whilst the RING Finger domain has intrinsic E3 Ligase activity like MDM2 the prototypical negative regulator of p53. The DWNN and RING Finger domains have resolved solution NMR structures, whilst the p53-binding domain has none. Thus, the first initiative undertaken was to model the RBBP6 p53-binding domain using I-TASSER and eThread-Modeller web-severs. Our results demonstrated that this domain mainly constitutes of alpha-helices and loop structures. Structural quality validations of both I-TASSER and eThread-Modeller models were further assessed using Swiss-Model and ProSA (Protein structure analysis) web-servers. Analyses were focussed on specific statistical parameters (Anolea, DFire, QMEAN, ProCheck and the ProSA Z-score). Results from these analyses show that the first I-TASSER model is the best possible representation of the RBBP6 p53-binding domain depicting minimal deviation from native state. Furthermore, screening and docking studies were performed using Schrödinger-Maestro v10.7: Glide SP and drug-like molecules that would potentially serve as agonist or antagonist of RBBP6 were identified. / MT 2018

A Thesis submitted to the Faculty of Health Sciences (Internal Medicine), University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017 / South Africa has the largest ARV treatment programme in the world, wherein highly active antiretroviral treatment (HAART) has improved the health related quality of life (HRQoL) in HIV/AIDS patients. On the contrary, cancers not previously associated with HIV/AIDS (non-Aids defining cancers; NADCs) have been shown to be increasing, compared to the AIDS defining cancers (ADCs). Lung cancer, as a NADC has been documented in the HIV/AIDS population as a leading malignancy. The poor understanding of the association between ARV drugs and lung cancer places a burden on public health, both globally and in South Africa (SA). Furthermore, the deregulation of the cell-cycle is one of the hallmarks of cancer, including lung cancer. The main aim of this study was to elucidate the effects of HAART components Efavirenz (EFV) and Lopinavir/ritonavir (LPV/r) on cell-cycle related genes in an in vitro lung cancer model. To achieve this, cellular based, molecular and Bio-Informatics approaches were employed. First, the cytotoxic effects of EFV (at 4, 13, 26, 50 μM) and LPV/r (at 10, 32, 50, 80 μM), for 24h, 48h and 72h on normal lung fibroblasts (MRC-5) and lung adenocarcinoma (A549) cells, were evaluated using the Alamar Blue (AB) assay. This was then followed by cell-impedance “xCELLigence” real-time cell analysis (RTCA) assay. This was done to determine the effects of EFV (at 4, 13, 50 μM) and LPV/r (at 10, 32, 80 μM) on cell viability, cell death and proliferation. Cell-cycle analysis using propidium iodide (PI) by Fluorescence-activated cell sorting (FACS) was done to quantify DNA present at each of the cell-cycle stages of the cell-cycle in response to ARV treatment. Subsequently, an apoptosis assay using Annexin V FITC and Propidium iodide (PI) dual staining by FACS was carried out to confirm and quantify the ARVs potential apoptotic effects. Then, 4′,6-diamidino-2-phenylindole (DAPI) staining was used to assess changes in nuclear morphology exerted by the ARVs’ effects. A more in depth interrogation of the cell-cycle was performed using a focussed gene array panel of some 84 human cell-cycle related genes. First, total RNA was isolated from both treated and untreated MRC-5 and A549 cells and reverse transcribed to cDNA for use as template in the PCR array reactions. From the array gene expression results, by convention a ±2 fold up-or-down-regulation was used as the basis of target selection. Following this, a real-time quantitative PCR (RT-qPCR) validation of selected genes of interest was done to quantify and confirm the PCR array results. This was followed by in-silico Bio-informatics analysis to map the molecular pathways regulated by the identified targets. For this purpose, STRING, Database for Annotation, Visualization and Integrated Discovery (DAVID), Reactome and Ingenuity Pathway Analysis (IPA) databases were used. Interestingly, double-edged oncogenic properties of both EFV and LPV/r at different concentrations were identified. The proliferative effects of EFV at 4, 13μM and LPV/r at10 μM, were elucidated, while 26, 50μM of EFV, and 32μM of LPV/r had slight inhibitory effects on cell proliferation. LPV/r at concentrations of 50 and 80μM exerted cytotoxic effects on the cells, as demonstrated by the AB and xCELLigence RTCA assays. Cell-cycle analysis using PI staining, particularly showed cell-cycle arrest at 32μM LPV/r, and a shift to G2/M by 13μM EFV, plasma relevant doses, compared to the untreated cells. An increasing apoptosis percentage was observed with increasing LPV/r concentrations, that is, 80μM LPV/r raised the apoptosis percentage almost two-fold compared to 32μM. This was coupled by necrosis, observed in a time-dependant manner. DAPI staining confirmed loss of nuclear integrity post ARV exposure, suggesting that both EFV and LPV/r impose damage to the genomic DNA. To further assess the observed changes in nuclear morphology, the effects of EFV and LPV/r on the expression of an arrayed panel of human cell-cycle genes in cancer and normal lung cells was determined. Significantly differentially expressed targets were identified and further quantified and confirmed by RT-qPCR. Such targets included ATM, p53, cyclin-dependant kinase inhibitors (CDKIs), such as, p21, aurora kinase B (AURKB), Mitotic Arrest Deficient-Like 2 (MAD2L2) and the apoptosis related gene, caspase 3 (CASP3). Bio-Informatics analyses revealed close and direct protein-protein interactions (PPIs) between these targets, notably, with change in interaction between the gene products involved in DNA repair mechanisms, observed between ARV treated and untreated groups, as illustrated by STRING interactions. DAVID, Reactome and IPA analysis showed changes in expression of genes related to stress and toxicity and DNA damage response genes. In particular, ATM, p53 and its downstream targets such as GADD45A (growth arrest and DNA damage inducible alpha) gene were up-regulated by ARV treatment, while cyclin/CDK activity was down-regulated, resulting in reduced cell proliferation. Thus in summary, both EFV and LPV/r altered the expression levels of cell-cycle related genes, influencing overall cellular health, acting to either inhibit or stimulate cell proliferation. This suggests EFV’s and LPV/r’s proliferative and inhibitory roles in the proliferation of lung cells. Moreover, future directions can include the transfection of lung cells with HIV provirus followed by treatment of the cells with the same ARVs under study. This could be substantiated by including HIV positive patient samples on and off ARV drug treatment with lung cancer, including HIV negative patients with cancer as one of the controls. / MT2018

Les anticorps monoclonaux (Acm) ont révolutionné la prise en charge de nombreuses maladies inflammatoires chroniques. L’administration par voie systémique des Acm ne semble pas optimale pour le traitement des maladies respiratoires. Ce travail a eu pour objectif d’étudier le comportement des anticorps après leur administration par voie pulmonaire. Le cetuximab, un anticorps anti-EGFR, a été étudié dans un modèle murin de tumeur pulmonaire bioluminescente et chez le macaque. Le G6-31, un anticorps anti-VEGF, a été étudié dans un modèle murin transgénique de tumeur pulmonaire spontanée. Les résultats montrent que les anticorps administrés par voie pulmonaire sont efficaces, persistent durablement dans le poumon et passent peu dans le compartiment sanguin. L’administration par voie pulmonaire d’Acm pourrait donc augmenter l’index thérapeutique de ces médicaments. Ces résultats ouvrent la voie pour l’administration par voie pulmonaire d’Acm dans les pathologies respiratoires en général. / Monoclonal antibodies (mAbs) modified profoundly the treatment of many chronic inflammatory diseases. Systemic administration of mAbs does not seem to be optimal for the treatment of respiratory diseases. This work aims at studying the fate of mAbs after pulmonary delivery. The lung delivery of Cetuximab, an anti-EGFR antibody, was assessed in an orthotopic mouse model of lung tumor and in macaques. G6-31, an anti-VEGF antibody, was studied in a transgenic murine model of spontaneous lung tumors. The results showed that mAbs, after pulmonary delivery, were pharmacologically effective, persisted durably into the lung and passed slowly into the bloodstream. Pulmonary delivery of mAbs might increase the therapeutic index of these drugs. Altogether, these results open the way for the pulmonary administration of mAbs in respiratory disorders

O câncer é uma doença que pode ser caracterizada pelo crescimento desordenado de células malignas, que podem se disseminar pelo organismo tornando-se agressivas e até mesmo incontroláveis. O câncer de pulmão é uma doença caracterizada por uma baixa sobrevida, em torno de 15% em cinco anos. O presente trabalho avaliou o perfil epidemiológico de câncer de pulmão na região de Governador Valadares e os fatores de risco associados, determinando a frequência do câncer de pulmão no período de 2010 a 2015. É um estudo observacional, analítico e retrospectivo com característica quantitativa que foi realizado a partir do banco de dados do Núcleo de Especialistas em Oncologia, foram analisados 7035 prontuários de pacientes preenchidos eletronicamente pelas equipes de saúde. A amostra selecionada corresponde a 227 pacientes com diagnóstico de câncer de Pulmão e suas sub localizações anatômicas sendo aplicado em dados estatísticos e encontrando alguns resultados relevantes como: relação entre sexo e Histórico familiar onde 65 pacientes afirmaram que possuíam casos de câncer na família. Outro fator é o consumo de bebida alcoólica e câncer de pulmão onde 27,65% entram em contato com a bebida alcóolica. Foi verificado que 161 pacientes entraram em contato com o tabaco diretamente ao longo da vida desenvolvendo o câncer de pulmão. Outro dado importante que 176 pacientes vieram do Sistema Único de Saúde, tornando-se uma importante via de atendimento aos pacientes com câncer de pulmão. / Cancer is a disease that can be characterized by the disordered growth of malignant cells, which can spread through the body becoming aggressive and even uncontrollable. Lung cancer is a disease characterized by a low survival rate, around 15% in five years. The present study aims to evaluate the epidemiological profile for lung cancer in the region of Governador Valadares and associated risk factors, determining the frequency of lung cancer in the period from 2010 to 2015. It is an observational, analytical and retrospective study with a quantitative trait which was carried out from the database of the Nucleus of Specialists in Oncology. 7035 medical records of patients, filled electronically by health teams were analyzed. The selected sample corresponds to 227 patients diagnosed with Lung cancer and its sub-anatomical locations being applied in statistical data and finding some relevant results such as: relationship between sex and family history where 65 patients stated that they had cases of cancer in the family. Another factor is the consumption of alcoholic beverage and lung cancer where 27.65% come in contact with the alcoholic beverage. It was verified that 161 patients came into contact with tobacco directly throughout their lives developing lung cancer. Another important fact is that 176 patients came from the Unified Health System, becoming an important route of care for patients with lung cancer.