Dermatofitoses são comumente causadas por fungos que parasitam pele e unha de humanos, cuja propagação depende do contato entre os hospedeiros infectados e não infectados. Muitos fatores contribuem para a patogenicidade dos dermatófitos, dentre eles, a capacidade de se instalar no ambiente ácido da pele se reveste de importância. Sendo assim, para ser bem sucedido, o dermatófito precisa ter capacidade aderente, germinação e penetração rápida das hifas e, portanto, dispor de uma maquinaria metabólica que atue de forma eficiente em pH ácido. A fim de identificar genes supostamente expressos nos passos iniciais da infecção, submetemos a linhagem H6 do dermatófito T. rubrum ao pH ácido por 30 minutos e 1 hora e isolamos dessas condições experimentais os transcritos com elevada expressão, empregando a metodologia de Biblioteca Subtrativa Supressiva (SSH). Obtivemos um total de 234 unigenes cujos transcritos revelaram ampla diversidade funcional. Esses transcritos estão envolvidos em 13 processos celulares diferentes, tais como, metabolismo, defesa e virulência, síntese de proteínas e transporte celular. Desses, confirmamos por Northern blotting, os genes que expressam as proteínas carboxipeptidase S1, acetoamidase, aconitase, dessaturase, a proteína TINA, transportador de aminoácidos, fator de alongamento alfa 1, proteína ribossomal L10, e uma proteína hipotética. Nesses experimentos também foi utilizada a linhagem de T. rubrum pacC-1, que tem o seu gene pacC rompido, com o objetivo de verificar se estes genes isolados seriam regulados pela proteína PacC. O gene pacC codifica uma proteína homóloga ao regulador transcricional PacC/Rim101p da conservada via de sinalização do pH. Verificamos que o gene pacC se expressa preferencialmente em pH 8.0 e que embora o padrão de processamento da proteína PacC seja dependente do pH a forma íntegra da proteína PacC foi identificada tanto em pH ácido como alcalino. Por outro lado, o mutante pacC-1 apresentou diminuida capacidade infectiva em fragmentos de unha humana quando comparado com a linhagem selvagem. Além disto, a atividade queratínolitica do mutante também se mostrou diminuída quando comparada ao controle, confirmando o papel da proteína PacC na capacidade infectiva do T. rubrum. / Dermatophytosis is commonly caused by fungi that parasite human skin and nail, whose propagation depends on the contact between infected and noninfected hosts. Many factors contribute to the pathogenicity of the dermatophytes. Among them, the capacity to install in the skin´s acid ambient bears great importance. Thus, in order to be successful, the dermatophyte needs to have adhering capacity, fast germination and penetration of hyphae and, therefore, needs to afford a metabolic machinery which acts efficiently in acid pH. In order to identify genes supposedly expressed in the initial steps of infection, we submitted the strain H6 of the dermatophyte T. rubrum to the acid pH for 30 minutes and 1 hour and isolated, from this experimental conditions, the transcripts with high expression, employing the suppression subtractive hybridization (SSH). We obtained a total of 234 unigenes whose transcripts revealed a wide functional diversity. These transcripts are involved in 13 different cell processes, such as metabolism, defense and virulence, protein synthesis and cell transport. Among these, we confirmed through Northern blotting the genes which express the proteins carboxipeptidase S1, acetamidase, aconitase, fatty acid desaturase, NIMA interactive protein (TINA), amino acid permease, elongation factor 1-alpha, 60S ribosomal protein L10 and a hypothetical protein. In these experiments, we also used the T. rubrum pacC- 1 strain, which has its pacC gene disrupted, aiming at verifying whether these isolated genes would be regulated by the PacC protein. The pacC gene encodes a protein homologous to the PacC/Rim101p transcriptional regulator of the conserved route of pH signaling. We verified that the pacC gene is expressed preferentially in both pH, and that although the processing pattern of the PacC protein is dependent on the pH, the full form of the PacC was identified as alkaline. On the other hand, the pacC-1 mutant presented diminished infecting capacity in human nail fragments when compared to the wild strain. Moreover, the keratinolytic activity of the mutant also seemed diminished when compared to the control, confirming the role of the PacC protein in the infecting capacity of T. rubrum.

A regulação da expressão gênica é essencial para os fungos se adaptarem às adversidades ambientais, como alterações no pH extracelular, escassez de nutrientes, força iônica e oscilações de temperatura. A resposta adaptativa ao pH ambiente é bem caracterizada em fungos modelo como Aspergillus nidulans, e envolve a via de transdução de sinal constituída pelos produtos dos genes pal e pacC. No dermatófito Trichophyton rubrum, o gene pacC foi inativado, e a linhagem mutante apresentou uma diminuição na atividade das queratinases, indicando que, de alguma forma, este gene está envolvido na regulação da atividade queratinolítica deste dermatófito, e consequentemente na sua virulência e patogenicidade. Além disto, a glicosilação protéica é uma importante forma de regulação pós traducional, estruturando e estabilizando glicoproteínas que podem ser da via secretória, da parede ou da membrana celular. O processo de glicosilação protéica sofre influência do pH extracelular e da fonte nutricional. Foi ainda relatado que este tipo de regulação pós traducional também sofre influência dos genes palB e pacC, indicando que estes genes tenham um papel na glicosilação de enzimas secretadas. O objetivo deste trabalho foi analisar a influência do pH e da fonte nutricional na expressão de genes que codificam enzimas de N- e O-manosilação, e sua possível modulação pela proteína PacC no dermatófito T. rubrum. Para tanto, foi analisado, por PCR em tempo real, o perfil transcricional destes genes nas linhagens H6 (controle) e pacC-1, utilizando-se como fonte de carbono glicose, glicose e glicina ou queratina em vários tempos de cultivo, em pH 5,0 ou 8,0. A análise da expressão gênica revelou que quando a linhagem controle é cultivada em queratina em pH 5,0 ocorre um aumento da expressão da O-manosiltransferase, comparado com o cultivo em glicina com glicose e glicose. Porém, nestas mesmas condições o gene da N-manosiltransferase da linhagem mutante apresenta maiores níveis de expressão que os da linhagem controle. Em pH 8,0 pode-se notar grande semelhança entre os perfis de expressão apresentados por estes dois genes. Os resultados obtidos indicam que o gene pacC tem um papel importante no sensoriamento de nutrientes em meio ácido, modulando a expressão destas transferases, nas condições avaliadas. Estas enzimas podem ativar proteínas que atuam na hidrólise da queratina, ou mesmo formar glicoproteínas de parede celular que são essenciais na adesão do fungo à célula do hospedeiro, sugerindo um papel das manosiltransferases no processo infeccioso. / Gene expression regulation is essential for fungi to adapt to environmental adversities, such as changes in the extracellular pH, nutrient starvation, ionic strength, and temperature. The adaptive response to ambient pH is well characterized in model fungi such as Aspergillus nidulans, and involves the signal transduction pathway consisting of the products of the pal and pacC genes. In the dermatophyte Trichophyton rubrum, the pacC gene was inactivated and the mutant strain showed a decreased activity of keratinases, indicating that, somehow, this gene is involved in the regulation of the keratonolytic activity of this dermatophyte, and consequently in its virulence and pathogenicity. Moreover, protein glycosylation is an important form of post-translational regulation, playing a role in protein folding and stability of glycoproteins of the secretory pathway, cell wall or membrane. The process of protein glycosylation is influenced by extracellular pH and nutritional source. It has also been reported that this type of post-translational regulation is also influenced by the palB and pacC genes, indicating that these genes have a role in glycosylation of secreted enzymes. The objective of this study was to analyze the influence of the pH and nutritional source in the expression of the genes coding for the N-and O-manosylation enzymes, and their possible modulation by PacC in the dermatophyte T. rubrum. To this end, the transcriptional profile of these genes was analyzed, by Real Time PCR, in the H6 (control) and pacC-1 strains, using glucose, glucose with glycine, or keratin as the carbon source, in several culture times, at pH 5.0 or 8.0. Gene expression analysis showed that when the control strain is grown in keratin at pH 5.0 there is an increased expression of the O-manosyltransferase encoding gene, compared to the cultivation in glucose and glucose with glycine. However, at the same conditions the gene coding for the N-manosyltransferase presented higher levels of expression in the mutant strain in relation to the control strain. At pH 8.0 there is a great similarity between the expression profile of these two genes. The obtained results indicate that pacC gene plays an important role in nutrient sensing at acidic pH by modulating the expression of these transferases in the conditions evaluated. These enzymes can activate proteins that play roles in the hydrolysis of keratin, or even forming cell wall glycoproteins that are essential for the adhesion of the fungus to the host cell, suggesting a role of the manosyltransferases in the infectious process.

v angličtině Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Pohanová Supervisor: Assoc. Prof.. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Chelation of ferrous ions by xanthen-3-one derivatives Iron is an essential element important for proper function of cells. Imbalance of iron levels can lead to serious diseases. Since there is no excretory mechanism, the homeostasis is regulated at the level of absorption in the intestine. The iron overload, which leads to tissue damage due to catalysis of the formation of free radicals, occur because of genetic disorders such as hemochromatosis or owing to frequent administration of transfusions. Rational therapy for iron overload is the administration of the chelators. The aim of this study was to evaluate the ability of derivatives of 2,6,7- trihydroxyxanthen-3-one (synthesized at the University of Sarajevo - Dr. Durić) to chelate iron in 4 (patho) physiologically relevant pH conditions. The ferrozine spectrophotometric method was used to determine the degree of chelation. Measurements showed dependency of the chelating effect on pH: lowering pH resulted in the decrease of the effect. At pH 7.5, most of the substances showed 100% ferrous ion chelation in the stoichiometric ratio of 1...

The presented diploma thesis deals with a targeted modification of the structural properties of hydrogels, which is closely related to the application properties of these materials (eg transport). Due to the desired pharmacological applications of the use of these materials for targeted drug release, hydrogels based on chitosan and polyvinyl alcohol as substances with good biocompatibility were selected for the study. The combination of these polymers ensured swelling (controlled by the presence of chitosan) and viscoelastic (influenced by the presence of polyvinyl alcohol) properties, which were characterized by a wide range of analytical and physicochemical methods (swelling, tensile strength, rheology, atomic force microscopy or specific surface area, etc.). Information on structural properties played a crucial role not only for a detailed description of the studied materials in terms of whether the structural properties can be changed in a targeted manner, but also served as an explanation for the different release of the active substance diclofenac from the hydrogel matrix. With regard to the literature search, the effect of pH and crosslinking was chosen to modify the properties. From the measured results it was found that even a slight change in pH has an effect on the transport or release of the active substance. The results obtained in this work may be useful in the formation of hydrogel matrices with drugs depending on the intended medical applications.

The interaction between protein A and immunoglobulin G (IgG) was studied at a variety of pH values using a surface plasmon resonance (SPR) device, which provides real time kinetic data without labelling or molecular alteration. This study was carried out due to the large scale use of Protein A affinity chromatography for the purification of IgG for pharmaceutical purposes, and is one of the most costly steps in the purification process. The results produced were largely in line with those produced in previous literature with binding remaining strong between pH 7.4 and 5.0, although the association rate decreased as pH decreased. Below pH 5.0, the rate of IgG elution markedly increased, with pH 3.5 showing near full elution seconds after the association phase of the SPR interaction finished. Problems were encountered with non-specific binding between the SPR sensor chip and IgG occurring under a variety of conditions, requiring various remedies. However, no complete interactions were successfully carried out under pH 5.0, so the results obtained below this value were obtained by binding at pH 7.4 and then elution at the desired pH. The data showed binding behaviour that was most successfully explained by a three-site model, each with a binding ratio of 1:1. The binding ratio is questionable given that Protein A and IgG typically bind at a ratio of 1:2 but may be explained by the sites being independent of one another and thus no secondary attachment is observed. A variety of models were fitted to the data but only two- and three-site models fitted the experimental data, with the three-site model being a more accurate and robust fit across pH changes. A multiple site model seems intuitively correct given the six different binding sites that Protein A has for interaction with IgG. The models produced have potential applications in a larger model of Protein A affinity chromatography, although a number of additional factors would need to be taken into account, such as mass transfer effects and the IgG concentration gradient.

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