Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Oxidative stress plays an important role in the pathogenesis of CVD. Heart failure is the end point of many forms of CVD. The purpose of this study is to identify novel cardiac specific indicators of oxidative injury useful for early and convenient diagnosis of heart failure.To determine the most suitable method for identification of non-invasive oxidative injury indicators in general, human diploid fibroblasts (HDFs) were treated with H2O2 for collection of mRNA, cell lysates and conditioned media to perform cDNA microarray and LC-MS/MS based Multidimensional Protein Identification Technology (MudPIT) analyses. Electron Spray Ionization (ESI)-LC-MS/MS analysis of the conditioned media led to the finding of IGFBP-6 as a non-invasive biomarker of cell oxidative injuy in vitro and in vivo. The data obtained from this study indicate that proteomic analysis of conditioned media is useful to identify non-invasive biomarkers valuable for diagnosis or management of diseases.Cardiomyocyts (CMCs) and Cardiac fibroblasts (CFs) in culture were used to identify cardiac specific indicators of oxidative stress. Increased level of Cystatin C was detected in the conditioned medium of CMCs due to H2O2 treatment. In vivo models of oxidative stress were used to validate the increase of Cystatin C. Cystatin C levels increased in the plasma of mice with doxorubicin induced cardiomyopathy and coronary artery occlusion induced myocardial infarction (MI). These data indicate that Cystatin C can be a potential indicator of CMC oxidative injury in vitro and in vivo.Cystatin C is a cysteine protease inhibitor. The finding that oxidative stress induces Cystatin C led us to investigate a novel pathway regulating cardiac extracellular matrix (ECM) with CFs in culture, increased levels of ECM protein and decreased levels of Cathepsin B (CTB) protein and activity were detected upon Cystatin C treatment. With coronary artery occlusion induced MI mouse model, increased levels of Cystatin C and ECM protein and decreased levels of CTB protein and activity were detected in the infarcted area of the myocardium. These data indicate that Cystatin C serves as a potential fibrotic factor during myocardial remodeling.

Cytochrome P4501A (CYP1A), a xenobiotic metabolizing enzyme found in allvertebrates, is highly induced following exposure to a number of organic contaminants.Several populations of teleosts residing in highly contaminated areas have been found toexhibit resistance to the toxic effects of contaminants, a condition characterized byreduced expression of CYP1A.Within this work I demonstrated that expression of CYP1A mRNA, protein, andactivity in caged rainbow trout (Oncorhynchus mykiss) was an effective biomarker ofpolychlorinated biphenyl (PCB) contamination. Furthermore, through the use of bothlaboratory and field studies, I demonstrated that several species inhabiting a PCBcontaminated site exhibited either acquired (Ameiurus natalis) or natural (Lepomiscyanellus) resistance to the CYP1A inducing effects of PCBs. Further studiescharacterized the response of several other Lepomis species to CYP1A inducingcompounds, demonstrating that the natural resistance of L. cynaellus is a characteristicshared by at least two other members of the genus. Lepomis species were relativelyinsensitive to CYP1A induction following PCB exposure yet exhibited highly inducedCYP1A levels following exposure to another CYP1A inducer, the model polyaromatichydrocarbon benzo[a]pyrene (BaP), suggesting a number of species within the genusLepomis may display natural resistance to certain classes of CYP1A inducingcompounds.Additional studies using responsive and resistant populations of killifish wereused to examine the consequences of resistance on fish physiology. Thyroid hormones,known to be altered by PCBs in mammals, were variable but did not differ significantlybetween responsive and resistant fish following PCB exposure. Treatment with PCBssuppressed production of the egg yolk precursor protein vitellogenin in primaryhepatocytes of responsive fish. Studies examining the developmental impacts of toxicantexposure demonstrated altered aspects of development in PCB responsive but notresistant Fundulus heteroclitus embryos exposed to polybrominated diphenyl ethers(PBDEs), compounds structurally related to PCBs. PBDE exposure in juvenile Ictaluruspunctatus failed to induce CYP1A or uridine diphosphate glucuronyltransferase(UDPGT) activity indicating PBDEs do not impact these commonly measuredtoxicological endpoints. The findings of this work describe novel pollutant responses in anumber of species with varying degrees of pollutant sensitivity and contribute to theunderstanding of toxicant induced alterations in teleost physiology.

One percent of children in North America may be affected by fetal alcohol spectrum disorder (FASD). FASD remains difficult to diagnose because confirmation of maternal alcohol use is a diagnostic criterion, and women consuming alcohol during pregnancy are reluctant to divulge this information for fear of stigmatization and losing custody of the child. Consequently, using a biomarker to assess alcohol exposure would provide a tremendous advantage. Recently, the measurement of fatty acid ethyl ester (FAEE) in hair has provided a powerful tool for assessing alcohol exposure. My thesis fills a translational gap of research between the development of the FAEE hair test and its application in the context of FASD. The guinea pig has been a critical model for FASD research, in which FAEE hair analysis has previously distinguished ethanol-exposed dams/offspring from controls. My first study, reports a positive dose-concentration relationship between alcohol exposure and hair FAEE, in the human, and the guinea pig. Humans also displayed over an order of magnitude higher FAEE incorporation per equivalent alcohol exporsure, suggesting that the test will be a sensitive clinical marker of fetal alcohol exposure. My second study utilized multi-coloured rats to investigate the potential of a hair-colour bias, as has been reported for other clinical hair assays; no evidence of bias is reported here. My third study is the first to examine the clinical use of the FAEE hair test in parents at high risk of having children with FASD. Over one third of parents tested positive for excessive alcohol use. Parents were investigated by social workers working for child protection services, and my fourth study reports that hair FAEE results agree with social worker reports. Individuals highly suspected of abusing alcohol were at a significantly greater risk of testing positive, whereas individuals tested based on other reasons (such as to cover all bases) were negatively associated with testing positive. The last study of my thesis, confirmed an association between alcohol and drug use by parents at high risk for having children with FASD, posing an added risk to children. This work helps bridge a gap in translational research, suggesting that the FAEE hair test has potential for use in FASD diagnosis and research.

Zugl.: Hannover, Med. Hochsch., Diss., 2007.

Ulm, Univ., Diss., 2008.

Universiẗat, Diss., 2004--Köln.

University, Diss., 2004--Bremen. / Enth. 6 Sonderabdr. aus verschiedenen Zeitschr.

The diagnostic evaluation of ever smokers with pulmonary nodules represents a growing clinical challenge due to the implementation of lung cancer screening. The high false-positive rate of screening frequently results in the use of unnecessary invasive procedures in patients who are ultimately diagnosed as benign, clearly highlighting the need for additional diagnostic approaches. We previously derived and validated a bronchial epithelial gene-expression biomarker to detect lung cancer in ever smokers. However, bronchoscopy is not always chosen as a diagnostic modality. Given that bronchial and nasal epithelial gene-expression are similarly altered by cigarette smoke exposure, we sought to determine if cancer-associated gene-expression might also be detectable in the more readily accessible nasal epithelium. Nasal epithelial brushings were prospectively collected from ever smokers undergoing diagnostic evaluation for lung cancer in the AEGIS-1 (n=375) and AEGIS-2 (n=130) clinical trials and gene-expression profiled using microarrays. The computational framework used to discover biomarkers in these data was formalized and implemented in an open-source R-package. We identified 535 genes in the nasal epithelium of AEGIS-1 patients whose expression was associated with lung cancer status. Using matched bronchial gene-expression data from a subset of these patients, we found significantly concordant cancer-associated gene-expression alterations between the two airway sites. A nasal lung cancer classifier derived in the AEGIS-1 cohort that combined clinical factors and nasal gene-expression had significantly higher AUC (0.81) and sensitivity (0.91) than the clinical-factor model alone in independent samples from the AEGIS-2 cohort. These results support that the airway epithelial field of lung cancer-associated injury extends to the nose and demonstrates the potential of using nasal gene-expression as a non-invasive biomarker for lung cancer detection. The framework for deriving this biomarker was generalized and implemented in an open-source R-package. The package provides a computational pipeline to compare biomarker development strategies using microarray data. The results from this pipeline can be used to highlight the optimal model development parameters for a given dataset leading to more robust and accurate models. This package provides the community with a novel and powerful tool to facilitate biomarker discovery in microarray data.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system (CNS) that exhibits considerable variation in its clinical course. Recently, the World Health Organization (WHO) published a classification system for tumors of the CNS that combines histological features with molecular parameters to determine tumor grade. The incorporation of molecular biomarkers that carry both prognostic and predictive value adds another level of objectivity to the glioma grading system and will help guide clinical decision. As such, the assessment of biomarkers has become an integral part of tumor evaluation in neuro-oncology. This curriculum will discuss the clinical relevance of the most recently studied biomarkers with prognostic and predictive value in the evaluation of GBM. Biomarkers regularly used for the assessment of GBM include the IDH mutations, MGMT methylation status, and EGFRvIII. Furthermore, this review will offer a perspective on experimental approaches currently under investigation for treatment of GBM. LITERATURE REVIEW FINDINGS: MGMT methylation of the promoter region is associated with better treatment response from temozolomide (TMZ), an alkylating therapeutic. Treatment benefit was most prominent in the elderly population and therapy should be individualized for that age group. Patients with GBM characterized by IDH1/IDH2 mutations carry a better overall prognosis primarily due to their higher sensitivity to chemo- and radiotherapy. The prognostic value of EGFRvIII remains controversial, although it may be associated with a worse prognosis. Nonetheless, EGFRvIII provides an ideal target for targeted molecular therapies as it is only found on tumor cells. PROPOSED METHODS: A curriculum aimed at educating primary care providers (PCPs) about the most clinically significant biomarkers in GBM will be developed. The curriculum will be in a PowerPoint format, and the hour-long lecture will be presented at continuing medical education national conferences. A pre- and post-test consisting of the same 10 multiple- choice questions will be administered on a voluntary basis to help evaluate knowledge acquisition from the curriculum. Results will be evaluated with a paired t-test analysis. The tests will be will be administered through Poll Everywhere, a smartphone survey application. CONCLUSION: There is increasing evidence to suggest that therapies should be individualized according to specific biomarkers with predictive value. PCPs are in a position where they are often the first providers to suspect the diagnosis of a brain tumor. Therefore, it is imperative for PCPs to be aware of the latest development in the field of neuro-oncology so that they may appropriately counsel patients.