Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Programmed Cell Death 4 (PDCD4) is a known tumour suppressor, lost in carcinomas of the breast, prostate, colon, lung and ovary. This study found significantly reduced levels of PDCD4 mRNA and protein in both primary patient oral squamous cell carcinomas (OSCCs) and OSCC cell lines. Moreover, lower PDCD4 mRNA levels were significantly correlated with nodal metastasis (P=0.019). To determine the functional significance of PDCD4 down-regulation in OSCC we asked whether PDCD4 played a role in invasion. In fact, over-expression of PDCD4 decreased invasion of OSCC lines. We then sought to determine a mechanism for PDCD4 down-regulation in OSCC. Previous studies in breast and colon carcinomas suggested that reduced PDCD4 expression was due to over-expression of miR-21. Interestingly, miR-21 was inversely correlated to PDCD4 mRNA (P=0.002) and PDCD4 protein (P<0.001) levels in OSCC patient samples. Moreover, we found that miR-21 directly regulated PDCD4 protein expression in OSCC cell lines. This is the first report in OSCC that demonstrates that PDCD4 is down-regulated by miR-21 and may play a role in OSCC invasion.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Lung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.NyckelordLung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.Nyckelord

This dissertation investigated locational risks to ecosystem health associated with proximity to industrial complexes. The study was performed at the behest of ranchers and citizens living and working down-prevailing wind from the Formosa Plastics, Inc. and ALCOA facilities located in Calhoun County, Texas. Concerns expressed were for potential genotoxicity resulting from exposure to complex chemical mixtures released by the facilities. Exposure assessment of the marine environment was performed with sediments and oysters from Lavaca Bay being analyzed. Numerous chemicals were found to be present at concentrations considered likely to result in adverse responses in exposed populations. Bayesian geostatistical analysis was performed to determine if the concentrations were affected by a spatial process. Mercury and polycyclic aromatic hydrocarbons were the most notable of the chemicals found to be present at elevated concentrations and affected by a spatial process. Evaluation of maps generated from spatial modeling revealed that proximity to ALCOA resulted in elevated risks for exposure to harmful concentrations of pollutants. Genotoxicity was measured in two sentinel species. Oysters (Crassostrea virginica) were utilized for evaluation of the marine environment and cattle (Bos taurus and Bos taurus crossbred cattle) were chosen for evaluation of the terrestrial environment. Chromosomal aberration analysis was performed on oyster hematocytes. Analysis of the results failed to demonstrate the presence of an important generalized spatial process but some specific locations close to the ALCOA plant had elevations in this measure of genotoxicity. Stress as measured by the lysosomal destabilization assay was also performed on oyster hematocytes. These results were found to be affected by a significant spatial process with the highest degree of destabilization occurring in close proximity to ALCOA. Genotoxicity in cattle was evaluated with the single cell gel electrophoresis assay and chromosomal aberration analysis. Bayesian geostatistical analyis revealed the presence of important spatial processes. DNA-protein cross-linkage was the most notable with a strong indication of increased damage down-prevailing wind from the industrial complexes. Results indicated that proximity to industrial facilities increased the risk for harmful exposures, genotoxicity, and lysosomal destabilization.

No description available.

Cave (Petrochelidon fulva) and cliff swallows (Petrochelidon pyrrhonota) were collected along the Rio Grande and evaluated as potential indicators of environmental contamination. The Rio Grande receives toxic substances from agricultural, industrial, municipal, and non-point sources; consequently, high levels of contaminants have been detected in birds, mammals, fishes and sediments. Swallows were obtained from 8 sites between Brownsville and El Paso, as well as from a reference site in Burleson County, 320 miles north of the nearest site of the Rio Grande. Blood samples were analyzed by flow cytometry, a technique that allows the detection of DNA damage in blood and other tissues. Plasma samples were analyzed for thyroid hormones using a radioimmunoassay technique. Organochlorines and trace metal analysis was limited to a few samples. DDE and PCB levels were below levels known to cause reduced hatching, embryo mortality, and deformities, Hg, Pb, and As were below detection, and Se, Ni and Cr concentrations were lower than levels known to cause harm in birds. Neither species showed sex-related differences in chromosome damage. Cave swallows from the Del Rio area had the highest levels of DNA variation, which may be indicative of DNA damage, possibly from PAHs exposure. Previous studies indicate that sediment samples from tributaries near Del Rio have high levels of chromium compared to other sites along the Rio Grande. A significant increase in DNA variation between sampling years was detected in cave swallows from Llano Grande Lake. Wildlife samples collected from Llano Grande Lake have recorded high levels of DDE and PCBs; in addition, this urban/agricultural contaminant sink appears to be affected by PAH exposure. T3 levels were below the detection limit of the radioimmunoassay. There were no gender related differences in T4 levels in cave swallows. Cave swallows sampled from Laredo had significantly higher T4 levels than those from birds at other sites during 1999. It was not possible to determine thyroid hormone disruption in plasma samples. Thyroid hormone and flow cytometry data were useful in establishing baseline data. Areas of concern based on genotoxic data include Llano Grande Lake, Del Rio, and El Paso.

This dissertation investigated locational risks to ecosystem health associated with proximity to industrial complexes. The study was performed at the behest of ranchers and citizens living and working down-prevailing wind from the Formosa Plastics, Inc. and ALCOA facilities located in Calhoun County, Texas. Concerns expressed were for potential genotoxicity resulting from exposure to complex chemical mixtures released by the facilities. Exposure assessment of the marine environment was performed with sediments and oysters from Lavaca Bay being analyzed. Numerous chemicals were found to be present at concentrations considered likely to result in adverse responses in exposed populations. Bayesian geostatistical analysis was performed to determine if the concentrations were affected by a spatial process. Mercury and polycyclic aromatic hydrocarbons were the most notable of the chemicals found to be present at elevated concentrations and affected by a spatial process. Evaluation of maps generated from spatial modeling revealed that proximity to ALCOA resulted in elevated risks for exposure to harmful concentrations of pollutants. Genotoxicity was measured in two sentinel species. Oysters (Crassostrea virginica) were utilized for evaluation of the marine environment and cattle (Bos taurus and Bos taurus crossbred cattle) were chosen for evaluation of the terrestrial environment. Chromosomal aberration analysis was performed on oyster hematocytes. Analysis of the results failed to demonstrate the presence of an important generalized spatial process but some specific locations close to the ALCOA plant had elevations in this measure of genotoxicity. Stress as measured by the lysosomal destabilization assay was also performed on oyster hematocytes. These results were found to be affected by a significant spatial process with the highest degree of destabilization occurring in close proximity to ALCOA. Genotoxicity in cattle was evaluated with the single cell gel electrophoresis assay and chromosomal aberration analysis. Bayesian geostatistical analyis revealed the presence of important spatial processes. DNA-protein cross-linkage was the most notable with a strong indication of increased damage down-prevailing wind from the industrial complexes. Results indicated that proximity to industrial facilities increased the risk for harmful exposures, genotoxicity, and lysosomal destabilization.