INTRODUCTION: Recent epidemiological studies have shown that Diesel Engine Exhaust (DEE) exposure is associated with lung cancer. Well recognized exposures, such as smoking, have long been known to cause lung cancer, and the mechanisms by which the disease occurs have been closely investigated. However, there is very little information regarding the mechanisms by which chronic DEE exposure leads to a disease outcome. It has also been shown that transcriptomic changes in the deeper portions of the airway may be detectable in the more proximal parts. The goal of this study was to assess transcriptomic alterations in the nasal epithelium of DEE exposed factory workers to better understand the physiologic effects of DEE and how chronic exposure may lead to disease. METHODS: Nasal epithelium brushings were obtained from 41 subjects who work in a factory with DEE exposure, and 38 comparable control subjects who work in factories without DEE exposure. The median Elemental Carbon (EC) levels of exposed individuals was 60.7g/m3, with a range of 17.2-105.4 g/m3, while the median of EC levels of unexposed controls was 10.87g/m3, with a range of 9.89-12.55g/m3. RNA was isolated from nasal epithelial cells, and profiled for gene expression using Affymetrix Human Gene 1.0ST microarray chips. Linear modeling was used to detect differential expression between DEE exposure and controls. Pathway enrichment in differentially expressed genes was assessed using EnrichR. GSEA provided comparisons between the genes known to be differentially expressed due to smoking, and the genes that were found in our data to be differentially expressed due to smoking or DEE. A linear modeling approach was further used to investigate the effects of the interaction between smoking status and DEE exposure, and boxplot analysis was used to explore the interaction effect. RESULTS: We found 225 genes whose expression is associated with DEE exposure at FDR q < 0.25, after adjusting for smoking status. Within this set of genes, we observed increased expression of genes involved in the oxidative stress response, cell cycle, and protein modification, as well as genes associated with the AhR pathway and the Nrf2-mediated xenobiotic metabolism response. Additionally, decreased expression of genes involved in transmembrane transport, such as CFTR and the solute carrier family genes was also found. Furthermore, we discovered 8 genes at FDR q < 0.25 that have altered expression due to the interaction of DEE and smoking status, suggesting a synergistic relationship between the effects of these exposures on some aspects of the physiological response. For these genes, the effects of DEE were generally more dramatic in never smokers. CONCLUSIONS: The transcriptomic alterations we identified may help provide insight into the underlying mechanisms of DEE carcinogenicity. The relationship between cigarette smoke exposure and DEE exposure may provide more information about how chronic DEE exposure leads to lung cancer and other respiratory diseases.

Recurrent airway obstruction (RAO) and Rhodococcus equi (R. equi) pneumonia are two equine respiratory diseases. RAO is an allergic asthma like disease of the middle-aged horses while the R. equi pneumonia affects only young foals. Respiratory disease is considered among the major causes of economic loss to the equine industry and tops the priority list for research that will focus on preventative and diagnostic facets of such disease. The objective of this research was to investigate the effect of antigen exposure and remission (via allergen avoidance and/or drug) on chronically affected RAO horses. Additionally, we also wanted to understand the changes in equine neonatal immune system due to R. equi exposure and identify molecular biomarkers for early disease screening. Various biological samples (lung tissue for the RAO study and blood leukocytes and nasal epithelial cells for the R. equi study) were used to extract ribonucleic acid (RNA). Complimentary deoxyribonucleic acid (cDNA) obtained from RNA was used to perform microarray hybridization experiments. Our findings suggest that compared to control horses allergen exposure leads to an elevated protein synthesis and inflammation that contributes to aggravation of symptoms and airway changes. We found that allergen avoidance controls inflammation and causes an improvement in lung function and other chronic features of RAO. The drug administration led to an accelerated remission in the chronic RAO features; a complete remission could however not be achieved. Hence it appears that although not a complete resolution, but allergen avoidance and drugs will help in a better management of chronic RAO symptoms. Our results suggest that the neonatal immune system is capable of initiating a protective immune response through birth up to 8 weeks of age. However there are also processes present that may be counter-productive to the host. Induction of such suppressive mechanisms may be a result of bacterial modulation of the host immune response or a result of immature host immune system. We also identified molecular biomarkers that will have the potential to screen foals for R. equi pneumonia soon after birth and before the onset of clinical symptoms. The research findings of this study will improve the current understanding of the two equine diseases.

Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa.

In a normal microarray classification problem there will be many genes, on the order of thousands, and few samples, on the order of tens. This necessitates a massive feature space reduction before classification can take place. While much time and effort has gone into evaluating and comparing the performance of different classifiers, less thought has been spent on the problem of efficient feature space reduction. There are in the microarray classification literature several widely used heuristic feature reduction algorithms that will indeed find small feature subsets to classify over. These methods work in a broad sense but we find that they often require too much computation, find overly large gene sets or are not properly generalizable. Therefore, we believe that a systematic study of feature reduction, as it is related to microarray classification, is in order. In this thesis we review current feature space reduction algorithms and propose a new, mixed model algorithm. This mixed-modified algorithm uses the best aspects of the filter algorithms and the best aspects of the wrapper algorithms to find very small yet highly discriminant gene sets. We also discuss methods to evaluate alternate, ambiguous gene sets. Applying our new mixed model algorithm to several published datasets we find that our new algorithm outperforms current gene finding methods.

Wood formation (xylogenesis) is a critical developmental process for all woody land plants. As an initial step to understand the molecular basis for temporal and spatial regulation of xylogenesis and the effect of the expression of individual genes on physical and chemical properties of wood, microarray and realtime RTPCR analyses were performed to monitor gene expression during xylogenesis under various developmental and environmental conditions. The specific objectives established for this study were: Objective 1. Microarray analysis of genes preferentially expressed in differentiating xylem compared to other tissues of loblolly pine (see Chapter II); Objective 2. Microarray analysis of seasonal variation in gene expression for loblolly pines (Pinus taeda L.) from different geographical sources (see Chapter III); Objective 3. Realtime RTPCR analysis of loblolly pine AGP and AGPlike genes (see Chapter IV). Based on the results from this study, candidate genes may be further studied for association with significant traits, used for genetic modification of wood properties, or included in future studies to further examine the molecular mechanisms of wood formation.

Many tests have been developed for comparing means in a two-sample scenario. Microarray experiments lead to thousands of such comparisons in a single study. Several multiple testing procedures are available to control experiment-wise error or the false discovery rate. In this dissertation, individual two-sample tests are compared based on accuracy, correctness, and power. Four multiple testing procedures are compared via simulation, based on data from the lab of Dr. Rajesh Miranda. The effect of sample size on power is also carefully examined. The two sample t-test followed by the Benjamini and Hochberg (1995) false discovery rate controlling procedure result in the highest power.

<p>Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.</p>

Glycomics is the systematic study of glycosylation in the context of a whole cell or organism. Glycosylated proteins are estimated to make up 50% of all proteins and cover the outside of the cell. Functional roles in glycosylation have been noted in pathogenesis, metastasis, and embryogenesis. However, the structure of these carbohydrates has been difficult to study due to the chemical nature of carbohydrates. Lectins, carbohydrate binding proteins excluding antibodies and enzymes, can be utilized to study glycosylation in a high throughput manner using a microarray format. Glycans, the carbohydrates attached to a protein or lipid, are not synthesized from a template. They are added co- or post-translationally by a concerted set of enzymes in the secretory pathway. In addition, the glycan structures may be altered by metabolism or trafficking. Cell type specific glycosylation has long been hypothesized due to observations of bacteria homing to tissues. We use lectin microarray technology to define the glycosylation in a subset of the NCI-60, a set of cell lines from different tissues. Using a customized gene expression microarray, we identify cell type dependent glycosylation genes and observe evidence of cell type dependent spliceforms for an O-glycosylated mucin. Data from the lectin microarray and a published gene expression data set were integrated using Generalized Singular Value Decomposition (GSVD), a linear matrix decomposition method. We have successfully decomposed the data into 3 cell type dependent meta patterns that segregate by glycosylation family. Correlation projection of the genes and subsequent gene ontology enrichment suggests that genes in different pathways covary with the types of glycosylation. An inverse relationship was revealed for the N- glycosylation pattern between the SVD of the lectins and the GSVD of the genes and lectins together. Whereas, the relationship was correlative for O-glycosylation, which was clearly illustrated in biplots. This work argues that types of glycosylation are regulated by different mechanisms in different cell types. / text

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa.