This work analyzes problems of friction and lubrication of total hip replacement (THR). At first, experimental study of friction coefficient under various conditions was conducted using hip joint simulator based on principle of pendulum. After that, lubrication film development was observed, using colorimetric interferometry with head from THR and glass cup with respect of geometry and clearance to cups made originally for THR. With respect to research study [25] it was found that increasing contact conformity leads to increased film thickness. Also protein adsorption on articulating surfaces was observed. Thanks to adsorption 80 nm film thickness was measured even if relative surface speed was zero. Furthermore protein aggregation was observed on the border of contact zone, similar aggregations were observed by Myant et al. [29] or Vrbka et al. [25]. These aggregations are formed when lubricant is under shear stress and it can positively affect film thickness. If the relative motion of articulating surfaces is not sufficient, the protein layer can`t be refreshed enough and film thickness start decreasing. Coefficient of friction 0,13 was measured when lubricating film development was observed. Close to this value are material combinations metal on polyethylene and metal on metal.

MSCAGR (Animal Science) / Department of Animal Science / The study evaluated the nutritive value of orange pulp- buffalo grass (OPBG) silage as a po-tential pig feed. In experiment 1, OPBG was ensiled in 58 one-litre jars, split into Reno-zyme® enzymes (containing α-amylases and β-endo 1, 3; 1, 4 beta glucanase) (denoted OPBGE) and 18 without enzyme (denoted OPBG). Samples were collected from the lab on days 7, 15 and 30 in a three by two factorial design (period by treatment). In Experiment 2, 12 South African Windsnyer-type (SAWIP) (27.2±3.9 kg) and 12 Large White- Landrace crosses (LW x LR) (28±9.8 kg) were fed diets containing different levels of bulk-ensiled OPBG ad libitum for 30 days, in a 2 X 3 (breed by level of OPBG (control, low (15% OPBG) and high (30% OPBG))) factorial arrangement. The apparent total tract digestibility (ATTD) of the pigs was measured during week 3 of feeding. The average body gain ABG average daily feed intake (ADFI), average daily gain (ADG), average gain to feed ratio (AG: F) were measured weekly. Serum glucose (GLU) , blood urea nitrogen (BUN), creatinine (CREAT), cholesterol, (CHOL) and triglycerides (TG) were measured during week 4 of the experiment. The levels of CP, EE, NDF, water soluble carbohydrates (WSC), lactic acid (LA) and pH were different (P<0.05) for OPBGE and OPBG on day 7. However the OPBGE concentra-tions of DM, ASH, CP, NDF, HEMI, WSC, LA and pH were higher (P<0.05) compared to OPBG. The OPBGE was lower (P<0.05) on EE and ADF, compared to OPBG on day 7. The levels of DM, ASH, CP, NDF, ADF, HEMI, LA and WSC were different (P<0.05) for OPBGE and OPBG on day 30. However the OPBGE levels of DM, CP, NDF, ADF, LA and WSC were higher (P<0.05) compared to OPBG. ASH, EE and HEMI were lower (P<0.05) on day 30. There were significant diets x day interactions (P<0.05) for DM, ASH, CP, EE, NDF, ADF, HEMI, WSC, and LA, but not for pH. The aerobic stability study was inconclusive. The digestibility levels of OM and NDF were different (P<0.05) for LW x LR and SAWIP, where LW x LR ATTD on OM and NDF was higher (P<0.05) compared to SAWIP for all diets. The LW x LR ADG was higher (P<0.05) compared to SAWIP for all diets. There was a diet x breed interaction effect (P <0.05) on ADG whereby the BUN and CREAT were different (P <0.05) for LW x LR and SAWIP. The SAWIP BUN was higher (P<0.05), but the CREAT and TG were lower (P<0.05) compared to LW x LR for all diets fed. In conclusion, OPBG inclu-sion in pig feed enhanced the quality of feed, digestibility, performance and blood metabo-lites profile. / NRF

La leucémie lymphoïde chronique (LLC) est le lymphome avec phase circulante le plus fréquent chez l’adulte dans les pays occidentaux. Elle est caractérisée par une grand hétérogénéité dans son évolution naturelle avec des formes indolentes ne nécessitant jamais de traitement spécifique et des formes agressives requérant une chimiothérapie rapidement après le diagnostic. Dans ce travail de thèse, nous avons posé la question du rôle des anomalies moléculaires et en particulier des gènes des immunoglobulines dans l’histoire naturelle de la maladie, tant au plan mécanistique que pour le pronostic. Nous avons étudié trois remaniements atypiques impliquant un gène des immunoglobulines et un partenaire inconnu dans la LLC/lymphome lymphocytique. Les points de cassure ont pu être identifiés et nous ont permis de mettre en évidence l’implication dans 2 cas d’ARN longs non codants en 17q25 et 8q24. De plus, deux cas ont des points de cassure dans une région chromosomique restreinte (espacés de 200 kb en 17q25). Il pourrait s’agir d’un locus important dans la lymphomagénèse, de même que pour la région 8q24 contenant MYC et de nombreux gènes non codants jusqu’ici peu explorés. Par ailleurs, dans l’ère du séquençage haut débit, de nombreux marqueurs pronostiques moléculaires sont décrits dans la LLC. Nous avons démontré que l’électrophorèse des protéines sériques normale (immunoglobulines polyclonales sans hypogammaglobulinémie) au diagnostic, un marqueur simple et peu couteux, reste dans l’ère du NGS, un marqueur indépendant de bon pronostic dans la LLC. Sa combinaison avec un statut IGHV muté identifie un groupe de patients qui n’auront probablement jamais besoin de traitement spécifique. Ce travail nous a conduit à mettre au point un outil performant de détection des anomalies de nombre de copies par séquençage haut débit. Celui-ci permet la mise en évidence de disomies uniparentales dont la signification pronostique n’est actuellement pas établie dans la LLC. Ces anomalies pourraient être le reflet d’une instabilité chromosomique et il serait intéressant d’étudier leur impact pronostique dans la LLC. / Chronic lymphocytic leukemia (CLL) is the most frequent lymphoma with leukemic phase in the elderly in Western countries. It is characterized by a great heterogeneity in its natural history with indolent forms never requiring any specific treatment and aggressive forms needing chemotherapy rapidly after diagnosis. In this work of thesis, we asked the question of the role of molecular abnormalities, and in particular of the immunoglobulin genes in the natural history of the disease, at mechanistic level and for prognosis. We studied three atypical rearrangements implicating an immunoglobulin gene and an unknown partner in CLL/lymphocytic lymphoma. The breakpoints have been identified and the implication of long non coding RNA was highlighted in two cases in 17q25 and 8q24. Moreover, two cases harboured breakpoints in a restricted chromosomic region (200 kb spaced in 17q25). It could be an important locus in lymphomagenesis, as is 8q24 region containing MYC and numerous other non coding genes poorly characterized by now. Furthermore, in high throughput sequencing (HTS) era, many molecular prognosis markers have been described in CLL. We demonstrated that normal serum protein electrophoresis (polyclonal immunoglobulin without hypogammaglobulinemia) at diagnosis, a simple and unexpensive marker, stays in HTS era an independent good prognosis marker in CLL. Its combination with unmutated IGHV genes status identifies a group of patients who will probably never require any specific treatment. This work led us to develop an efficient tool to detect copy number variations by THS. This tool allows to highlight uniparental disomy whose prognosis signification is not established in CLL. These abnormalities could reflect chromosomal instability and it could be interesting to study their prognosis impact in CLL.

Einleitung: Die Pathogenese chronisch entzündlicher Atemwegserkrankungen ist mit zunehmender Schädigung und fibrotischen Umbauvorgängen und daraus resultierenden Einschränkungen physiologischer Funktionen verbunden. Bei der Recurrent Airway Obstruction (RAO) des Pferdes sind solche strukturellen Veränderungen häufig assoziiert mit Verdickung der Atemwegswand, subepithelialer Fibrose und Obstruktion der Atemwege. Bei RAO besteht die Standardtherapie darin, die Bronchokonstriktion mit β2-Agonisten und die Entzündung mit Glucocorticoiden aufzuhalten. In den letzten Jahrzehnten konnte jedoch gezeigt werden, dass nicht alle Patienten mit der empfohlenen Therapie ausreichend behandelt werden können und neue Ansatzpunkte der Therapie gefunden werden müssen. Daher konzentrieren sich neuere Forschungsarbeiten auf strukturelle Alterationen in den Atemwegen, das sogenannte Airway-Remodelling. Zielsetzung: In der vorliegenden Arbeit sollen zunächst primäre equine Bronchialfibroblasten (EBF) isoliert, charakterisiert und kultiviert werden. Im nächsten Schritt sollen in diesen Zellen die β2-Rezeptorexpression und –eigenschaften charakterisiert werden. Anschließend soll die Eignung dieser Zellen als Zellmodell zur Untersuchung der zellulären Proliferation und Transformation sowie der Regulation der de-novo-Synthese von extrazellulärer Matrix und deren Beeinflussung durch β2-Agonisten und Glucocorticoide allein oder in Kombination sowie TGF-β überprüft werden. Material und Methoden: Mittels enzymatischer Verdauung mit 0,25 % Trypsin werden die EBF aus den Bronchien von 10 gesunden Schlachtpferden isoliert und in DMEM kultiviert. Diese Zellen werden morphologisch, immunzytochemisch und funktionell in deren Eigenschaften in An- oder Abwesenheit von fetalem bovinem Serum (FBS) oder Pferdeserum (HS) charakterisiert. Die Dichte und Subtypverteilung von β-Adrenozeptoren wird mittels Radioligandbindungsstudien mit [125I]-(-)-Iodocyanopindolol in Gegenwart von Rezeptorsubtyp-selektiven β-Antagonisten (β2: ICI 118,551 und β1: CGP 20712A) in EBF untersucht. Der Einfluss von β2-Agonisten auf die Proliferation und Differenzierung der EBF sowie die Kollagensynthese wird mittels [3H]-Thymidineinbauassay, Bestimmung von Gesamtprotein und α-Smooth Muscle Aktin (α-SMA) mit Lowrymethode und Western Blot-Analyse bzw. [3H]-Prolininkorporationsassay ermittelt. Die statistische Signifikanz wird über einen t-Test für verbundene Stichproben oder eine One-Way-ANOVA mit nachfolgendem Dunnett’s Test ermittelt und das Signifikanzniveau wird P ≤ 0,05 festgelegt. Ergebnisse: EBF können im DMEM-Medium nur in Gegenwart von Serum langfristig wachsen und kultiviert werden. In serum-freiem Medium und unter vorübergehendem Serumentzug können EBF nicht anhaften bzw. lösen sie sich ab. Die Effekte von FBS und HS auf EBF sind allerdings unterschiedlich. FBS fördert die Zellproliferation und -verdopplungsrate sowie die Zellpassage bis zur Passage 20 signifikant besser als HS (max. 9 Passagen). Unter FBS entwickeln EBF eine für Fibroblasten charakteristische spindelförmige Morphologie aber eine schwache α-SMA-Expression, während unter HS die Zellen eine atypische, polygonale Morphologie zeigen, jedoch mit signifikant hohem α-SMA und Proteingehalt. Radioligandenbindungsstudien zeigen, dass EBF lediglich den β2-Adrenozeptorsubtyp mit einer maximalen Rezeptorendichte (Bmax) von 5037 ± 494 Bindungsstellen/Zelle exprimieren. Die Behandlung der EBF mit β-Agonisten (Clenbuterol, Salbutamol, Isoproterenol) führt konzentrationsabhängig zur Abnahme dieser Anzahl der Rezeptoren mit unterschiedlicher Wirkungsstärke (Clenbuterol > Salbutamol > Isoproterenol), wobei Dexamethason diese nicht verändert. Diese Agonisten sowie auch Dexamethason hemmen die Proliferation der EBF, und dies kann in Gegenwart von ICI 118,551 aber nicht von CGP 20712A gehemmt werden, was auf den Einfluss des β2-Adrenozeptors hinweist. β2-Agonisten und Dexamethason gemeinsam resultieren in einer verstärkten Hemmung der EBF-Proliferation. Transforming Growth Factor-β1 (TGF-β1) stimuliert die Transformation von EBF zu Myofibroblasten mit einer erhöhten Expression von α-SMA in EBF, welches eher durch Dexamethason als durch Clenbuterol gehemmt wird. Die Kollagensynthese wird nur durch Dexamethason signifikant gehemmt. Schlussfolgerung: Das erstmalig etablierte EBF-Kulturmodell dient der Erforschung von Signalwegen und neuen Arzneimitteltargets im Zusammenhang mit der Pathogenese der equinen RAO insbesondere dem Atemwegs-Remodelling. So kann gezeigt werden, dass die Verwendung von β2-Agonisten allein oder in Kombination mit Glucocorticoiden eine Hemmung der Proliferation und Transformation von EBF in vitro bewirkt. Dies deutet auf einen zusätzlichen therapeutischen Nutzen von Clenbuterol und Glucocorticoiden hin und impliziert die Notwendigkeit eines frühzeitigen Einsatzes dieser Pharmaka bei der RAO des Pferdes.

Graphene (G) and nanocrystalline diamond (NCD) are carbon allotropes and promising nanomaterials with an excellent combination of their properties, such as high mechanical strength, electrical and thermal conductivity, possibility of functionalization and very high surface area to volume ratio. For these reasons, G and NCD are employed next to electronics in biomedical applications, including implant coating, drug and gene delivery and biosensing. For a fundamental characterization of cell behavior on G and NCD, we studied osteoblast adhesion and proliferation on differently treated G and NCD. Generally, both G and NCD exhibited better properties for osteoblast cultivation than control tissue culture polystyrene. Better cell adhesion but lower cell proliferation were observed on NCD compared to G. The most surprising finding was that hydrophobic G with nanowrinkled topography enhanced cell proliferation extensively, in comparison to hydrophilic and flat G and both NCDs (hydrophobic and hydrophilic) with slightly higher roughness. Promoted cell proliferation enables faster cell colonization of G and NCD substrates, meaning faster new tissue formation which is beneficial in biomedical applications. Furthermore, it was shown that osteoblast adhesion was promoted in the initial absence of fetal bovine...

Alzheimer disease (AD) is the most frequent dementia. The prevalence is approximately 10% in 65 years old people. The current treatment is only progression protective, therefore it is crucial to find a new diagnostic approach for diagnosing AD in early stage. We analysed a set of 55 patients by the drop coating deposition Raman spectroscopy with the goal to verify previously published high sensitivity of the AD spectroscopic diagnosis in cerebral spinal fluid (CSF) and to find a new diagnostic method for blood serum (BS). We optimized measurement conditions for BS. The results were evaluated by the cluster analysis and the principal component analysis. The small set of samples exhibited high sensitivity in both CSF and BS but that distinctly decreased in the whole set. The results for CSF were affected by the choice of the analysed spectral interval. The best for AD diagnose was the interval containing peaks at 980, 1080 and 1249 cm-1.The results for BS have been the most sensitive in the whole spectral range. They have low sensitivity but high specificity for AD (92%). The usage of neural networks has conversely high sensitivity and low specificity in both sets of samples of BS and CSF. Powered by TCPDF (www.tcpdf.org)

In this doctoral thesis, a novel method for the determination of primary bile acids cholic acid and chenodeoxycholic acid is presented. Bile acids play various vital roles in the mammalian body. Moreover, their determination is extremely helpful in liver and biliary disease diagnosis and management. These saturated organic compounds lack strong chromophores and fluorophores in their structure, and thus are usually hard to detect in spectroscopy. For this reason, either instrumentally advanced but expensive methods, such as mass spectrometry, or less reliable enzymatic methods are commonly employed in bile acids quantitation. Hence, the demand for simple and reliable methods for their determination is strong. Bile acids are also known to be virtually inert for direct electrochemical oxidation. Herein, a simple method for their chemical activation for electrochemical oxidation on bare electrode materials was developed, optimized and applied to cholic acid and chenodeoxycholic acid determination. The activation is based on a dehydration reaction of a primary bile acid with 0.1 mol L-1 HClO4 in acetonitrile (water content 0.55%) that introduces double bond(s) into the originally fully saturated steroid core. This naturally increases the electron density in the structure, and thus allows electrochemical...

This work documents the first case of idiopathic AA amyloidosis in humans caused by mutation in the promoter region of SAA1 gene. Knowledge of the mechanism of the disease may be an indication for targeted treatment in the future. Mutations in the SAA1 promoter should be considered in all cases of idiopathic forms of AA amyloidosis in which neither the immune nor the inflammatory component of the disease are clearly present.

Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...

Development of methods for studying drug delivery systems is of great significance for the improvement of topical formulations. Active compounds for topical drug delivery are often formulated into gels and creams, that can be applied onto skin surfaces. It is important to know the extent of the permeability of the active compounds, in order to determine the medical effect. This study examines the possibilities of using an animal-free skin equivalent for penetration and permeation experiments, i.e. a silk scaffold integrated with viable human dermaland epidermal cells. Mammalian cell culturing together with silkconstruct formation, constituted the upstream bioprocess and acquisition of the skin equivalents. Permeability of fluorescently labelled Bovine Serum Albumin and Sodium Fluorescein salt was assessed, using a Franz- cell setup incorporated with the skin equivalents. Furthermore, fluorescence analysis and SDS-PAGE was performed on the collected samples, along with cryosectioning and image analysis of the skin equivalents. The results indicate variations in tissue integrity, leading to both high and low permeability. Fluorescence intensity can be correlated with the amount of sample liquid passing through. The model is still under development, hence more research is needed to draw a conclusion regarding the cellular composition of the skin equivalents, and how it influences permeability. / NextBioForm