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...

Conjugation of reactive intermediates of drugs with proteins or DNA may result in toxic effects such as hepatotoxicity, agranulocytosis, allergies, tumors, etc. From 1975 to 1999, 2.9% of drugs were withdrawn from the market due to such severe adverse drug reactions. Thus, formation of chemically reactive intermediates is a widely discussed problem in drug development processes. Early detection of potentially toxic compounds is required for drug discovery and drug development. Conjugation of such electrophilic compounds with glutathione (GSH) is one of the most important detoxifying reactions in vivo. Processing of these GSH-conjugates ultimately leads to the formation of renally cleared mercapturic acids, which may also be oxidized to sulfoxides. Thus, mercapturic acids may be generated and detected in vitro and non-invasively in vivo in urine to assess the reactivity of a compound in early stages of drug development processes. Therefore, the aim of this work was to develop and evaluate a HPLC-MS/MS screening method for simple and rapid detection and characterization of known and unknown mercapturic acids and application of the method to several different matrices. Based on the common constant neutral loss (CNL) of 129 Da of all mercapturic acids tested (in negative ion mode), a CNL survey scan was performed using a linear ion trap instrument and was combined with two enhanced product ion (EPI) scans with different collision energies to characterize the detected signals. The CNL resulted from the cleavage between the sulfur and the carbon atom in the N-acetyl-L-cysteine moiety. After optimization of the experimental parameters, the detection limits of the reference substances in rat urine ranged from 0.3 to 15.5 pmol on column (i.e. 20 ng/ml to 800 ng/ml). For in vitro evaluation of the method, the model compounds acetaminophen, diclofenac, bifonazole, clozapine, troglitazone, carbamazepine, and bisphenol A were screened for formation of reactive intermediates and, hence, detection of the corresponding mercapturic acids. To determine possible species- and tissue-specific toxicities, the model compounds were incubated with stimulated neutrophils and with liver microsomes from rats and humans. Species-specific differences were observed in incubations of acetaminophen and diclofenac with rat and human hepatic microsomes. Tissue-specific differences in biotransformation of the model compounds in incubations with human neutrophils and human liver microsomes were observed for diclofenac, carbamazepine, clozapine, and bifonazole. The developed HPLC-MS/MS method was also evaluated in vivo by analysis of rat and human urine. Drug-related mercapturic acids were detected in urine of rats orally treated with acetaminophen (20 mg/kg and 640 mg/kg b.w.) or diclofenac (10 mg/kg and 20 mg/kg b.w.). Human urine samples were analyzed before and after oral administration of a clinically used dose of 500 mg and 50 mg of acetaminophen. Besides detection of the mercapturic acid of N-acetylbenzoquinoneimine (AAP-MA), a second mercapturic acid with m/z 327 occurred dose-dependently in rat and human urine samples after administration of acetaminophen. Further investigations on identification of this metabolite using authentic compounds and comparing their MS/MS mass spectra demonstrated oxidation of AAP-MA to stereoisomeric sulfoxides in vivo. For diclofenac, a novel mercapturic acid with m/z 441 was detected in rat urine samples that was identical to a metabolite obtained in incubations with human neutrophils before. The in vivo formation of this diclofenac metabolite is described here for the first time. In addition, three endogenously formed mercapturic acids were detected and identified. In conclusion, the results of the in vitro and in vivo evaluation demonstrate the advantages of the rapid and generic HPLC-MS/MS screening method for the detection of mercapturic acids, that can be obtained with a minimum of sample preparation and a high throughput in diverse matrices. / Konjugation reaktiver Intermediate mit Proteinen oder DNA kann zu toxischen Effekten wie Hepatotoxizität, Neutropenie, Allergien, Tumoren u.a. führen. Zwischen 1975 und 1999 wurden 2.9% der zugelassenen Arzneistoffe wegen Auftretens solcher unerwünschten, toxischen Nebenwirkungen vom Markt genommen. Daher stellen Substanzen, die reaktive Intermediate bilden können, ein großes Problem in der Arzneistoffentwicklung dar. Aus diesem Grund ist die pharmazeutische Forschungsindustrie daran interessiert, solche potenziell toxischen Substanzen bereits in frühen Phasen der Arzneistoffentwicklung zu erfassen. Elektrophile, reaktive Intermediate sind instabil und reagieren schnell mit nukleophilen Substraten. Die Konjugation reaktiver Intermediate mit Glutathion stellt hierbei einen der Hauptmechanismen der Detoxifizierung im Organismus dar. In vivo können enzymatisch geregelte Reaktionen das Glutathionaddukt abbauen und so zur Bildung renal ausscheidbarer Merkaptursäuren führen, die auch zu den entsprechenden Sulfoxiden oxidiert werden können. Man kann Merkaptursäuren aber auch direkt durch Konjugation mit N-Acetyl-L-cystein gewinnen. So können reaktive Intermediate in vitro generiert und als Merkaptursäuren detektiert und nicht-invasiv auch in vivo erfasst werden. Ziel dieser Arbeit war es, eine HPLC-MS/MS-Screening-Methode zur einfachen und schnellen Detektion und Charakterisierung von bekannten und unbekannten Merkaptursäuren als Biomarker für die Bildung reaktiver Metabolite in verschiedenen Matrices zu entwickeln und zu evaluieren. Für alle untersuchten Merkaptursäuren und deren Sulfoxide war ein Neutralverlust von 129 Da (im negativen Ionenmodus) charakteristisch. Dieser entstand durch Spaltung der Schwefel-Kohlenstoff-Bindung im Merkaptursäureanteil und diente als Basis für die Entwicklung der HPLC-MS/MS-Methode. Dafür wurde ein CNL-Scan auf 129 Da im negativen Ionenmodus durchgeführt. Der CNL-Scan konnte unter Verwendung der vorhandenen Ionenfalle mit zwei Produkt-ionen-Scans (EPI) mit unterschiedlichen Kollisionsenergien kombiniert und für eine Charakterisierung der detektierten Signale verwandt werden. Nach Optimierung der Instrument- und HPLC-Parameter wurden für die einzelnen Referenzsubstanzen Nachweisgrenzen im Bereich von 0.3 bis 15.5 pmol on column (entspricht einem Bereich von 20 ng/ml bis 800 ng/ml) in Rattenurin bestimmt. Für die In-vitro-Evaluierung der CNL-Screening-Methode wurden die Modellsubstanzen Paracetamol, Diclofenac, Troglitazon, Bifonazol, Clozapin, Carbamazepin und Bisphenol A auf die Bildung reaktiver Intermediate hin untersucht, die durch Zusatz von N-acetylcystein abgefangen wurden. Um eventuell Aufschluß über gewebe- oder speziesspezifische Toxizitäten von Arzneistoffen zu bekommen, wurden die Modellsubstanzen in stimulierten neutrophilen Granulozyten und in Ratten- und Humanlebermikrosomen inkubiert. Speziesspezifische Unterschiede in der Bildung von reaktiven Intermediaten zwischen Inkubationen mit Ratten- und Humanlebermikrosomen wurden bei Paracetamol und Diclofenac beobachtet. Organspezifische Unterschiede in der Bildung von reaktiven Intermediaten zwischen Inkubationen mit neutrophilen Granulozyten und humanen Lebermikrosomen wurden bei Diclofenac, Carbamazepin, Clozapin und Bifonazol gefunden. Die HPLC-MS/MS-Screening-Methode wurde durch Messungen von Ratten- und Humanurinproben auch in vivo evaluiert. Arzneistoffbezogene Merkaptursäuren wurden in Urinproben von Ratten gemessen, die über eine Schlundsonde Paracetamol (20 mg/kg und 640 mg/kg K.G.) bzw. Diclofenac (10 mg/kg und 20 mg/kg K.G.) zugeführt bekommen hatten. Humanurin wurde nach Gabe einer therapeutischen Dosis von 500 mg Paracetamol und einer subtherapeutischen Dosis von 50 mg analysiert. Neben der bekannten Merkaptursäure des N-acetylbenzochinonimins (NAPQI) wurde ein weiterer Metabolit (m/z 327) dosisabhängig in den Urinproben von Ratte und Mensch detektiert. Durch nähere Untersuchungen zur Identifizierung dieses Metaboliten anhand von Referenzsubstanzen und deren Massenspektren konnte nachgewiesen werden, dass das Merkapturat des NAPQI zu stereoisomeren Sulfoxiden oxidiert wurde. Bei den Diclofenac-Proben wurde zum ersten Mal ein Metabolit mit m/z 441 in Rattenurin detektiert und charakterisiert, der nur in Inkubationen mit stimulierten neutrophilen Granulozyten, jedoch nicht mit Lebermikrosomen gebildet wurde. Mit der entwickelten HPLC-MS/MS Screening Methode konnten weitere, vom Arzneistoff unabhängige Merkaptursäuren im Urin detektiert und charakterisiert werden. Schließlich zeigen die Ergebnisse zur In-vitro- und In-vivo-Evaluierung die Vorteile dieser schnellen und generischen HPLC-MS/MS-Screening-Methode zur Detektion von Merkaptursäuren, die mit minimaler Probenvorbereitung und hohem Probendurchsatz für verschiedene Matrices eingesetzt werden kann.