LUNG CANCER IN NEPAL: THE ROLE OF TRADITIONAL TOBACCO PRODUCTS AND COMBUSTION RELATED HOUSEHOLD AIR POLLUTION

The burden of chronic diseases such as cancer is increasing in low and middle income countries around the globe. Nepal, one of the world’s poorest countries, is no exception to this trend, with lung cancer as the leading causes of cancer deaths. Despite this, limited data is available on the environmental and behavioral risk factors that contribute to the lung cancer etiology in Nepal. The objectives of this dissertation are to: 1) investigate the ethnic differences in consumption of local tobacco products and their role in lung cancer risk in Nepal; 2) evaluate urinary metabolite of 1,3-butadiene as a biomarker of exposure to combustion related household air pollution (CRHAP); 3) investigate the association between CRHAP exposure and lung cancer risk using urinary metabolite of 1,3-butadiene as a biomarker of exposure; 4) investigate the association between CRHAP exposure and lung cancer risk using questionnaire based measure of exposure. Lung cancer cases (n=606) and frequency matched controls (N=606) were recruited from B.P. Koirala Memorial Cancer Hospital. We obtained biological samples and information on lifestyles including cooking habits and type of fuels used. We used liquid chromatograph tandem mass spectrometer (LC-MS/MS) to quantify urinary metabolites of 1,3-butadiene in urine samples. We employed a combination of logistic and linear regression models to detect any exposure-disease associations while controlling for known confounding variables. Overall, we found that ethnic groups in Nepal use different tobacco products that have different differing cancer potency -we observed the highest odds ratios for the traditional tobacco products. The biomarker analysis showed strong evidence that monohydroxybutyl mercapturic acid is associated with biomass fuel use among participants. However, we did not find significant association between urinary MHMBA and lung cancer risk. When we used questionnaire based measure of exposure to household air pollution, we observed significant, dose-response associations between CRHAP exposure and lung cancer risk, particularly among never-smokers. Our results show that important role of local tobacco products in lung cancer risk in Nepal. Furthermore, we demonstrate that CRHAP exposure is a risk factor for lung cancer risk, independent of tobacco smoking.

Characterization of Borrelia burgdorferi Gene Product BBD18 for Its Role(s) in Pathogen Biology and Infectivity

bbd18 is a differentially expressed Borrelia burgdorferi gene that is transcribed at almost undetectable levels in spirochetes grown in vitro but dramatically upregulated during tick infection. The gene also displays low yet detectable expression at various times in tissues of murine hosts. As the gene product bears no homology to known proteins, its biological significance remains enigmatic. To understand the gene function, we created isogenic bbd18-deletion mutants as well as genetically-complemented isolates from an infectious wild-type B. burgdorferi strain. Compared to parental isolates, bbd18 mutants - but not complemented spirochetes - displayed slower in vitro growth. The bbd18 mutants also reflect significantly reduced ability to persist or remain undetectable both in immunocompetent and SCID mice, yet were able to survive in ticks. This suggests BBD18 function is essential in mammalian hosts but redundant in the arthropod vector. Notably, although bbd18 expression and in vitro growth defects are restored in the complemented isolates, their phenotype is similar to the mutants - being unable to persist in mice but able to survive in ticks. Despite low expression in cultured wild-type B. burgdorferi, bbd18 deletion downregulated several genes. Interestingly, expression of some, including ospD and bbi39, could be complemented, while that of others could not be restored via bbd18 re-expression. Correspondingly, bbd18 mutants displayed altered production of several proteins, and similar to RNA levels, some were restored in the bbd18 complement and others not. To understand how bbd18 deletion results in apparently permanent and noncomplementable phenotypic defects, we sought to genetically disturb the DNA topology surrounding the bbd18 locus without deleting the gene. Spirochetes with an antibiotic cassette inserted downstream of the gene, between bbd17 and bbd18, were significantly attenuated in mice, while a similar upstream insertion, between bbd18 and bbd19, did not affect infectivity, suggesting that an unidentified cis element downstream of bbd18 may encode a virulence-associated factor critical for infection.

Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation

Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.