Cancer, group of diseases characterized by an uncontrolled cell growth, represents one of the great challenges of modern clinical research. Currently, the standard treatment of the cancer disease relies mainly on the whole body exposition to various factors, which targets the dividing cells, combined with surgical resection of the tumor. Unfortunately, this treatment is sometimes accompanied by numerous severe side-effects (e.g., nausea, loss of hair, infertility etc.). Therefore, in the past 40 years enormous resources and effort have been invested into finding a way how to specifically target and destroy the cancerous cells. This goal has been primarily addressed by the search for molecules, mainly proteins, which are predominantly expressed in the cancerous tissues compared to the healthy cells. Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), represents such a target since it is highly expressed in a prostate carcinoma as well as in a solid tumor neovasculature. Additionally, GCPII is widely used as a model target molecule for proof-of-principle studies on targeted drug delivery. GCPII thorough biochemical characterization is essential for its appropriate use. Therefore, our laboratory has been investigating GCPII from various perspectives for more...

Em 1978, o trabalho realizado por Stephenson e Zamecnik demonstrou a capacidade de um oligonucleotídeo de impedir a expressão de uma proteína específica. Atualmente, duas tecnologias são mais utilizadas para este propósito: os oligodeoxiribonucleotídeos antisense e o RNA de interferência (siRNA), que se aproveitam da capacidade de anelação entre as fitas complementares. A maior diferença entre as duas técnicas é a maquinaria proteica recrutada, isso é, o complexo RISC atua no funcionamento do siRNA, e a protease RNase H atua na clivagem da fita de RNA quando hibridizada com DNA. Apesar da grande aplicabilidade destas tecnologias, tanto para doenças metabólicas quanto para canceres, o veículo de entrega e proteção dessas sequências é de fundamental importância, visto que a aplicação desses oligonucleotídeos livres está sujeita à rápida degradação e ineficiência. A modificação das bases é uma das estratégias para conferir maior estabilidade às sequências, porém estas tem sido relacionadas a um aumento da toxicidade. Nessa dissertação, a quitosana, um polissacarídeo catiônico é utilizado para síntese de nanopartículas e encapsulamento dos oligodeoxiribonucleotídeos antisense (ASO). Para isso, foram realizadas modificações na quitosana comercial como despolimerização, trimetilação ou conjugação com PEG, seguida da síntese das nanopartículas com a adição de tripolifosfato de sódio (TPP) pelo método de gelatinização ionotrópica. A estabilidade das nanopartículas foi medida em função do tempo, da variação de temperatura e da diferença de pH. Além disso, a toxicidade dessas nanopartículas foi analisada através da viabilidade celular em diferentes linhagens, NB-4, HepaRG, HTC e BHK-570. A expressão da proteína verde fluorescente (GFP) na célula NB-4 foi utilizada para avaliar a entrega do ASO desenhado, sendo sua fluorescência monitorada por microscopia confocal. Os resultados demonstram que as nanopartículas se mantiveram estáveis durante o período de tempo analisado, assim como com a temperatura variando de 22 a 45°C e em pH ácido. Cada linhagem celular respondeu de forma diferente ao tratamento com as nanopartículas sem ASO, sendo a linhagem saudável BHK-570 com a maior resistência. Ademais, todas as células apresentaram viabilidade reduzida quando tratadas com concentrações na ordem de 1011 nanopartículas/mL a base de quitosana trimetilada. A fluorescência das células NB-4 quando tratada com as nanopartículas com ASO diminuiu consideravelmente nas 18 primeiras horas, seguida de um aumento após 42 horas. Dessa forma, pode-se concluir que as nanopartículas de quitosana propostas nessa dissertação apresentaram uma excelente alternativa para a entrega de material genético, principalmente para o trato gastro-intestinal, devido à sua estabilidade em pH ácido. / The property of an oligonucleotide to interfere in the expression of a protein was observed in 1978 by Stephenson and Zamecnik. To perform such interference, there are today, two main techniques being explored: antisense oligodeoxyribonucleotides and interference RNA. In both cases, the particularity of their chemical structure is taken into account as soon as they can bind in a complementary manner to the messenger RNA and inhibit its translation. The great difference between these techniques is related to the proteases involved in the process, while for interference RNA the RISC machinery acts, for antisense oligodeoxyribonucleotides RNase H cleaves the RNA in the duplex DNA-RNA. Although these tools to edit the translation process are relevant to the treatment and even cure of metabolic disorders and cancers, it is still not effective when employed without a coating to protect the sequences before it reaches the destiny in vivo. Efforts have been made in developing modified bases to be more stable, but they show some toxicity. In this dissertation, chitosan, a natural cationic polyssacharide, is used to produce nanoparticles to protect the antisense oligodeoxyribonucleotide (ASO). For this reason, the commercial chitosan was modified, depolymerized, trimetilated or PEGlated and the nanoparticles were synthesized with sodium tripolyphosphate (TPP) by ionotropic gelation method. The stability along time, in different pHs and temperatures was assessed. The toxicity of nanoparticles without ASO was quantified by MTT tests in NB-4, HepaRG, HTC and BHK-570 cell lines. A green fluorescent protein (GFP) expressed by NB-4 cells was the target to evaluate the delivery efficiency of the ASO, and its fluorescence was measured by confocal microscopy. Results showed that nanoparticles were stable over time as well as in temperatures ranging from 22 to 45°C and in acidic pH. Each cell line responded in a different manner to the treatment, with the health cell BHK-570 showing higher resistance. Furthermore, all of them presented lower viability when treated with trimetilated chitosan nanoparticles in the highest concentrations (ca 1011 nanoparticles/mL). NB-4 cells presented a decrease in fluorescence in 18 hours of treatment followed by an increase after 42 hours. We conclude that chitosan nanoparticles are a good alternative to the delivery of genetic material even more in the gastro intestinal tract due to its great stability in acid pH values.

A leishmaniose é uma doença causada por parasitos do gênero Leishmania e transmitida por insetos flebotomíneos. O objetivo deste trabalho foi comparar o perfil da infecção por L. amazonenses em BALB/c, BALB/c nude e C57BL/6, e avaliar a expressão das proteínas CP, LACK, LRR17, metacaspase, PDI e STI, em amastigotas isolados de BALB/c e BALB/c nude. Observamos em 13 semanas o aumento mais precoce da espessura da pata em BALB/c do que em BALB/c nude. A carga parasitária nos animais nude foi superior à dos demais. Por outro lado, animais C57BL/6 controlaram a infecção a partir da sexta semana. A análise da expressão gênica nas lesões mostrou um padrão misto Th1/Th2 em todos os animais, e maior expressão de IFN-g, IL-4, IL1b e iNOS em BALB/c. Em baço e linfonodo observamos baixa expressão de CD3e e elevada expressão de iNOS em BALB/c nude. A análise por FACs mostrou em baço e linfonodo a esperada baixa porcentagem de células TCD4 e T CD8 em BALB/c nude, no entanto com capacidade de produzir IFN-g e IL-4. A LACK e a metacaspase estão aumentadas nos parasitas isolados de BALB/c nude. A metacaspase apresentou maior atividade também em nude. Não observamos diferença na expressão da LACK e metacaspase em amastigotas isolados de macrófagos infectados na presença de IFN-g e IL-4. / Leishmaniasis is caused by parasites of Leishmania genus, transmitted by phlebotomine sandflies. The aim of this study was to compare the profile of the infection by L. amazonenses in BALB/c, BALB/c nude and C57BL/6 and to analyze the expression of CP, LACK, LRR17, metacaspase, PDI and STI proteins, in amastigotes isolated from BALB/c and BALB/c nude lesions. We observed during 13 weeks an early increase in footpad thickness in BALB/c compared to BALB/c nude. Parasite loads in nude mice were higher than the others. C57BL/6 mice controlled infection since the sixth week. Gene expression analysis of the lesions indicated a mixed Th1/Th2 pattern in all mice, and higher expression of IFN-g, IL-4, IL1b and iNOS in BALB/c. In spleens and lymph nodes we observed lower expression of CD3e and high expression of iNOS in BALB/c nude. FACs analysis of spleens and lymph nodes showed low percentages of TCD4 and T CD8 cells in BALB/c nude, but producing IFN-g and IL-4. LACK and metacaspase are increased in parasites isolated from BALB/c nude, and metacaspase had higher enzymatic activity in amastigotes from nude. No differences in LACK or metacaspase in amastigotes isolated from macrophages infected in the presence of IFN-g and IL-4 were observed.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences The University of Alcalá, Faculty of Medicine, Department of biomedicine and biotechnology Student: Zuzana Chvojanová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: María Isabel Arenas Jimenéz Title of the diploma thesis: Study of the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A Bisphenol A (BPA) is one of the most widespread compounds in the world, producing over 6 billion metric tons per year. It is widely used as part of polycarbonate plastics and epoxy resins, from which reusable plastic bottles, food boxes and some medical equipment are made. It is also used to coat the inner layer of the cans. Previous studies have shown that BPA contributes to many chronic diseases in the human body, such as kidney disease - diabetic nephropathy. Podocytes - terminally differentiated cells of the Bowman's capsule in glomerulus - are an integral part of the filtration barrier, where they play an important role in preventing the plasmatic proteins from penetrating to the urine. Therefore, in this study, we looked at the effect of BPA on these cells and their particular proteins, using both in vivo and...

Epilobium hirsutum L. (Onagraceae) is a flowering, tall and perennial plant and native to Eurasia. It shows analgesic, anti-microbial and anti-proliferative activity, and it is used in our country as an alternative medicine. The pharmacological effect of Epilobium hirsutum L. could be explained by the presence of polyphenolics including steroids, tannins and flavonoids in the aerial parts. Viscum album L. (Loranthaceae) is a shrub that grows as an epiphyte on the branches of deciduous trees. It involves in the enhancement of macrophage phagocytic and cytotoxic mediated abilities as well as the strengthening the immune system. CYP24A1 and CYP27B1 are members of cytochrome P450 superfamily and the most important enzymes involved in the metabolism of vitamin D3. CYP27B1 and CYP24A1 are mitochondrial enzymes and also known as 25-hydroxyvitamin D3 1alpha-hydroxylase and 24-hydroxylase, respectively. CYP24A1 involves in 24-hydroxylation of 25-OH-D3 and 1,25-(OH)2D3 which is required for the catabolism of vitamin D3 compounds while CYP27B1 involves in 1&alpha / -hydroxylation of 25-OH-D3 into 1,25-(OH)2D3. In this study, in vivo effects of Epilobium hirsutum and Viscum album (subspecies growing on pine-trees-subsp. austriacum (Wiesb.) Vollmann) on rat liver CYP24A1 and CYP27B1 mRNA and protein expressions were investigated. To achieve this goal, 37.5 mg water extract of Epilobium hirsutum L./kg body weight/day was intraperitoneally injected to male rats for 9 days. To study the effect of Viscum album L., 10 mg water extract of Viscum album L./kg body weight/day was injected with the same conditions. After decapitation, livers were removed and S1.5 fractions were prepared. Effects of Epilobium hirsutum L. and Viscum album L. on rat liver mRNA and protein expressions were analyzed by qRT-PCR and western blotting, respectively. Epilobium hirsutum L. extract caused 31% and 18% decrease in rat liver CYP24A1 (p&lt / 0.0001) and CYP27B1 (p&lt / 0.05) protein expressions, respectively. The effect of Epilobium hirsutum L. on mRNA expression of CYP24A1 could not be observed, because CYP24A1 mRNA was almost undetectable in liver. Injection of Epilobium hirsutum L. to rats caused 2.7 fold increase in mRNA expression of CYP27B1 with respect to controls and normalized with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression as an internal reference (p&lt / 0.005). Viscum album L. caused 17% decrease in CYP24A1 protein expression (p&lt / 0.05). When rats injected with plant extract of Viscum album L., 18% decrease in CYP27B1 protein expression was observed (p&lt / 0.05). The effect of Viscum album L. on mRNA expression of CYP24A1 could not be observed since CYP24A1 mRNA was almost undetectable in liver. Injection of Viscum album L. to rats caused 3.8 fold increase in mRNA expression of CYP27B1 with respect to controls and normalized with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression as an internal reference (p&lt / 0.005). In conclusion, vitamin D3 metabolism may be affected by medicinal plants Epilobium hirsutum L. and Viscum album L. due to the changes in mRNA and protein expressions of CYP24A1 and CYP27B1 enzymes.

Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder affecting the cerebellum, pons and retina. SCA7 patients present with gait ataxia and visual impairment as the main symptoms. Anticipation, commonly observed in SCA7 families, is a phenomenon where an earlier age at onset and a more severe progression of disease is seen in successive generations. In order to identify the gene responsible for SCA7, we performed linkage analysis on a Swedish SCA7 kindred. Evidence for linkage of the SCA7 disease locus to a 32 cM region on chromosome 3p12-21.1, between markers D3S1547 and D3S1274, was established. A number of neurodegenerative disorders associated with anticipation are caused by expanded (CAG)n repeats in their respective disease genes. In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library for CAG repeat containing clones, mapping to chromosome 3. Four candidate clones were isolated and analysed, but could all be excluded as the SCA7 disease gene. In 1997, the SCA7 disease gene was identified and, as expected, shown to harbour a CAG repeat, expanded in SCA7 patients. Analysis of the SCA7 CAG repeat region in Swedish SCA7 patients demonstrated that CAG repeat size was negatively correlated to age at onset of disease. Furthermore, patients with larger repeats presented with visual impairment, whereas patients with smaller repeats presented with ataxia as the initial symptom. SCA7 is the most common autosomal dominant cerebellar ataxia in Sweden and Finland, but rare in other populations. In order to investigate if the relatively high frequency of SCA7 in these countries is the result of a founder effect in the region, a haplotype analysis was performed on all SCA7 families available. All 7 families shared a common haplotype of at least 1.9 cM surrounding the SCA7 locus. In addition, strong linkage disequilibrium was demonstrated for marker D3S1287 closely linked to the SCA7 gene, suggesting a founder effect for the SCA7 mutation in Sweden and Finland. The function of the SCA7 protein, ataxin-7, is not known and it does not show significant homologies to any previously known proteins. In order to gain insight into the function of ataxin-7 we analysed the expression of ataxin-7 in brain and peripheral tissue from SCA7 patients and controls. In brain, expression was found to be mainly neuronal with a nuclear subcellular localisation. Ataxin-7 expression was found throughout the CNS, not restricted to sites of pathology. We also confirmed previously reported findings of neuronal intranuclear inclusions (NIls) in the brains of SCA7 patients. Based on our findings, we conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression pattern in affected and non-affected tissue or the distribution pattern of aggregated protein.

Calcium plays important roles in both eukaryotic and prokaryotic cells. Its actions help to stabilize cell synthesis, growth and development. In this thesis, studies have been completed to determine effects of calcium and pH on bacterial cell growth and protein expression using the bacterial cell strain E.coli BL21(DE3). Our studies demonstrated the addition of calcium addition in the media does not affect growth but increases protein expression, while reducing the pH from 7 to 4 through the addition of 10mM EGTA in LB media inhibits both. Additionally, we report studies on the design, expression, and purification of fluorescent mCherry variants and their differences in their optical properties, including: extinction coefficients , quantum yields and pKa values. Also, we report progress in the crystallization of two GFP calcium sensors: G1 and D1, using 13 and15% PEG 4000 and 3350 respectively in 50mM HEPES buffer (pH 6.8-7.0) in an effort to optimize crystallization.

In this study the aim was to optimize the production of the Affibody fusion-protein Z03358- ABD094-(S4G)3-IL2 with regard to the amount of soluble protein produced. However, problems with reproducibility with this protein and the chosen expression system were encountered. Therefore, expression of the His-tagged Affibody His6-(Z05477)2 was evaluated using the same expression system as well as expression in another well characterized expression system. Both target proteins are of therapeutic interest. One of the proteins is an IL2 fusion protein (Z03358-ABD094-(S4G)3-IL2) that bind the platelet-derived growth factor receptor β (PDGFR-β). PDGF signaling is of interest in cancer treatment where, among other things, the effects of PDGF on tumor angiogenesis is researched. The His6-(Z05477)2 protein has a classified target but is developed as a therapeutic in the area of inflammation and autoimmune disease. Both model proteins are known to be difficult to purify due to low solubility. The two E. coli expression systems investigated and compared were BL21(DE3) and Lemo21(DE3). The fusion protein Z03358-ABD094-(S4G)3-IL2 was produced in BL21(DE3) in inclusion bodies with a yield of 4.95 g/l. An optimized process for the expression of His6-(Z05477)2 using BL21(DE3) was developed with a yield of 6.6 g/l soluble protein after expression at 30°C for 6 h.

Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities. Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement. Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen, v methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor. The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined. The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p&lt / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.

Anthropogenic climate warming is predicted to accelerate over the next century, with potentially dramatic consequences for wildlife. It is important to understand as well as possible how different organisms will respond to this stress. This project seeks to gain a better mechanistic understanding of the thermal biology of the Glanville Fritillary butterfly (Melitaea cinxia) at the latitudinal and elevational extremes of its range. Investigation of the temperatures at which adult butterflies took spontaneous flight revealed a significant difference between populations from the elevational extremes, with insects from high elevation taking flight at lower thoracic temperatures than those from low elevation. Contrary to expectation, there was no systematic effect of latitude on takeoff temperature. If these measures represent adaptation to climate, then these effects are not simple and the influences of elevation and latitude are not the same. Investigation of thermal tolerance across all life cycle stages found no difference in larval performance between the populations tested. There was however an effect of treatment. This suggests that in M. cinxia, even populations from different extremes of the range may not differ in their thermal tolerance. The effect of treatment suggests that there is temperature-induced plasticity. The adaptive significance of this has been explored to some extent. Investigation of heat shock protein expression between the latitudinal extremes finds no difference in Hsp21.4 expression when exposed to heat stress, however both Hsp20.4 and Hsp90 were upregulated in response to heat stress. For Hsp20.4, there were significant differences in expression between the populations. Finally, a species distribution model using maximum entropy techniques was conducted for M. cinxia, predicting both the current and future (2100) distributions of the species. The model closely matches the known current distribution, and predicts a clear northward range shift in response to climate change. / text