Les personnes vieillissantes doivent composer au quotidien avec des douleurs chroniques. Le but de ce travail est de mieux comprendre les mécanismes sous-jacents qui contribueraient aux douleurs chroniques liées au vieillissement et par là, ouvrir un chemin vers de nouvelles perspectives thérapeutiques. Les contrôles inhibiteurs diffus nociceptifs (CIDN) ont un rôle qui n’est pas des moindres dans le contrôle de la douleur. Des études expérimentales examinant l’effet analgésique de la contre stimulation hétérotopique nociceptive (HNCS), un protocole permettant de tester l’efficacité de ces CIDN, suggèrent que le recrutement des CIDN au sein de cette population était plus faible (i.e. moins d’inhibition) comparé à une population plus jeune. En revanche, les études examinant la sensibilisation centrale induite par sommation temporelle (TS) de la douleur rapportent des résultats mitigés. De plus, une composante importante influençant l’expérience de douleur, les ressources cognitives, dont l’inhibition cognitive, se voient aussi décliner avec l’âge. Premièrement, le recrutement des CIDN a été comparé entre des participants sains, jeunes et des plus âgés avec la HNCS, et le recrutement des mécanismes de sensibilisation centrale avec la TS. La stimulation électrique du nerf sural a été choisie pour permettre de quantifier la douleur, tout en prenant une mesure indicative de la nociception spinale qu’est le réflexe nociceptif spinal (RIII). Nos sujets ont aussi participé à une tâche cognitive (le Stroop), testant l’inhibition cognitive. Deuxièmement, l’efficacité des CIDN ainsi que de l’inhibition cognitive a été testée chez les jeunes et les aînés en imagerie par résonance magnétique (IRM), afin de vérifier la relation entre ces deux mesures psychophysiques et l’épaisseur corticale des régions qui y sont impliquées ainsi que l’effet de l’âge sur celles-ci. Les résultats suggèrent un moindre recrutement des CIDN chez les plus âgés lors de l’expérimentation de la HNCS. Également, les sujets âgés présentaient des capacités d’inhibitions cognitives plus faibles que les jeunes. En plus, une corrélation entre l’inhibition cognitive et la modulation du réflexe RIII par la HNCS a été mise en évidence. Pour l’expérience de TS, les résultats étaient comparables pour les deux groupes, suggérant que les mécanismes impliqués dans la régulation de la douleur ne subiraient pas l’effet de l’âge de la même manière. Pour l’étude de l’épaisseur corticale, on y trouve une diminution globale de l’épaisseur corticale liée à l’âge, mais aussi une corrélation de l’analgésie par la HNCS avec l’inhibition cognitive et également, une relation des deux avec l’épaisseur corticale du cortex orbitofrontal (OFC) latéral gauche, suggérant la possibilité d’une existence d’un réseau neuronal au moins partiellement commun du contrôle inhibiteur descendant sensoriel et cognitif. Ce travail montre que l’effet de l’âge sur les mécanismes centraux de la régulation de la douleur est loin d’être uniforme. Également, il montre une corrélation entre la modulation endogène de la douleur et l’inhibition cognitive, ces deux processus seraient associés à une même région cérébrale. Ces résultats pourraient contribuer à identifier d’autres méthodes thérapeutiques, ouvrant ainsi une nouvelle avenue vers d’autres options dans la prise en charge des douleurs chroniques chez les personnes vieillissantes. / Elderly have to deal daily with chronic pain. The diffuse inhibitory descending controls (DNIC) are much involved in the pain control. It was shown that the DNIC recruitment was lower in elderly than in young population, using the analgesic effect of the heterotopic noxious counter stimulation (HNCS), a protocol allowing testing the efficiency of the DNIC. However, the studies assessing the central sensitization by the temporal summation of pain after repetitive stimuli have reported mixed results, the effect of age is still unclear. In addition, there is an age-related decline of an important component, which has an influence on pain perception, the cognitive resources and especially the cognitive inhibition. The goal of this work is to understand the underling mechanisms of the chronic pain related to the normal aging, which can leads to a new therapeutic perspectives. First, the recruitment of the CIDN was compared between healthy young and elderly participants using the HNCS, and the central sensitization was tested using TS. The sural nerve electric shock stimulation was chosen, because it allows us to have two measures, the pain rating and the nociceptive spinal reflex (RIII), an indicative measure of the nociception. Our participants were also invited to participate in a cognitive task (the Stroop), to assess the cognitive inhibition. Second, the efficacy of the CIDN and the cognitive inhibition in elderly and young participants was tested, but this time in a magnetic resonance imaging session. We have wanted to test the association between these two-psychophysics measures, with the cortical thickness of the regions involved into these two processes, and the effect of the normal aging on them. The results have showed a less recruitment of the DNIC in elderly in the HNCS experiment. Also, the elderly have performed less than young in the cognitive inhibition. In addition, a correlation between the cognitive inhibition and the modulation of the RIII-reflex was found. For the TS experiment, no difference was shown between the two groups, suggesting that aging may affect differently the mechanisms involved in endogenous pain modulation. The morphometric study has showed a global diminution of the cortical thickness with aging. Furthermore, there was an association, between the HNCS analgesia and the performance in cognitive inhibition, and between theses two processes and the cortical thickness of the left lateral OFC. These results suggest an eventual existence of at least a partial neuronal common network for the sensorial and cognitive descending inhibitory control. This thesis has showed that the effect of normal aging on the central mechanisms of the pain regulation might be not uniform. Also, on one hand a correlation between the endogenous modulation of pain and cognitive inhibition was reported, and on the other hand that these two processes would be related to the same cortical area. Together, these findings would help to develop new therapeutic methods and offer more options to the management of pain in the elderly.

In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Radek Machan Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Determination of permeability and active transport of selected butyrylcholinesterase inhibitors in vitro European Medicine Agency (EMA) and Food and Drug Administration agency (FDA) emphasise drug membrane permeability and drug-drug interactions on ABC transporters expressed in physiological barriers should be investigated for compounds in preclinical studies or for those already clinically used but evidence free. In this work we aimed to assess the capability of several experimental butyrylcholinesterase inhibitors that had been designed to treat dementia to permeate blood-brain barrier and to elucidate role of ATP-binding (ABC) cassette transporters in this transport. For this purpose, we employed in vitro bidirectional transport study across monolayers formed by polarized and highly differentiated Caco-2 cells. The permeability values gained from measurements were similar to values of several commonly used drugs for treatment of CNS disorders (e.g. antidepressants, antiepileptics). In addition, the compounds showed values of efflux ratio (basolateral- to-apical/apical-to-basolateral) approximately one...

Doctor of Philosophy / Department of Human Nutrition / Tonatiuh Melgarejo / Background and Objective: Crohn’s disease (CD) patients express low levels of host defense peptides (HDPs) especially β-defensins, which may compromise intestinal barrier function. Antibiotic treatment for bacterial infections in CD is limited and rarely curative, making it necessary to find alternative therapeutic approaches. We therefore investigated to what extent an essential amino acid; L-isoleucine (L-ILE) might induce the expression of human β-defensins (HBDs) in colonic epithelial cells as an alternative approach to help patients with CD. Antimicrobial activity of HBD2 was also assessed against four bacterial isolates which can cause secondary infections in CD. Methods: HTB-37 Caco-2 cells were stimulated with L-ILE at a concentration of 0 - 500µg/ml for 6 hours. Total RNA was extracted using RNeasy Micro Kit (QIAGEN). Reverse transcription was carried out with Superscript ®III First-Strand Synthesis System. The cDNA was amplified using specific primers for HBD1-3. Antimicrobial activity of HBD2 was determined using the broth dilution assay. Results: HBD1 was constitutively expressed under all conditions. HBD2 was expressed in HTB-37 cells after stimulation with L-ILE. Below 25µg/ml L- ILE stimulation, no expression of HBD2 was observed. HBD2 exhibited antimicrobial activity against bacterial isolates tested, with a MIC of 32, 64 and 128 µg/ml for both strains of E. coli, S. aureus and P. aeruginosa respectively. Conclusions: Our results indicate that L-ILE stimulation of HTB-37 Caco-2 cells can induce HBD2 expression. Data collected from our in vitro studies might have major implications for modifying the intestinal microbiota towards a healthier state in CD patients. Promoting the expression of HBD2 by colonic cells may lead to a lower rate of infection in these patients. Future in vivo studies are warranted to determine the potential clinical use of intra colonic administration of L-ILE in CD patients. The observed antimicrobial activity of HBD2 against bacterial isolates provides evidence that it is a crucial component of mucosal epithelial defense against infections which can complicate disease symptoms in CD.

A determination of corrosion inhibitors in engine coolants represent a difficult analytical problem due to their different physicochemical properties. Nowadays a lot of instrument methods are needed to determine them. The aim of this work was the development of methods for their determination using only one instrumentation. Capillary zone electrophoreses was chosen as a suitable technique. Three electrophoretic methods for three groups of corrosion inhibitors were developed. Firstly, method for determination of inorganic anions was developed in range from 5 to 50 ppm with limit of detection around 1 ppm. Background electrolyte contains sodium chromate, CTAB and CHES solution. Analytes were detected indirectly at 450 nm. Another method was for determination of organic acids anions. They were determined in range from 5 to 500 ppm. Limit of detection for each analyte was around 1 ppm. Measurement was made with PDC a CTAC water solution. Indirect detection was used for this determination at 350 nm. Last developed method can be use for determination of aryltrizoles in range from 5 to 500 ppm. Limit of detection was around 1 ppm. As a background electrolyte was used sodium tetraborate solution. The developed methods were validated and their suitability for determination of corrosion inhibitors in real...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Jan Juhász Supervisor: Prof. MUDr. Radomír Hrdina, CSc. Title of diploma thesis: Treatment of lower extremity peripheral artery disease The lower extremities ischemia is a disease caused most often by atherosclerosis during which the lumen in lower limb arteries becomes narrow. Its prevalence is increasing, especially in the developed countries. The disease can be asymptomatic and symptomatic. The symptoms are very unpleasant and decrease patient's quality of life. Advanced stages of the disease may be life threatening. Therefore, it is vital to timely and correctly diagnose the illness. During the therapy, it is possible to use pharmacological as well as non-pharmacological procedures, and, preferably, a combination of the two types of treatment. The pharmacotherapy can be divided into several parts. The prevention of atherosclerotic complications makes use of preventive measures and antiplatelet therapy to reduce the cardiovascular risk. The symptoms therapy focuses on improving patients' quality of life by prescribing the vasoactive medications cilostazol, naftidrofuryl or pentoxifylin. The critical limb ischemia therapy uses prostaglandin analogues alprostadil, iloprost, limaprost or...

This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...

Os objetivos deste estudo foram comparar os níveis de inibição pré-sináptica (IPS) durante a iniciação do passo entre indivíduos com doença de Parkinson (DP) com bloqueio motor (BM), sem-BM (S-BM) e indivíduos saudáveis pareados pela idade (ISPI) e, possíveis correlações entre os níveis de IPS com as variáveis comportamentais (amplitude e tempo do ajuste postural antecipatório [APA]) e os escores do New Freezing of Gait Questionnaire (NFOG-Q). A amostra foi composta por 22 indivíduos com BM e 12 S-BM (estágio 3 da DP avaliados no estado on da medicação). Dezesseis ISPI também foram incluídos no estudo. Todos atenderam os critérios de inclusão. Os indivíduos foram avaliados em dois dias. No primeiro dia, todos responderam uma anamnese e o Mini Exame do Estado Mental. Para os indivíduos com DP foi aplicada a escala de estadiamento da DP e a parte III da Escala Unificada de Avaliação da DP (UPDRS) e somente os indivíduos com BM foram avaliados com o NFOG-Q. No segundo dia, todos os indivíduos realizaram a tarefa iniciação do passo na plataforma de força AMTI para avaliação da amplitude e do tempo do APA com ou sem a evocação do reflexo-H na condição teste ou condicionado. O estimulador portátil Nicolet® Viking Quest da CareFusion foi utilizado para evocar o reflexo-H teste e condicionado. O estímulo elétrico foi evocado quando a amplitude do APA ultrapassasse 10 a 20% da linha de base. Todos indivíduos realizaram a tarefa iniciação do passo em 3 condições: 1) sem estímulo; 2) estímulo teste estimulação no nervo tibial; e 3) estímulo condicionado estimulação do nervo fibular antes (intervalo de 100 ms) da estimulação do nervo tibial. Nas três condições, os indivíduos foram instruídos a realizarem 15 passos, logo, 15 estímulos nas condições 2 e 3 de maneira aleatória. A ANOVA one way mostrou diferenças significantes nos valores de IPS entre os três grupos, onde os ISPI apresentaram valores maiores de IPS do que os outros dois grupos e, indivíduos S-BM apresentaram valores maiores IPS comparado aos indivíduos com BM (P<0,001), porém, todos os indivíduos deste último grupo apresentaram facilitação. Adicionalmente, amplitudes maiores e tempos menores do APA foram observados somente para os ISPI (P<0,05), além disso, não foram observadas diferenças entre essas variáveis para os dois grupos de DP (P>0,05). Em relação às correlações, especialmente para os indivíduos com BM, observamos que valores maiores de facilitação estão: fortemente associados com os escores maiores do NFOG-Q (r = -74; P<0,0001); moderadamente associados com as amplitudes menores do APA (r = 0,54; P<0,004); e fracamente associados com os tempos maiores do APA (r = -0,42; P<0,244). Por fim, houve uma forte associação entre as amplitude menores do APA e escores maiores do NFOG-Q (r = -,075; P<0,0001). Em conclusão, indivíduos com BM apresentam facilitação (ausência de IPS) durante a iniciação do passo e associada com deficiências na amplitude e tempo do APA e a severidade do BM. Isso indica que a medula espinhal tem participação na iniciação do passo e pode ser fortemente influenciada por alterações sensório-motoras / The aims of this study were to compare the presynaptic inhibition levels (PSI) in the step initiation between subjects with Parkinson\'s disease (PD) with freezing of gait (FoG), non-FOG (non-FoG), and age-matched healthy controls (HC) and, possible correlations between the PSI levels with the behavioral variables and the New Freezing of Gait Questionnaire (NFOG-Q) scores. Twenty individuals with FoG, 12 individuals with non-FoG (stage 3 of PD assessed in the clinically defined on state), and 16 HC met the inclusion criteria. Subjects visited the laboratory for two days. On the first day, all of the subjects answered the anamnesis and the Mini-Mental State Examination. However, only in the subjects with PD were assessed the severity of disease and the motor symptoms (Unified Parkinson´s Disease Rating Scale part III [UPDRS-III], but the FoG severity was assessed only in the subjects with FoG. On the second day, all of the subjects performed the step initiation on the force platform (AMTI) to assess amplitude and time of anticipatory postural adjustment with or without evoking H-reflex in the test or conditioned condition. The constant-current stimulator (Nicolet® Viking Quest portable EMG apparatus, CareFusion) was used to evoke the H-reflex. The electrical stimulus was evoked when the APA amplitude exceeded 10 to 20% of the baseline. All of the subjects performed the step initiation in 3 conditions: 1) without stimulation of the nerve; 2) test stimulus stimulation of the tibial nerve; and 3) conditioning stimulus - stimulation of the fibular nerve before (100 ms interval) stimulation of the tibial nerve. At three conditions, subjects were instructed to perform 15 steps, thus, 15 stimuli at conditions 2 and 3 in a random order. The ANOVA one way showed significant differences in the PSI values between the three groups, where the ISPI presented higher values of PSI than the other two groups, and S-BM individuals had higher PSI values compared to individuals with BM (P<0.001), however all of the individuals with FoG presented facilitation. In addition, larger amplitudes and smaller times of APA were observed only for ISPI (P<0.05); in addition, no differences were observed between these variables for the two PD groups (P>0.05). In relation to correlations, especially for individuals with FoG, we observed that greater values of facilitation are: strongly associated with the higher NFOG-Q scores (r = -0.74; P<0.0001); moderately associated with the smaller APA amplitudes (r = 0.54; P<0.004); and weakly associated with higher APA times (r = -0.42, P<0.244). Finally, there was a strong association between the smaller APA amplitudes and higher NFOG-Q scores (r = -0.75, P<0.0001). In conclusion, individuals with FoG presented facilitation (absence of PSI) in the step initiation which was associated with deficits in amplitude and time of APA and FoG severity. This indicates that the spinal cord has participation in the step initiation that can be strongly influenced by the abnormal sensory motor

O uso imprudente de antibióticos para o tratamento da mastite bovina pode levar ao aumento da resistência bacteriana e ao comprometimento da eficácia dos tratamentos atuais. Desta maneira, novas alternativas para o tratamento da mastite devem ser procuradas. As actinobactérias são capazes de produzir extratos brutos que contêm metabólitos secundários ativos, que inibem o crescimento de outras bactérias. Portanto, o presente estudo teve como objetivo avaliar a concentração inibitória mínima (CIM) de extratos brutos de actinobactérias para isolados de Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae e Streptococcus uberis, provenientes de 23 rebanhos leiteiros. Estes isolados foram identificados pela cultura microbiológica e por espectrometria de massas. Assim, foi realizada uma triagem, pelo bioensaio colorimétrico, de 15 extratos brutos, previamente testados e identificados, provenientes de actinobatérias isoladas de diferentes ambientes e plantas (caatinga, áreas de reflorestamento e do eucalipto) para 10 isolados de cada espécie causadora de mastite. Em seguida, os extratos Caat 1-54 e Caat P5-8 foram selecionados para determinação da CIM, a 50% (CIM50) e a 90% (CIM90). Para determinar a CIM dos extratos brutos, foram utilizados 20 isolados de cada espécie, totalizando 80 isolados. Foi utilizada a análise de sobrevivência para avaliação dos resultados. O extrato Caat 1-54 apresentou os menores valores de CIM50 e CIM90 para S. aureus (0,39 µg/mL e 6,25 µg/mL, respectivamente) e S. chromogenes (CIM50 = CIM90 = 0,78 µg/mL). O ceftiofur apresentou os menores valores de CIM tanto para Strep. dysgalactiae (CIM50 &le; 0,048 µg/mL; CIM90 = 100 µg/mL), como para Strep. uberis (CIM50 &le; 0,19 µg/mL; CIM90 = 0,39 µg/mL). Seguido do ceftiofur, o extrato Caat 1-54 foi o que apresentou menores valores CIM, com apenas uma diluição seriada de diferença entre as CIM50 das duas espécies (1,56 e 0,78 µg/mL para Strep. dysgalactiae e Strep. uberis, respectivamente). O extrato Caat P5-8 apresentou os maiores valores de CIM50 e CIM90 para todas as espécies estudadas (CIM &ge; 25 µg/mL). Os resultados obtidos indicam o potencial dos extratos brutos provenientes de actinobactérias para o tratamento da mastite bovina causada por S. aureus, S. chromogenes, Strep. dysgalactiae e Strep. uberis / The reckless use of antibiotics for the treatment of bovine mastitis can increase bacterial resistance and harm the effectiveness of current treatments. Thus, new alternatives for mastitis treatment must be searched. Actinomycetes are capable to produce crude extracts with secondary active metabolites that inhibit the growth of other bacteria. Therefore, the aim of this study was to evaluate the minimum inhibitory concentration (MIC) of crude extracts obtained from actinomycetes for Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae and Streptococcus uberis isolated from 23 dairy herds. These bacteria were identified by microbiology culture and mass spectrometry. Thus, the colorimetric bioassay was performed. In this screening 15 crude extracts previously tested and identified, obtained from actinomycetes from different Brazilian environments and plants (caatinga, reforestation areas and eucalyptus) were used for 10 isolates of each mastitis pathogens species. Then the Caat 1-54 and Caat P5-8 extracts were selected to the MIC 50% (MIC50) and 90% (MIC90) determination. Twenty mastitis pathogens isolates of each bacteria species were used. Survival analysis was used to evaluate the results. Caat 1-54 extract presented the lowest values of MIC50 e MIC90 for S. aureus (0.39 µg/mL and 6.25 µg/mL, respectively) and S. chromogenes (MIC50 = MIC90 = 0.78 µg/mL). On the other hand, ceftiofur presented the lowest MIC values for both Strep. dysgalactiae (MIC50 &le; 0.048 µg/mL; MIC90 = 100 µg/mL) and Strep. uberis (MIC50 &le; 0.19 µg/mL; MIC90 = 0.39 µg/mL). After ceftiofur, Caat 1-54 presented the highest efficiency against Strep. dysgalactiae and Strep. uberis and MIC50 for both species differed by only one serial dilution (1.56 e 0.78 µg/mL for Strep. dysgalactiae and Strep. uberis, respectively). Caat P5-8 extract presented the highest MIC50 and MIC90 values for all pathogens (MIC &ge; 25 µg/mL). Results indicated the potential of crude extracts from actinomycetes to treat bovine mastitis caused by S. aureus, S. schromogenes, Strep. dysgalactiae and Strep. uberis