Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical analysis Candidate Michaela Beranová Supervisor doc. PharmDr. Miroslav Miletín, Ph.D. Title of thesis Preparation of fluorescent azaphthalocyanines for labeling of oligonucleotide probes I. Azaphthalocyanines (AzaPc) are macrocyclic compounds with large system of conjugated bonds and are therefore used as dyes and pigments. Their potential as diagnostic and therapeutic means is also under intensive examination. The aim of this thesis is a synthesis of precursors for cyclotetramerization of asymmetric fluorescent tetrapyrazinoporphyrazines (TPyzPz) via solid phase method bearing two various moieties, one suitable for binding to solid phase and the other intended for binding of the final TPyzPz to oligonucleotides. At the beginning of the thesis, basic terms related to AzaPc are defined. Also the main principles of synthesis of TPyzPz as well as possible modifications of their final structure are discussed. An important part of the thesis is the description of the typical properties of TPyzPz, the influence of the structure on the individual parameters and the fields of their use. Afterwards the preparation of each precursor with two different substituents for cyclotetramerization is...

Neste trabalho investigou-se o desenvolvimento de biofilmes anaeróbios em um sistema de laboratório chamado de \"Modified Robbins Device\" (MRD). O objetivo específico foi o de comparar a organização das células anaeróbias, particularmente daquelas que são comuns em lodos de esgoto, sobre superfícies hidrofílicas (vidro) e hidrofóbicas (polipropileno). A hibridação in situ com sondas fluorescentes complementares ao RNAr 16S específicas para domínio e grupos e a microscopia confocal de varredura a laser foram utilizadas para verificar a composição microbiana dos biofilmes, bem como do inóculo. Foram realizados dois tipos de experimentos, um com culturas puras de metanogênicas e outro com células oriundas de lodo granulado anaeróbio. As culturas puras de metanogênicas, Methanobacterium formicicum (DSM 1535), Methanosaeta concilii (DSM 3671) e Methanosarcina barkeri (DSM 800) foram usadas como inóculo para a formação dos biofilmes no interior do MRD durante 9 dias. Os resultados mostraram que as três espécies colonizaram ambas as superfícies após o segundo e sétimo dia de ensaio. No segundo experimento, o MRD foi inoculado com um consórcio microbiano anaeróbio e a formação do biofilme foi estudada durante 22 dias. As amostras dos biofilmes bem como aquelas retiradas do frasco-reservatório de células apresentaram composição microbiana semelhante, ambas foram dominadas por Archaeae metanogênicas hidrogenotróficas relacionadas com membros da família Methanobacteriaceae, já que foram detectadas com a sonda MB1174. Este grupo contribuiu com cerca de 44 a 90% do total de células coradas com DAPI e foi morfologicamente semelhante à Methanobacterium e Methanobrevibacter. As células detectadas com a sonda específica para membros da ordem Methanomicrobiales (MG1200) representaram cerca de 2 a 18,0% do total de células coradas com DAPI no frasco-reservatório e de 0,1 a 2,0% nas amostras dos biofilmes. Estas células foram ) morfologicamente semelhantes à Methanospirillum, também uma metanogênica hidrogenotrófica. Não foram detectadas células pertencentes à família Methanosarcinaceae, pois a hibridação com a sonda MSMX860 foi negativa. Células que hibridaram com a sonda específica para o Domínio Bacteria (EUB338) representaram cerca de 2 a 18% do total de células coradas com DAPI. Os resultados mostraram que as Archaeae metanogênicas hidrogenotróficas que foram predominantes no inóculo também dominaram os biofilmes que se desenvolveram em ambas as superfícies, vidro e polipropileno. Os dados desse trabalho sugerem que a hidrofobicidade do material suporte não influenciou o desenvolvimento e a composição microbiana dos biofilmes anaeróbios, considerando as condições específicas dos ensaios realizados. / In this study the development of anaerobic biofilms using a laboratory system called modified robbins device (MRO) were investigated. We were especially interested in comparing the organization of anaerobic cells, particularly those that are very common in domestic sewage sludge, in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescence in situ hybridization (FISH) with domain and group speci fie probes that target intracell ular 16S rRNA and confocal laser scanning microscopy (CLSM) were used to investigate the microbial composition of both the inoculum and anaerobic biofilms. Two sets of experiments were carried, one with pure methanogenic organisms and the other with cells from a mesophilic anaerobic granular sludge. The pure methanogenic cultures, Methanobacterium formicicum (OSM 1535); Methanosaeta conci/ii (OSM 3671) and Methanosarcina barkeri (OSM 800) were used to seed the MRD to allow the development of biofilms over 9 days. The results showed that ali the three species were colonizing both surfaces after 2 and 7 days of experimental period. In the second experiment, the biofilm reactor was seeded with a microbial anaerobic consortium and biofilm forrnation was studied during 22 days. Biofilm and culture vessel samples showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MBI174). This group accounted for 44 to 90% of the OAPI-stained cells and morphologically resembled Methanobacterium and Methanobrevibacter. Cells detected with the Methanomicrobiales specific probe (MG 1200) accounted for 2 to 18.0% of the OAPI-stained cells in the culture vessel and 0.1 to 2.0% in the biofilm samples. These cells were morphologically similar to Methanospiriltum, also a hydrogenotrophic methanogen. No cells were detected by the Methanosarcinaceae specific probe (MSMX860). Cells which hybridized to the Bacteria specific probe (EUB338) accounted for the remaining 3 to 18% of the DAPI-stained cells. The results showed that the hydrogenotrophic methanogenic Archaea cells predominated in the inoculum and the biofilms that developed on both surfaces, glass and polypropylene. Our data suggest that the hydrophobicity of the support material did not influence the development and the microbial composition of anaerobic biofilms, considering specific conditions of the experiments.

Neste trabalho investigou-se o desenvolvimento de biofilmes anaeróbios em um sistema de laboratório chamado de \"Modified Robbins Device\" (MRD). O objetivo específico foi o de comparar a organização das células anaeróbias, particularmente daquelas que são comuns em lodos de esgoto, sobre superfícies hidrofílicas (vidro) e hidrofóbicas (polipropileno). A hibridação in situ com sondas fluorescentes complementares ao RNAr 16S específicas para domínio e grupos e a microscopia confocal de varredura a laser foram utilizadas para verificar a composição microbiana dos biofilmes, bem como do inóculo. Foram realizados dois tipos de experimentos, um com culturas puras de metanogênicas e outro com células oriundas de lodo granulado anaeróbio. As culturas puras de metanogênicas, Methanobacterium formicicum (DSM 1535), Methanosaeta concilii (DSM 3671) e Methanosarcina barkeri (DSM 800) foram usadas como inóculo para a formação dos biofilmes no interior do MRD durante 9 dias. Os resultados mostraram que as três espécies colonizaram ambas as superfícies após o segundo e sétimo dia de ensaio. No segundo experimento, o MRD foi inoculado com um consórcio microbiano anaeróbio e a formação do biofilme foi estudada durante 22 dias. As amostras dos biofilmes bem como aquelas retiradas do frasco-reservatório de células apresentaram composição microbiana semelhante, ambas foram dominadas por Archaeae metanogênicas hidrogenotróficas relacionadas com membros da família Methanobacteriaceae, já que foram detectadas com a sonda MB1174. Este grupo contribuiu com cerca de 44 a 90% do total de células coradas com DAPI e foi morfologicamente semelhante à Methanobacterium e Methanobrevibacter. As células detectadas com a sonda específica para membros da ordem Methanomicrobiales (MG1200) representaram cerca de 2 a 18,0% do total de células coradas com DAPI no frasco-reservatório e de 0,1 a 2,0% nas amostras dos biofilmes. Estas células foram ) morfologicamente semelhantes à Methanospirillum, também uma metanogênica hidrogenotrófica. Não foram detectadas células pertencentes à família Methanosarcinaceae, pois a hibridação com a sonda MSMX860 foi negativa. Células que hibridaram com a sonda específica para o Domínio Bacteria (EUB338) representaram cerca de 2 a 18% do total de células coradas com DAPI. Os resultados mostraram que as Archaeae metanogênicas hidrogenotróficas que foram predominantes no inóculo também dominaram os biofilmes que se desenvolveram em ambas as superfícies, vidro e polipropileno. Os dados desse trabalho sugerem que a hidrofobicidade do material suporte não influenciou o desenvolvimento e a composição microbiana dos biofilmes anaeróbios, considerando as condições específicas dos ensaios realizados. / In this study the development of anaerobic biofilms using a laboratory system called modified robbins device (MRO) were investigated. We were especially interested in comparing the organization of anaerobic cells, particularly those that are very common in domestic sewage sludge, in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescence in situ hybridization (FISH) with domain and group speci fie probes that target intracell ular 16S rRNA and confocal laser scanning microscopy (CLSM) were used to investigate the microbial composition of both the inoculum and anaerobic biofilms. Two sets of experiments were carried, one with pure methanogenic organisms and the other with cells from a mesophilic anaerobic granular sludge. The pure methanogenic cultures, Methanobacterium formicicum (OSM 1535); Methanosaeta conci/ii (OSM 3671) and Methanosarcina barkeri (OSM 800) were used to seed the MRD to allow the development of biofilms over 9 days. The results showed that ali the three species were colonizing both surfaces after 2 and 7 days of experimental period. In the second experiment, the biofilm reactor was seeded with a microbial anaerobic consortium and biofilm forrnation was studied during 22 days. Biofilm and culture vessel samples showed nearly the same microbial composition, both were dominated by hydrogenotrophic methanogenic Archaea related to the Methanobacteriaceae as detected by the specific probe (MBI174). This group accounted for 44 to 90% of the OAPI-stained cells and morphologically resembled Methanobacterium and Methanobrevibacter. Cells detected with the Methanomicrobiales specific probe (MG 1200) accounted for 2 to 18.0% of the OAPI-stained cells in the culture vessel and 0.1 to 2.0% in the biofilm samples. These cells were morphologically similar to Methanospiriltum, also a hydrogenotrophic methanogen. No cells were detected by the Methanosarcinaceae specific probe (MSMX860). Cells which hybridized to the Bacteria specific probe (EUB338) accounted for the remaining 3 to 18% of the DAPI-stained cells. The results showed that the hydrogenotrophic methanogenic Archaea cells predominated in the inoculum and the biofilms that developed on both surfaces, glass and polypropylene. Our data suggest that the hydrophobicity of the support material did not influence the development and the microbial composition of anaerobic biofilms, considering specific conditions of the experiments.

Marginale Parodontitis, die multikausale Erkrankung des Parodonts ist eine Infektionskrankheit, modifiziert durch Wirtsfaktoren und äußere Einflüße. Die als pathogene Mischflora bezeichnete Kombination kommensaler Mikroorganismen spielt die primäre Rolle in der Ätiopathogenese der Parodontitis. In der Aufstellung des Studienziels wurden einzelne Bakterienarten (T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula und C. ochracea) ausgewählt, die eventuell als "Markerkeime" in der aggressiven Form der Parodontitis betrachtet werden können. Dazu wurde eine Kontrollgruppe untersucht, die eine gesunde parodontale Flora besitzt. Die angewandte Nachweismethode basiert auf der PCR-Amplifikation von 16S rDNA und darauffolgender dot-blot Hybridisierung mit Oligonukleotidsonden. Die entsprechenden Sonden wurden hergestellt, optimiert und evaluiert. Für die epidemiologische Untersuchung wurde subgingivale Plaque von vier Parodontaltaschen und einer Kontrollstelle von 45 Patienten mit aggressiver Parodontitis, sowie an fünf Stellen von 21 Senioren entnommen. Die Prävalenz der einzelnen Bakterienarten wurde mit Hilfe des Chi-Quadrat Test verglichen. Obgleich eine hohe interindividuelle Variabilität der Kolonisationsmuster zu beobachten war, konnten T. forsythensis, P. gingivalis, C. rectus und F. nucleatum signifikant häufiger in den Parodontaltaschen als an den gesunden Stellen nachgewiesen werden und können deswegen als "Leitkeime" der aggressiven Parodontitis angesehen werden. A. actinomycetemcomitans konnte nur bei einzelnen Patienten mit aggressiver Parodontitis festgestellt werden. Die Ergebnisse für P. intermedia und E. corrodens ließen keine eindeutige Assoziation sowohl mit der aggressiven Parodontitis als auch mit dem gesunden Parodontalzustand zu. Bei Senioren wurde C. ochracea besonders häufig nachgewiesen. Die Ergebnisse dieser Studie bewiesen die erfolgreiche Einsetzbarkeit der hergestellten Oligonukleotidsonden. / A multifactorial risk pattern of periodontitis has been recognized, where in addition to host and environmental factors, a pathogenic microbiota plays a primary role. The purpose of the current research was to analyze the prevalence of periodontitis-associated microorganisms in patients with aggressive periodontitis and periodontally healthy elders by using molecular-biologic detection methods like eubacterial PCR-amplification of 16S rDNA in combination with dot-blot hybridization. The oligonucleotide probes for the detection of T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula and C. ochracea were designed and evaluated. The PCR products of 42 cultivated target and closely related bacteria were used for the optimization of hybridization conditions. For the epidemiological study, subgingival plaque was sampled from four pockets and one healthy site of 45 aggressive periodontitis patients as well as from five sites of 21 elderly. The differences in the prevalence of bacterial species were analyzed by the chi-square test. The data revealed frequent colonization by T. forsythensis, P. gingivalis, F. nucleatum and C. rectus in patients with aggressive periodontitis, however individual variations were obvious. These species could be predominantly identified in periodontal pockets, but were significantly less common in the healthy sites of the periodontitis patients and in the elderly. These putative pathogens can be conclusively determined as the key-bacteria in patients with aggressive periodontitis. No direct association for P. intermedia and E. corrodens with aggressive periodontitis or periodontal health could be seen. A. actinomycetemcomitans could be detected in only a few patients, reducing its suspected importance in the etiology of aggressive periodontitis. C. ochracea was highly prevalent in the well-maintained elderly, suggesting its association with healthy flora. The results of the study confirmed the reliability of the oligonucleotide probes in a specific and sensitive detection of the respective oral species.