BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Non-clinical safety assessment is essential during the drug development process in the pharmaceutical industry, and involves numerous, detailed in vitro and in vivo toxicology tests (general, reproductive and genetic), and safety pharmacology studies. The testis is a common organ for adverse drug effects leading to attrition of potential compounds. It would, therefore, be useful to detect testicular toxicity as early as possible in the drug development process. Histopathology is the standard method for assessing testis toxicity, but a biomarker for ‘early warning’ detection of testicular toxicity would be far more useful in non-clinical toxicology studies. The aim of this thesis was to evaluate the feasibility of this approach. It is thought that proteins can leak from seminiferous tubules into testicular interstitial fluid following testicular damage, due to either loss of integrity of the blood-testis barrier (BTB) or germ cell damage. A potential biomarker protein could, therefore, leak out of seminiferous tubules into interstitial fluid and then into blood following toxicological insult to the testis. A suitable biomarker protein must be testis specific, abundant, and not normally be present in blood. It may also need to have a low molecular weight. To investigate if proteins do leak out of seminiferous tubules following testicular damage, three known testicular toxicants which affect different aspects of the testis were used; cadmium chloride causes disruption to the blood-testis barrier and spermatogenesis, methoxyacetic acid (MAA) specifically causes a loss of pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) causes Sertoli cell vacuolation and subsequent germ cell disruption. Adult male Wistar rats were treated with various doses of these toxicants to give mild and moderate responses. Samples were collected 24 hours later. Testicular damage was investigated by immunohistochemistry for well-known germ cell markers (DAZL, VASA) and using a general antibody to seminiferous tubule proteins. The integrity of the BTB was evaluated using immunofluorescent co-localisation of occludin, ZO-1, claudin-11, N-cadherin and β-catenin, and a biotin tracer. Protein leakage was investigated using analysis of interstitial fluid samples by 1D gel electrophoresis and staining with Coomassie-based dye or Western blotting for germ cell proteins and with the general antibody to seminiferous tubule proteins. Protein leakage from seminiferous tubules into interstitial fluid was observed with high dose cadmium chloride treatment. This was coincident with a loss of integrity of the BTB. No leakage was observed with MAA treatment which caused a specific loss of pachytene spermatocytes, or DNB which caused Sertoli cell vacuolation. With both treatments the BTB did not appear to be damaged suggesting that protein leakage occurs only following loss of integrity of the BTB. This was further investigated using treatments reported to specifically disrupt the BTB, namely intra-testicular administration of glycerol or transforming growth factor-β3, with samples collected 48 hours later. The damage caused was very localised, although BTB disruption with glycerol treatment caused some protein leakage. The presence of germ cell proteins in interstitial fluid samples before and after the development of the BTB during normal development was also evaluated, although most proteins of interest were not expressed in germ cells of the immature testis before BTB formation. Finally, five potential biomarker candidate proteins (ADAM3, Calpastatin, DAZL, FABP9, VASA) were selected and investigated using samples from the testicular toxicant studies. Smaller molecular weight proteins were thought to be more likely to leak out of seminiferous tubules, however, VASA, a large molecular protein (76kDa) was shown to leak into interstitial fluid following high dose cadmium chloride treatment. However, FABP9 (low molecular weight) was found to be the most promising biomarker for loss of BTB integrity. The results suggest that a biomarker could only be detected if there is a loss of integrity of the BTB and severe disruption of spermatogenesis, thus conferring no real advantage over present histopathology-based toxicity evaluations. Therefore, an automated immunohistochemistry and image analysis method was investigated as a refined method for detection of testicular toxicity at the end of a toxicology study, and shown to have promise.

The current study focuses on finding differential expression between circulating microRNAs in plasma from patients with abdominal aortic aneurysms compared to un-diseased individuals by using a qPCR-based array. In addition, we evaluated the expression of deregulated microRNAs in human tissue samples as well as microarray data from two independent mouse models of aneurysm development. Fifteen miRNAs were found to be significantly differentially expressed, with four of them surviving multiple testing. Interestingly all four of them were substantially different in murine aneurysm development.

Niemann-Pick type Cl (NPC1) disease is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium levels. Since the lysosomal system is involved in regulating aspects of transition metal ion homeostasis and its intracellular compartmentalization, we have investigated whether there are metal ion metabolism defects and haematological abnormalities in NPC1 disease. We have identified multiple haematological changes, including decreased haematocrit, haemoglobin and mean corpuscular haemoglobin volume in mice.

<strong>Background</strong>. Pathological processes in Parkinson’s disease (PD) start long before the first symptoms appear and by the time the disease is clinically established the results of neurodegeneration may be irreversible. Efforts to prevent or stem disease progression need to start in early disease and good characterization and new markers of early PD are urgently needed. <strong>Objectives</strong>. This thesis aims to characterize early disease stages in three projects. Firstly, clinical features of PD within 3 years of diagnosis will be explored in an incident cohort of patients and controls, using a range of tools to cover the whole breadth of clinical presentation of PD. Secondly, functional imaging studies in PD published so far will be examined through a meta-analysis to identify the most robust functional imaging markers. Thirdly, a functional MRI resting-state study in early PD will be performed to identify reproducible differences between patients and matched control subjects. <strong>Results</strong>. The cohort analysis found that age was a strong predictor of disease severity, independent of disease duration, while gender was seen to affect disease severity depending on the body region. A meta-analysis of all published functional imaging studies across all disease stages showed abnormal activations in the Basal Ganglia but also in a wide range of motor and non-motor brain areas. Dopamine supplementation normalized activations in the Basal Ganglia and some other areas, while other circuits remained resistant to medication suggesting non-dopaminergic abnormality. In the resting-state study, the Basal Ganglia Network showed greatly reduced connectivity in early PD compared to controls, which normalized on administration of dopaminergic medication. Reduced BGN connectivity was also validated on a separate group of PD subjects achieving very good separation of patients from controls. <strong>Conclusions</strong>. The effect of gender and age on early presentation of PD has potential significance for early diagnosis and choice of outcome measures for clinical trials. Within the realm of imaging, traditional task-based fMRI studies fail to show a clear and reproducible pattern of activations making this method unfeasible for early diagnostic testing. In contrast, resting-state fMRI connectivity in the Basal Ganglia Network appears to be a promising and reliable method even in the early stages of PD. Clinical profiling and resting imaging changes offer avenues for developing future biomarkers in early PD.

Genome-wide microarray technology has facilitated the systematic discovery of diagnostic biomarkers of cancers and other pathologies. However, meta-analyses of published arrays using melanoma as a test cancer has uncovered significant inconsistences that hinder advances in clinical practice. In this study a computational model for the integrated analysis of microarray datasets is proposed in order to provide a robust ranking of genes in terms of their relative significance; both genome-wide relative significance (GWRS) and genome-wide global significance (GWGS). When applied to five melanoma microarray datasets published between 2000 and 2011, a new 12-gene diagnostic biomarker signature for melanoma was defined (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, and WNT4). Of these, CXCL13, COL11A1, PTPRF and SHC4 are components of the MAPK pathway and were validated by immunocyto- and immunohisto-chemistry. These proteins were found to be overexpressed in metastatic and primary melanoma cells in vitro and in melanoma tissue in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin. One challenge for the integrated analysis of microarray data is that the microarray data are produced using different platforms and bio-samples, e.g. including both cell line- and biopsy-based microarray datasets. In order to address these challenges, the computational model was further enhanced the stratification of datasets into either biopsy or cell line derived datasets, and via the weighting of microarray data based on quality criteria of data. The methods enhancement was applied to 14 microarray datasets of three cancers (breast, prostate, and melanoma) based on classification accuracy and on the capability to identify predictive biomarkers. Four novel measures for evaluating the capability to identify predictive biomarkers are proposed: (1) classifying independent testing data using wrapper feature selection with machine leaning, (2) assessing the number of common genes with the genes retrieved in independent testing data, (3) assessing the number of common genes with the genes retrieved in across multiple training datasets, (4) assessing the number of common genes with the genes validated in the literature. This enhancement of computational approach (i) achieved reliable classification performance across multiple datasets, (ii) recognized more significant genes into the top-ranked genes as compared to the genes detected by the independent test data, and (iii) detected more meaningful genes than were validated in previous melanoma studies in the literature.

No description available.

Master of Science / Biomedical Sciences / Gregory F. Grauer / Chronic kidney disease (CKD) is a potentially life-threatening disease that reportedly affects 10% of dogs and 30% of cats over the age of 15. There is no cure available for CKD, but medical management is available for patients with this disease. Research has focused on earlier detection of CKD with the goal of instituting medical management and monitoring as early in the disease course as possible. Symmetric dimethylarginine (SDMA) has recently emerged as a novel renal excretory biomarker that may aid in early detection of CKD in cats and dogs. SDMA is non-protein bound and is freely filtered by the glomerulus, is not secreted or reabsorbed, and has greater than 90% excretion by the kidneys, making it a potential target for measurement of glomerular filtration rate (GFR). Previous studies have demonstrated a close parallel between SDMA and serum creatinine (sCr), which is the currently favored serum biomarker for assessment of GFR. Research has also demonstrated a correlation between SDMA and GFR. Serum concentrations of SDMA increase above normal when GFR is decreased by 25-40%; much earlier than the 75% decrease in GFR typically required for sCr to increase above its reference interval. The studies reported here demonstrate a potential use for the SDMA:sCr ratio as a predictor of volume responsive azotemia. Furthermore, longitudinal assessment of older dogs and cats for early detection of CKD showed that SDMA was a more sensitive indicator of CKD than sCr. The evaluation of SDMA reported in this thesis presents a novel perspective on SDMA and its use clinically.

No description available.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Daniel Diviš Supervisors: Prof. Dr. Florence Apparailly, Directrice de Recherche Prof. PharmDr. Petr Pávek, Ph.D. (formal tutor) Title of diploma thesis: The role of miR-150 in the physiopathology of oligoarticular juvenile idiopathic arthritis Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatoid disease affecting children, and its pathological mechanisms are still poorly understood. Innate and adaptive immunity including myeloid cells play a major role in these processes. Epigenetic deregulations along with non-coding microRNAs have been reported in many inflammatory diseases. Moreover, preliminary results obtained by the research group of Prof. Florence Apparailly showed accumulation of intermediate monocytes along with the high expression of miR-150 in the synovial fluid of children affected by oligoarticular JIA. Based on these findings a hypothesis has been postulated suggesting that miR-150 could have a role in the pathogenesis of this disease and in the regulation of monocyte differentiation and function. To study the impact of miR-150 on monocytes from the peripheral blood of healthy donors, transfection experiments were performed to neutralize miR-150. The...