>Magister Scientiae - MSc / Tuberculosis (TB), a deadly disease second to HIV/AIDS, is a global health problem. Diagnosis of active tuberculosis is tedious and requires expensive procedures since there is no recognizable method for sole detection of active TB. Although this is a deadly disease, treatment drug toxicity is also an issue that also causes fatalities in diagnosed patients. Therefore, a rapid sensitive and specific diagnostic method is imperative for TB drug management. In this study spectroscopic and/or electrochemical biosensors were developed for the detection and quantification of TB treatment drugs. The biosensors were constructed with electroactive layers of graphene oxide coupled to silver nanoparticles and/or zinc oxide nanoparticles. These nanoparticles coupled with graphene oxide sheets were covalently attached onto the enzymes such as Cytochrome P450-2D6 to achieve the electrochemical detection of the TB treatment drugs and obtain the required electron transfer between the electrode surface and enzyme. The surface morphology of graphene oxide, nanoparticles as well as the green synthesized nanocomposites were achieved using High-Resolution Transmission Electron Microscopy (HRTEM), Atomic Force Microscopy (AFM), and High- Resolution Scanning Electron Microscopy (HRSEM) while the elemental analysis were obtained using Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-Ray (EDX), Raman spectroscopy and X-Ray diffraction (XRD). Additionally, the optical properties of the developed nanocomposites where further characterised using Small Angle X-ray Scattering (SAXS), Photoluminescence Spectroscopy (PL) and Ultraviolet Spectroscopy (UV-vis). The electrochemical studies were obtained using cyclic voltammetry (CV) and showed an increase in electron conductivity for the green synthesized zinc oxide nanoparticles coupled with graphene oxide (ZnONPs/GO) and silver nanoparticles coupled with graphene oxide (AgNPs/GO) nanocomposite which was an indication that they were suitable as platforms towards biosensor development. Furthermore, amperometric technique was also used for biotransformation of the TB treatment drugs (Ethambutol and Pyrazinamide) in standard solutions of 0.1 M phosphate buffer (pH 7.0). Furthermore, the sensitivity value of 0.0748 μA/μM was determined for the ethambutol biosensor while a value of 0.1715 μA/μM was determined for the pyrazinamide biosensors. Very good detection limits were obtained for the standard solutions of ethambutol and pyrazinamide where a value of 0.02057 nM was determined for ethambutol at concentration linear range of 50 μM – 400 μM. Additionally, a value of 0.8975 x 10-2 nM was determined for pyrazinamide at the concentration linear range of 100 μM – 300 μM. The determined limit of detections have provided a clear indication that these biosensors have potential of being used in human samples since these values are below the peak serum concentrations of these drugs in TB diagnosed patients as reported in literature. This was further confirmed by the limit of quantification values determined for each biosensor where a value of 0.8975 x 10-2 nM was determined for pyrazinamide and a value of 0.02057 nM was determined for ethambutol.

Masters Research - Fuculty of Health Sciences / Tuberculosis (TB) is one of the leading causes of mortality in the developing world. Mycobacterium tuberculosis, the causative organism, infects approximately a third of the world’s population. With high rates of HIV infection, particularly in sub-Saharan Africa, TB rates are inevitably soaring. Treatment regimens are based on multi-drug therapy taken over a prolo nged period, leading to poor patient compliance, in turn resulting in the development of multi-drug resistant TB (MDR-TB) which is difficult to treat. The need for new anti-TB drugs that can decrease the period of treatment or the number of doses and that will be effective against latent and MDR-TB is desperate. An added advantage would be the ability of a new class of anti-TB drugs to have a novel target to avoid potential crossresistance to existing drugs. Various approaches have been taken to antituberculosis drug development, including the high-throughput screening of plants, which represent an enormous, largely untapped resource of novel compounds. A further parameter to increase the chances of finding promising lead compounds is to focus research on plants that have traditionally been used to treat symptoms associated with TB. Traditional herbalists prescribing plant-based treatments have long played an important role in the provision of primary healthcare, especially in rural areas where most of the population is poor and unable to afford modern drugs. The aim of this study is to research literature resources pertaining to medicinal plants in southern Africa used to treat symptoms associated with TB, collect these plants, prepare methanol and acetone extracts for the antimicrobial and antimycobacterial testing, identify a plant with promising activity, and to isolate the active principle/s. These compounds in turn would be identified structurally and tested for activity against a range of micro-organisms, including mycobacteria, as well as for cytotoxicity. Twenty-three plant parts from nineteen different species were collected and 46 extracts prepared. These extracts were tested against a range of Gram-positive and –negative bacteria and fungi using the disc diffusion and broth micro-dilution methods. The effects of these extracts were also tested against non-pathogenic mycobacteria using the broth microdilution method, and against M. tuberculosis using the radiometric BACTEC 460 ABSTRACT V method. Te n of the nineteen species exhibited activity against two or more of the eleven organisms tested, namely Xerophyta retinervis bark, Helichrysum odoratissimum leaves, Ozoroa paniculosa bark, Eriocephalus africanus leaves, Siphonochilus aethiopicus roots, Conyza scabrida leaves, Syzigium cordatum bark, Tetradenia riparia leaves, Datura stramonium leaves and Dioscorea sylvatica tubers. The acetone extract of O. paniculosa was further pursued for the isolation of its active principles as it exhibited potent activity against Enterococcus faecalis, Pseudomonas aeruginosa, M. tuberculosis and Mycobacterium aurum with MIC’s of less than 1mg/ml. Previous work on related species has indicated anti-cancer, anti-helminthic, anti- malarial and anti-schistosomiasis activities, but no antimicrobial or antimycobacterial properties have been researched. Moronic, anacardic, and ginkgolic acids have previously been isolated from related species. Bio-assay guided fractionation led to the isolation of a known C15:1 anacardic acid (compound 1) and two HPLC fractions (HPLC2 and HPLC3) of which the predominant components of HPLC3 is the saturated analogue of anacardic acid. NMR data of HPLC2 suggest it is also a C15:1 anacardic acid, although the location of its double bond is as yet unkown. These compounds were cytotoxic to CHO cells at 44.9 – 64μg/ml. Compound 1 and HPLC2 were moderately active against M. tuberculosis with MIC’s of 125μg/ml, while HPLC3 had increased activity with an MIC of 31.3μg/ml, work not previously reported. Similarly, HPLC3 had the greatest activity a gainst M. smegmatis with an IC50 value of 22.1μg/ml. M. aurum had higher IC 50 values ranging from 98.4 to 112.5μg/ml for all three compounds. Compound 1 had potent activity against a range of Grampositive bacteria with IC50 values of 1.3, 2.1 and 6.5μg/ml against Bacillus cereus, E. faecalis and Staphylococcus aureus respectively. Furthermore, this compound had good activity against one of two drug-resistant strains of S. aureus tested with IC50’s of 6.9 and 43.2μg/ml. The saturated anacardic acid (HPLC3) in general had poorer activity against Gram-positives than its unsaturated analogues, consistent with reported literature. The anacardic acids had decreased activity against yeast and Gram-negative organisms tested, with IC50 values of around 80μg/ml against Candida albicans, 19 ->250μg/ml for Serratia odorifera, 26 - >250μg/ml for Klebsiella pneumoniae and 17 – 68μg/ml for ABSTRACT VI Pseudomonas aeruginosa, results comparable to those previously reported. The saturated anacardic acid appeared to have better activity than its unsaturated analogues against Gram- negatives and yeast. The current literature suggests that lipophilic compounds have greater activity against M. tuberculosis. Results obtained in this project are in support of these findings, as the saturated anacardic acid, believed to be the major constituent of HPLC3 based on NMR data available, had increased activity against this organism compared to the unsaturated and less hydrophobic compound 1 and HPLC2. It is possible that these findings implicate anacardic acid in the disruption of the mycobacterial cell wall.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Science in Medicine (Pharmaceutical affairs) Johannesburg, 1998 / Tuberculosis control in South Africa continues to be a major challenge with 90 000 new cases reported in 1995. Major contributory factors towards these epidemic proportions are patient non-compliance and the occurrence of drug resistant tuberculosis. Fixed drug combinations of anti-tuberculosis drugs have been reported to reduce the possibility of resistance arising to any one of the drugs in combination and to improve compliance. However, the combination anti-tuberculosis drugs available at present still suffer the disadvantage of patients having to take 6 tablets per day. Therefore, the purpose of this project was to investigate a formulation that would reduce this disadvantage. / IT2018

A Pirazinamida (Z), droga de primeira linha usada no tratamento da tuberculose, necessita ser hidrolisada pela enzima bacteriana pirazinamidase (PZase) para que o seu metabólito ativo, o ácido pirazinóico (POA), possa agir. O principal mecanismo molecular de resistência a esta droga envolve mutações no gene pncA, que codifica a PZase. Com base nestas informações e tendo em vista a ausência de estudos acerca de resistência à Z em isolados clínicos de M. tuberculosis em nosso país, o presente trabalho propôs caracterizar as mutações envolvendo o gene pncA, bem como relacioná-las com os resultados do teste de atividade da enzima PZase e da concentração inibitória mínima (CIM) de Z. A caracterização molecular dos isolados foi realizada por \"Spoligotyping\", sendo que, todos os isolados testados foram confirmados como pertencentes à espécie M. tuberculosis. A CIM foi realizada por três metodologias: técnica em microplaca utilizando o Alamar Azul como revelador (MABA), método de microdiluição em caldo (BMM), e método das proporções em Lowenstein-Jensen. Os resultados obtidos dão conta de uma boa associação entre as metodologias, e a determinação da CIM pelo método MABA mostrou-se uma nova e segura opção a ser utilizada para Z. A maioria dos isolados clínicos de M. tuberculosis resistentes à Z (88%), apresentaram também atividade de PZase negativa, bem como mutações no gene pncA. Algumas exceções foram encontradas, já que 12% dos isolados clínicos resistentes não apresentaram mutações no gene pncA e tiveram atividade da PZase positiva, sugerindo a existência de outro mecanismo envolvido com resistência à Z. Das 22 mutações encontradas no gene pncA, 9 estão sendo descritas apenas neste estudo. Registrou-se também a presença de 5 isolados clínicos apresentando fenótipo de monorresistência à Z. / Pyrazinamide (Z), a first-line antituberculous drug, is a prodrug that must be activated by bacterial pyrazinamidase (PZase) to the active form pyrazinoic acid, which kills M. tuberculosis. Many studies have shown that mutation in the gene encoding PZase (pncA) is the major mechanism of Z-resistance in M. tuberculosis. Based on this information and taking into consideration the absence of studies concerning Z-resistance in Brazilian M. tuberculosis strains, this study was aimed at characterizing pncA mutations and investigating its correlation with Z-resistance and PZase activity. The molecular characterization carried out by Spoligotyping revealed that all tested strains belong to M. tuberculosis species. The minimal inhibitory concentration (MIC) of Z was determined by three methods: microplate Alamar Blue assay (MABA), broth microdilution method (BMM) and method of proportions on Lowenstein-Jensen medium. The results showed a good association between the 3 methods, and MABA for MIC determination signalized a new and safe option to be used for Z. Most of Z-resistant strains (88%) presented pncA mutations as well as loss of PZase activity. Some exceptions were found since 12% of Z-resistant strains presented neither pncA mutations nor loss of PZase activity, what suggests the existence of another Z-resistance mechanism. Nine of 22 mutations found in pncA gene were described only in this study. During the course of this investigation were identified 5 Z-monoresistant M. tuberculosis strains.

Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-17Bitstream added on 2014-06-13T19:27:32Z : No. of bitstreams: 1 baldan_hm_me_arafcf.pdf: 708297 bytes, checksum: eb9f42b90c2538af491182c884f6a251 (MD5) / Este estudo teve como objetivo avaliar os efeitos da administração simultânea de PYR sobre os parâmetros farmacocinéticos da INH e a produção de seus metabólitos, em um grupo de animais sob tratamento com INH+PYR e outro com INH+RMP+PYR, bem como de avaliar o comportamento dos biomarcadores de hepatotoxicidade, as transaminases AST e ALT, nesses grupos. Na primeira fase do protocolo experimental foi coletado sangue da cauda dos animais (ratos Wistar, machos, peso~250g) para análise dos biomarcadores como valores basais, em seguida realizou-se a administração, por gavagem, de doses múltiplas de INH (100mg/Kg) (grupo I), INH+PYR (350mg/Kg) (grupo II), INH+PYR+RMP (100mg/Kg) (grupo III), PYR (grupo IV) e água estéril (grupo V controle) por 21 dias. Para os animais que receberam INH (grupo I, grupo II e grupo III) construiu-se a curva de sua concentração plasmática x tempo. Amostras seriadas de sangue foram coletadas em 10 tempos diferentes entre 0-24h; para cada tempo de coleta foram empregados 5 ratos. As amostras foram desproteinizadas com ácido tricloroacético a 10%; para análise dos metabólitos acetilados promovendo a hidrólise com HCl 6M; em seguida, a INH e a Hz foram derivatizadas com cinamaldeído. As amostras foram analisadas por HPLC usando coluna Resolve TM C18 e detector UV-VS, operando a 340nm. Os parâmetros farmacocinéticos da INH apresentam diferenças estatisticamente significativas (p<0,05, teste de Tukey) entre os grupos, e sua média e EPM foram: Grupo INH vs Grupo INH+PYR: t1/2 = 1,4 (0,070) vs 1,0 (0,100); K = 0,51 (0,024) vs 0,77 (0,100); t1/2 = 3,4 (0,026) vs 7,2 (0,910); Kel = 0,21 (0,015) vs 0,10 (0,018); ClT/F = 0,68 (0,016) vs 0,90 (0,047); Vd/F = 3,32 (0,19) vs 9,52 (1,52); AUC0-24 ss INH = 146,36 (3,25) vs 111,96 (5,60); AUC0-24 ss Hz = 2,87 (0,07) vs 4,60 (0,09)... / The aim of the present study was to evaluate the hepatotoxicity effect, pharmacokinetic response and biotransformation of isoniazid of rats treated with i) isoniazid (INH), ii) INH + pyrazinamide (PYR) and iii) INH + PYR + rifampicin (RMP). Daily doses of the tuberculostatic drugs were administrated intragastrically to the animals (Wistar rats) for one period of 21 days as follow: INH (100mg/Kg) (group I), INH (100mg/Kg) + PYR (350mg/Kg) (group II), INH (100mg/Kg) + PYR (350mg/Kg) + RMP (100mg/Kg) (group III), PYR (350mg/Kg) (group IV) and sterile water (group V, control). The serum levels of the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined before the administration of the drugs (basal) and after the 21 days treatments. On day 21, blood samples were obtained before and 0,25; 0,5; 0,75; 1; 1,5; 3; 6; 12 and 24 hours after the dose (five animals for each point). The blood samples were deproteinized with 10% trichloroacetic acid, derivazed by 1% cinnamaldehyde. For the determination of the acetylated metabolites acetylisoniazid (AcINH) and acetylhydrazine (AcHz) a previous hydrolysis with 6 M hydrochloride acid was performed. Then, the samples were centrifuged and the supernatant analyzed by liquid chromatograph. The results are presented as mean and SEM. The pharmacokinetic parameter of the INH and its metabolites AcINH and hydrazine (Hz) were compared between the groups (p < 0.05, Turkey s test). The results significantly different were: Group INH vs Group INH+PYR: t1/2 = 1,4 (0,070) vs 1,0 (0,100); K = 0,51 (0,024) vs 0,77 (0,100); t1/2 = 3,4 (0,026) vs 7,2 (0,910); Kel = 0,21 (0,015) vs 0,10 (0,018); ClT/F = 0,68 (0,016) vs 0,90 (0,047); Vd/F = 3,32 (0,19) vs 9,52 (1,52); AUC0-24 ss (INH) = 146,36 (3,25) vs 111,96 (5,60); AUC0-24 ss (Hz) = 2,87 (0,07) vs 4,60 (0,09)... (Complete abstract, click electronic address below)

>Magister Scientiae - MSc / The tuberculosis (TB) disease to this day remains one of the world’s prominent killerdiseases. Pyrazinamide (PZA) is one of the most commonly prescribed anti- tuberculosis (anti-TB) drugs due to its ability to significantly shorten the TB treatment period from the former nine months to the current six months duration. However, excess PZA in the body causes hepatotoxicity and damages the liver. This hepatotoxicity, together with the resistance of the bacteria to treatment drugs, poor medication and inappropriate dosing, greatly contribute to the high incidents of TB deaths and diseases that are due to side effects (such as liver damage). This brings about the calls for alternative methods for ensuring reliable dosing of the drug, which will be specific from person to person due to inter-individual differences in drug metabolism. A novel biosensor system for monitoring the metabolism of PZA was prepared with a Ni-PPI-PPy star copolymer and cytochrome P450 2E1 (CYP2E1) deposited onto a platinum electrode. The nanobiosensor system exhibited enhanced electro-activity that is attributed to the catalytic effect of the incorporated star copolymer. The biosensor had a sensitivity of 0.142 µA.nM-1, and a dynamic linear range (DLR) of 0.01 nM-0.12 nM (1.231 – 7.386 ng/L PZA). The limit of detection of the biosensor was found to be 0.00114 nM (0.14 ng/L) PZA. From the HPLC peakconcentration (Cmax) of PZA determined 2 h after drug intake is 2.79 – 3.22 ng.L-1,which is very detectable with the nanobiosensor as it falls within the dynamic linear range.

Charles University, Pharmaceutical Faculty in Hradec Králové Department: Department of Pharmaceutical Chemistry and Pharmaceutical analysis Candidate: Martina Čečetková Supervisor: PharmDr. Jan Zitko, Ph.D. Title of Diploma Thesis: Pyrazinamide derivatives as potential antimicrobial compounds Even in 21st century, tuberculosis still remains a serious and global health threat. Tuberculosis is one of the 10 most common causes of death, the most burdened are developing countries, but this disease infects up to 1/3 of population worldwide. Due to ineffective treatment of tuberculosis in developing countries, the prevalence of tuberculosis which does not respond to standard treatment is increasing. It is necessary to develop new drugs effective against multidrug-resistant tuberculosis and prevent further spread of the disease. The design of final structures is based on previously synthesized molecule 6- chloro-N-(4-(4-fluorophenyl)thiazol-2-yl)pyrazine-2-carboxamide, which structure comes from first line antitubercular pyrazinamide (PZA) and 4-arylthiazol-2-amine scaffold with formerly identified antimycobacterial activity. This starting compound exhibits high activity against M. tuberculosis described by MIC = 0,78 µg/mL and low cytotoxicity. The object of study was to determine effect of substitution...

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-18T14:46:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) PRISCILA ROCHA. DISSERTAÇÃO.DIGITAL.pdf: 900577 bytes, checksum: 21461faf83aca3f0a9ae61d40c9c66e3 (MD5) / Made available in DSpace on 2016-08-18T14:46:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) PRISCILA ROCHA. DISSERTAÇÃO.DIGITAL.pdf: 900577 bytes, checksum: 21461faf83aca3f0a9ae61d40c9c66e3 (MD5) Previous issue date: 2015-08-26 / Objetivo: Descrever as reações adversas decorrentes do uso dos fármacos antituberculose do novo esquema preconizado pelo Ministério da Saúde (rifampicina, isoniazida, pirazinamida e etambutol) utilizando comprimidos em dose fixa combinada. Métodos: Estudo descritivo utilizando dados coletados em prontuários médicos de pacientes com tuberculose pulmonar tratados com esquema básico no serviço de referência para tuberculose da Paraíba no período de 2010-2014. Resultados: A amostra foi composta por 257 pacientes com tuberculose pulmonar e houve reação adversa em 71 pacientes (27,63%) totalizando 118 eventos. Esses eventos dividiram-se em reações menores e maiores respectivamente 47,9% e 43,7%, e em 8,5% dos casos, ocorreram ambas as reações. O sistema mais comprometido foi o gastrointestinal em 68,4%, seguido pelo osteoarticular em 13,56%. A hepatotoxicidade foi isoladamente a reação adversa mais comum (30,5%). A toxicidade visual ocorreu em apenas um paciente (0,38%). Houve necessidade de suspender o tratamento em 7,39% e de troca de esquema terapêutico em 1,95%. A frequência de reações adversas observada com o novo esquema foi inferior a de outros estudos nacionais e semelhante aos estudos que utilizaram o esquema antigo. Conclusão: As reações adversas foram principalmente alterações gastrointestinais, sendo a mais comum a hepatotoxicidade. Houve apenas um caso de alteração visual. Na maioria dos casos, não houve necessidade de suspensão nem troca do tratamento. / Objective: To describe the adverse reactions due to the use of antituberculosis drugs under the new regimen recommended by the Health Ministry (rifampicin, isoniazid, pyrazinamide and ethambutol) using tablets in fixed doses combination. Methods: A descriptive study using data collected from medical records of patients with pulmonary tuberculosis treated with basic outline reference service of tuberculosis in Paraíba during the period from 2010 to 2014. Results: The sample consisted of 257 patients with pulmonary tuberculosis, and adverse effects occurred in 71 patients (27.63%) totalizing 118 events. These events were divided into smaller and bigger reactions respectively 47.9% and 43.7%, and in 8.5% of the cases both reactions occurred. The most compromised system was the gastrointestinal at 68.4%, followed by osteoarticular at 13.56%. The hepatotoxicity alone was the most common adverse reaction (30.5%). The visual toxicity occurred in only one patient (0.38%). It was necessary to discontinue the treatment in 7.39% and change the regimen for 1.95%. The frequency of adverse reactions observed with the new scheme was lower than other national studies and similar to studies that used the old scheme. Conclusion: The adverse reactions were mainly gastrointestinal disorders, being hepatotoxicity the most common one, there was only one case of visual change. In most cases there was no need to suspend neither to change the treatment.

A Pirazinamida (Z), droga de primeira linha usada no tratamento da tuberculose, necessita ser hidrolisada pela enzima bacteriana pirazinamidase (PZase) para que o seu metabólito ativo, o ácido pirazinóico (POA), possa agir. O principal mecanismo molecular de resistência a esta droga envolve mutações no gene pncA, que codifica a PZase. Com base nestas informações e tendo em vista a ausência de estudos acerca de resistência à Z em isolados clínicos de M. tuberculosis em nosso país, o presente trabalho propôs caracterizar as mutações envolvendo o gene pncA, bem como relacioná-las com os resultados do teste de atividade da enzima PZase e da concentração inibitória mínima (CIM) de Z. A caracterização molecular dos isolados foi realizada por \"Spoligotyping\", sendo que, todos os isolados testados foram confirmados como pertencentes à espécie M. tuberculosis. A CIM foi realizada por três metodologias: técnica em microplaca utilizando o Alamar Azul como revelador (MABA), método de microdiluição em caldo (BMM), e método das proporções em Lowenstein-Jensen. Os resultados obtidos dão conta de uma boa associação entre as metodologias, e a determinação da CIM pelo método MABA mostrou-se uma nova e segura opção a ser utilizada para Z. A maioria dos isolados clínicos de M. tuberculosis resistentes à Z (88%), apresentaram também atividade de PZase negativa, bem como mutações no gene pncA. Algumas exceções foram encontradas, já que 12% dos isolados clínicos resistentes não apresentaram mutações no gene pncA e tiveram atividade da PZase positiva, sugerindo a existência de outro mecanismo envolvido com resistência à Z. Das 22 mutações encontradas no gene pncA, 9 estão sendo descritas apenas neste estudo. Registrou-se também a presença de 5 isolados clínicos apresentando fenótipo de monorresistência à Z. / Pyrazinamide (Z), a first-line antituberculous drug, is a prodrug that must be activated by bacterial pyrazinamidase (PZase) to the active form pyrazinoic acid, which kills M. tuberculosis. Many studies have shown that mutation in the gene encoding PZase (pncA) is the major mechanism of Z-resistance in M. tuberculosis. Based on this information and taking into consideration the absence of studies concerning Z-resistance in Brazilian M. tuberculosis strains, this study was aimed at characterizing pncA mutations and investigating its correlation with Z-resistance and PZase activity. The molecular characterization carried out by Spoligotyping revealed that all tested strains belong to M. tuberculosis species. The minimal inhibitory concentration (MIC) of Z was determined by three methods: microplate Alamar Blue assay (MABA), broth microdilution method (BMM) and method of proportions on Lowenstein-Jensen medium. The results showed a good association between the 3 methods, and MABA for MIC determination signalized a new and safe option to be used for Z. Most of Z-resistant strains (88%) presented pncA mutations as well as loss of PZase activity. Some exceptions were found since 12% of Z-resistant strains presented neither pncA mutations nor loss of PZase activity, what suggests the existence of another Z-resistance mechanism. Nine of 22 mutations found in pncA gene were described only in this study. During the course of this investigation were identified 5 Z-monoresistant M. tuberculosis strains.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Lucie Kučerová Supervisor: doc. PharmDr. Jan Zitko, Ph.D. Consultant: PharmDr. Martin Juhás Title of diploma thesis: Design, synthesis and evaluation of pyrazinamide derivatives as potential antimicrobial compounds II Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis complex and is currently one of the most common causes of death from an infectious disease. Treatment of tuberculosis is long-term, combined and controlled to prevent resistance. Resistance is very serious and therefore treatment is always performed with more antituberculars at the same time. Finding new drugs and improving existing ones is a constant part of research. In the theoretical part I tried to summarize information about tuberculosis, its causative agent, diagnostics, possible prevention and treatment strategy. I have described the most commonly used antituberculars, especially the first- line antituberculars - pyrazinamide, from which the derivatives synthesized in my work are based. In the experimental part I described the procedures and reactions used for synthesis of the new compounds, which were formed by combining pyrazinamide with various amino acids. In this...