Šio baigiamojo darbo tikslas – įvertinti imunochromatografinio tyrimo metodo patikimumą, atliekant Žmogaus imunodeficito viruso aspektų analizę. Siekiant įvertinti imunochromatografinio tyrimo metodo patikimumą, ŽIV tyrimai buvo atliekami atrankiniu FIRST RESPONSE HIV CARD TEST 1-2.0 testu, pagrįstu nagrinėjamu metodu. Tyrimas atliktas Infekcinių ligų ir tuberkuliozės ligoninės, VšĮ Vilniaus universiteto ligoninės Santariškių klinikų filialo laboratorijoje. ŽIV tyrimai atlikti visiems 18 - 60 metų amžiaus pacientams, hospitalizuotiems į Infekcinių ligų ir tuberkuliozės ligoninės Žvėryno stacionarą, taip pat ambulatoriškai konsultuojamiems dėl nepatikslintos kilmės karščiavimo, bėrimo, limfadenopatijos, mononukleozinio sindromo, užsitęsusio arba besikartojančio viduriavimo bei virusinių hepatitų nuo 2013-01-01 iki 2013-12-31. Gauti teigiami mėginiai buvo siunčiami į Nacionalinę visuomenės sveikatos priežiūros laboratoriją, kurioje atlikti patvirtinamieji tyrimai dėl ŽIV. Ne visi teigiami mėginiai buvo patvirtinti, tačiau šio tyrimo metu buvo atskleista 14 naujų ŽIV atvejų per metus. Siekiant nustatyti gautų klaidingai teigiamų rezultatų susidarymo priežastį, buvo atrinkti pacientai, kuriems nebuvo patvirtinti atrankiniai ŽIV tyrimai. Išnagrinėjus pagrindines jų tuometines diagnozes, nustatyta kad daugiausiai klaidingai teigiamų rezultatų gaunama sergant šiomis infekcinėmis ligomis: rože, Epštein – Baro sukelta mononukleoze ir hepatitu A. / The main goal of this paper is to assess the reliability of the immunochromatographic method carrying out the analysis of human immunodeficiency virus aspects. In order to assess the reliability of immunochromatographic method, the HIV tests were performed using the selective FIRST RESPONSE CARD TEST HIV 1-2.0 test, based on the method in question. The assay was carried out in the Laboratory of National Tuberculosis and Infectious disease hospital in Vilnius. The HIV tests were carried out from 01-01-2013 till 31-12-2013 to all patients aged 18-60 years, who were hospitalized in the National Tuberculosis and Infectious disease hospital in Vilnius as well as to outpatient, who were consulted due to fever of unspecified origin, rash, lymphadenopathy, mononucleosis syndrome, prolonged or recurrent diarrhea and viral hepatitis. All positive samples were sent to National Public Health Surveillance Laboratory for final confirmation. Not all the samples were confirmed, but this assay revealed 14 new cases of HIV per year. In order to identify the reasons of false positive results, the patients, whose selective HIV tests were not approved, have been selected and after the examination of their diagnoses, it was found that the majority of false-positive results were received to patients with the following infections: rose, mononucleosis induced by Epstein - Barr virus and hepatitis A.

The primary function of vitamin D in humans is to maintain sufficient circulating calcium concentrations. Low vitamin D levels could result in excessive calcium resorption from bone. Vitamin deficiency may therefore decrease bone mineral density (BMD), resulting in an increased risk of fracture. This thesis sought to determine the association between vitamin D intake and bone health and to estimate circulating levels of vitamin D optimal for bone health without increasing the risk for non-bone disease. Furthermore, the thesis assessed the difference in performance between common serum vitamin D assays and the genetic influence of vitamin D status. In prospective population-based cohorts, blood concentrations <40 nmol/L (lowest 5%) increased the risk of fracture in elderly men. Low levels were further associated with a slight decrease in lumbar spine BMD. Both high (>98 nmol/L) and low (<46 nmol/L) vitamin D levels were associated with higher cancer and overall mortality. In another cohort, also of older men and women, no association was found between vitamin D levels and fracture. Low vitamin D levels were weakly associated with decreased total body BMD in men but not in women. Dietary intake of vitamin D over a 20-year period in more than 60,000 Swedish women was not associated with osteoporosis or fracture, regardless of calcium intake. During summer, dietary vitamin D intake and other life style habits are of minor importance for the variation in vitamin D levels relative to sun exposure and genes. In summer time, genes explain about half  of the variation in vitamin D levels, but none of the variance in winter time. The variability between vitamin D assays was substantial. Three assays classified 8, 22 and 43% of the same study population as vitamin D insufficient if <50 nmol/L was set as the insufficiency level. Based on the results in this thesis, low 25(OH)D levels and low dietary vitamin D intake are not a major cause of fractures in community-dwelling elderly Swedish women and men. Differences in assay performance and potential negative health outcomes of high 25(OH)D levels need to be considered.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.

Orientador: Nereu Carlos Prestes / Banca: Fernada da Cruz Landim-Alvarenga / Banca: Regina Kiomi Takahira / Banca: Wilter Ricardo Russiano Vicente / Banca: Mara Regina S. Balarin / Resumo: Os líquidos fetais possuem diversas funções que são vitais para o feto. Para a espécie eqüina até o presente momento, não está totalmente definida a composição bioquímica do líquido amniótico no decorrer da gestação. O presente trabalho teve como objetivo verificar a composição bioquímica do liquido amniótico e alantoideano das éguas em diferentes fases da gestação. Para isso analisou-se 60 amostras de fluidos fetais, empregando-se kits comerciais para se determinar a concentração bioquímica da Fosfatase Alcalina, Glicose, Proteínas Totais, Uréia, Creatinina, Cálcio, Cloreto, Sódio e Potássio durante a gestação. A concentração da alfa-fetoproteína foi avaliada empregando-se a eletroforese em gel de poliacrilamida. A concentração da Fosfatase Alcalina no liquido amniótico foi maior quando comparada ao liquido alantoideano nas três fases da gestação (p < 0,05). Para a glicose o valor médio entre os dois fluidos não apresentou variações (p < 0,05). Para a Proteína total o valor médio do liquido amniótico foi maior que o alantoideano (p < 0,05). A Uréia sofreu variações na concentração entre as fases, mas não há diferenças dos valores médios (p > 0,05) entre os fluidos. Para a Creatinina os valores presentes no liquido alantoideano são mais altos que os valores do liquido amniótico (p < 0,05). As concentrações dos íons Cloreto e Sódio apresentaram-se mais elevados (p < 0,05) no liquido amniótico. As concentrações dos íons Cálcio e Potássio foram mais elevadas nos líquidos alantoideanos (p<0,05). A eletroforese identificou duas bandas protéicas que podem ser a alfa-fetoproteína, ela parece aumentar a concentração durante o período gestacional. Porém, faltam estudos na espécie eqüina para a comparação dos resultados do presente trabalho. / Abstract: Fetal fluids play a vital role in the development of the fetus. The biochemical composition of the amniotic fluid along pregnancy in horses had not been described until this present study. Sixty samples of fetal fluids were collected and the concentrations of Alkaline Phosphatase (FA), Glucose, Total Proteins (PT), Urea, Creatinin, Calcium (Ca), Chloride (Cl), Sodium (Na) and Potassium (K) along pregnancy were determined using commercially available kits. The levels of alpha-fetoprotein (AFT) were measured by polyacrylamide gel electrophoresis. During the three stages of pregnancy the concentrations of FA in the amniotic fluid were higher than those determined in the allantoic fluid (p < 0,05). The glucose levels did not differ between the fluids (p < 0,05). The mean values for the concentrations of PT were higher in the amniotic fluid than in the allantoic (p < 0,05). The urea levels differ among the pregnancy stages, but there were no differences in the mean values of urea (p > 0,05) between the two fluids. The concentrations of creatinin obtained in the allantoic fluid were higher than those obtained in the amniotic fluid (p < 0,05). The concentrations of Cl and Na were elevated (p < 0,05) in the amniotic fluid. The levels of the ions Ca e K were higher in the allantoic fluid (p<0,05). Polyacrylamide gel electrophoresis identified two protein bands that could be alpha-fetoprotein, which appears to have its concentration increased during pregnancy. There is a need for more studies in the biochemical composition of fetal fluids in horses to compare the results obtained in this study. / Doutor

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Os produtos naturais apresentam um papel fundamental na descoberta de novos fÃrmacos. Nesse contexto, os produtos naturais marinhos apresentam um grande potencial quÃmico e farmacolÃgico. Resultados prÃvios demonstraram um potencial quÃmico e farmacolÃgico promissor do zoantÃdeo Protopalythoa variabilis encontrado no litoral cearense. Por outro lado, fortes indÃcios apontam que os microrganismos associados sejam os verdadeiros responsÃveis pelo elaborado arsenal de metabÃlitos secundÃrios isolados de invertebrados marinhos. Dessa forma surgiu o interesse em estudar o potencial biomÃdico da microbiota associada ao P. variabilis. Os esforÃos para isolar as bactÃrias renderam um total de 10 colÃnias. Destas, 3 apresentaram padrÃes morfolÃgicos tÃpicos de actinomicetos e 2 delas foram estudadas neste trabalho. A cepa BRA-035 foi identificada como pertencente ao gÃnero de actinomicetos Streptomyces, e seu extrato acetato de etila, resultante da fermentaÃÃo em meio A1, causou alta inibiÃÃo do crescimento celular contra a linhagem de carcinoma de prÃstata metastÃtico PC-3/M, com concentraÃÃo inibitÃria mÃdia (CI50 ) na ordem de pg/mL. O fracionamento bioguiado levou ao isolamento dos compostos glicopiericidina A, piercidina D, e piericidina A, sendo que esta Ãltima apresentou elevada atividade antiproliferativa em linhagens tumorais de cÃncer de ovÃrio (OVCAR-8), cÃncer de cÃlon (HCT-116) e PC-3/M, com CI50 encontradas na ordem de pico atà fento molar. Apesar de jà ter sido amplamente estudada, esta pode ser a primeira ocorrÃncia de atividade antiproliferativa de Piericidina A contra as linhagens testadas neste trabalho, assim como a ordem de grandeza de sua potÃncia. Outra cepa selecionada para estudo, a BRA-060, tambÃm foi identificada como pertencente ao gÃrero Streptomyces. Sua fermentaÃÃo foi otimizada em meio Marine Broth para produÃÃo de extrato acetato de etila com citotoxicidade contra a linhagem de leucemia promielocÃtica HL-60 em ng/mL. O fracionamento bioguiado deste extrato resultou no isolamento de trÃs dicetopiperazinas: a ciclo (L-Tyr-trans-L-Pro), a ciclo(L-Phe-cis-4-OH-D-Pro) e a ciclo(L-Phe-trans-4-OH-L-Pro). As dicetopiperazinas isoladas apresentaram potente atividade citotÃxica contra as linhagens de glioblastoma (SF-295), cÃncer de ovÃrio (OVCAR-8), HCT-116 e HL-60 e outra leceumia K-562, com valores de CI50 variando de nano a micro molar. Embora suas estruturas jà terem sido descritas pela literatura, nenhum trabalho foi feito quanto a suas atividades citotÃxicas, podendo este ser o primeiro relato sobre suas propriedades antitumorais. Esses resultados ressaltam a importÃncia da microbiota associada ao zoantÃdeo P. variabilis, encontrado no litoral cearense, como fonte de compostos com potencial anticÃncer. / Natural products exhibit a fundamental role in the discovery of new medicines. Marine natural products possess a huge chemical and pharmacological potential to yield new leads. Previous results support the promising chemical and pharmacological potential of the zoanthid Protopalythoa variabilis from the coast of Ceara State. On the other hand, strong evidences have indicated that associated microorganisms are the producers of the elaborate arsenal of secondary metabolites isolated from marine invertebrates. Then this study aimed to investigate the biomedical potential of associated microbiota to P. variabilis. The efforts to isolate associated bacteria yielded 10 different strains, and 3 of them showed typical actinomycete morphology. Here 2 of these actinomcetes had their anticancer potential studied. The strain BRA-035 belongs to the genus Streptomyces. BRA-035 culture using A1 broth yielded an acetate extract with potent cytotoxicity against a prostatic carcinoma cell line (PC-3/M), with inhibitory concetrantion mean (IC50) value in pg/mL. The bioguided fractionation led to the isolation of glucopiericidin A, piericidin D and piericidin A, and the later showed high antiprolifetative activity against ovarian cancer (OVCAR-8), colon cancer (HCT-116) and PC-3/M with IC50 values ranging from pico to fentomolar. Although Piericidin A has been widely studied, this might be the first report of cytotoxic activity against the lineages tested in this work, as well as the magnitude of its potency. Another strain selected for this study, BRA-060, was identified as belong to the genus Streptomyces also. The fermentation was optimized in marine broth to produce an acetate extract highly cytotoxic against a leukemia cell line (HL-60). The bioguided fractionation of these extracts yielded three diketopiperazines: cyclo (L-Try-trans-L-Pro); cyclo (L-Phe-cis-4-OH-D-Pro) and cyclo (L-Phe-trans-4-OH-D-Pro). The diketopiperazines isolated showed potent cytotoxic activity against glioblastoma (SF-295), ovarian cancer (OVCAR-8), HCT-116, HL-60 and another leukemia (K-562) The IC50 values ranging from nanomolar and micromolar. Althought their structures have already been described in the literature, there was no previous report of their cytotoxic acitivities. Altogether, these results emphasize the value of microbiota associated with the zoanthid P. variabilis, found on the coast of Cearà State, as a source of active compounds.

Made available in DSpace on 2015-04-20T12:31:52Z (GMT). No. of bitstreams: 1 Helenires Quiroz de Souza.pdf: 4253901 bytes, checksum: 8968bf2a9d9d6e181ad4528e0b805f1b (MD5) Previous issue date: 2006-05-31 / The Class Basidimycetes is formed by those fungi called mushroom ande ar-of-wood, beyond others groups less known. Due to the importance of this group of fungi, the biotechnological potential was investigated in this study. The mushroom were collected in forest areas of campus of University of Amazonas (UFAM), Bosque da Ciência and campus V8 (INPA), Reserva de Campina (INPA), located in BR 174, km 45, Manaus, Amazonas and Urucu (Coari/AM). After the isolation micelial, the fungi were evaluated as regards the growth at 280C, for 30 days in liquid cultures GPY and BD to determine the antimicrobial activity. The samples were filtered and the mycelium was dry to determine the biomass. The culture filtrates had been tested against Ralstonia solanacearum, agent of bacterial wilt of several vegetal species, Corynespora sp., Colletotrichum sp., Escherichia coli, Staphylococcus aureus, Bacillus cereus e Salmonella anatum. For an enzyme production study, the fungus had been cultivated in liquid culture and tested in solid medium for proteases, amylases, cellulases, pectinases and phenoloxidases. In another stage, the production of amylases and proteases in different nutritives sources was studied. Sixty samples of Basidiomycetes were collected from February to June 2003. The families Agaricaceae, Auriculariaceae, Cantharellaceae, Ganodermataceae, Hygrophoraceae, Polyporaceae, Russulaceae, Stereaceae, Tremellaceae e Tricholomataceae had been identified. The Reserva de Campina was the one that presented greater diversity of fungi. The results demonstrated inhibitory effect of culture filtrates against bacterial strains tested. The species E. coli, S. anatum and B. cereus, beyond inhibition halos, had presented halos of grown stimulus. It was not effect against the fungi Corynespora sp. and Colletotrichum sp.. The culture filtrate Trametes sp. presented greater activity of inhibition. The medium BD favored greater production of dry biomass. The production of protease an amylase was detected for all the isolated ones, four produced amylases, five produced phenoloxidases and one produced pectinase. For production of amylases, fungi cultivated in medium added of wheat bran had gotten biggest halos. For proteases, the biggest halos had been observed for the fungi grown in way with fish flour. / A classe Basidiomycetes é formada por aqueles fungos chamados de cogumelos e orelhas-depau, além de outros grupos menos conhecidos. Devido à importância deste grupo de fungos, o presente trabalho teve como objetivo investigar o seu potencial biotecnológico quanto à produção de antimicrobianos e enzimas. Os basidiomas dos fungos foram coletados em áreas de floresta do campus da UFAM, do Bosque da Ciência, Campus/V8 e Reserva de Campina do INPA, localizada na BR 174, km 45, Manaus/AM e Urucu em Coari/AM. Após o isolamento micelial, os fungos foram avaliados quanto ao crescimento radial em meio sólido. Para verificar a produção de antimicrobianos, os fungos foram cultivados em meio líquido BD e GPY durante 30 dias, em repouso a 28oC. Após esse período, o micélio foi seco para determinar a biomassa e os filtrados das culturas foram testados contra Ralstonia solanacearum, agente da murcha de várias espécies vegetais, Corynespora sp., Colletotrichum sp., Escherichia coli, Staphylococcus aureus, Bacillus cereus e Salmonella anatum. Quanto à produção de enzimas, os fungos foram cultivados em meio líquido e testados em meio sólido para proteases, amilases, celulases, pectinases e fenoloxidases. Em outra etapa, foi estudada a produção de amilases e proteases em diferentes fontes nutricionais. Foram coletados 60 fungos, de fevereiro a junho de 2003, e identificadas as famílias Agaricaceae, Auriculariaceae, Cantharellaceae, Ganodermataceae, Hygrophoraceae, Polyporaceae, Russulaceae, Stereaceae, Tremellaceae e Tricholomataceae. A Reserva de Campina foi a que apresentou maior diversidade de fungos. Todas as 18 amostras de filtrados de cultura dos Basidiomycetes, utilizadas nos bioensaios, apresentaram efeito inibitório contra um ou mais microrganismos-teste. As espécies E. coli, S. anatum e B. cereus, além de halos de inibição, apresentaram halos de estímulo de crescimento. Não foi observado efeito inibitório contra os fungos Corynespora sp. e Colletotrichum sp. O filtrado do fungo Trametes sp., cultivado em meio GPY, foi o que apresentou maior atividade de inibição. O meio de cultura BD favoreceu maior produção de biomassa seca. Dos dez fungos estudados, foi detectada a produção de proteases e celulases por todos os isolados, quatro produziram amilases, cinco fenoloxidases e um pectinase. Para a produção de amilases, os fungos cultivados em meio acrescido de farelo de trigo apresentaram os maiores halos. Para a produção de proteases, os maiores halos foram observados para os fungos crescidos em meio com farinha de peixe.

>Magister Scientiae - MSc / There are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.

The ability to generate a range of concentrations of various solutions rapidly and conveniently is an ongoing need in biotechnology. In this thesis we demonstrate how we took advantage of the full process control afforded by our recent custom high resolution 3D printer and resin advances to realize highly integrated and miniaturized microfluidic components for simultaneous on-chip serial dilution for dose-response assays. With judicious selection of mixed layer thicknesses and pixel-by-pixel dose control, we show that the diameter of 3D printed membrane valves can be reduced from 300 µm to 46 µm. We further introduce an entirely new kind of 3D printed valve, termed a squeeze valve, in which the active area is reduced still further to 15 µm x 15 µm. We demonstrate and characterize pumps based on each type of valve and introduce a short (<1 mm long) high aspect ratio channel that enables rapid diffusion-based mixing. We show that combining two pumps with this diffusion mixing channel results in a highly compact 1:1 mixer component. Connecting 10 of these components in series yields a miniature 10 stage 2-fold microfluidic serial dilution module that from two solution inputs simultaneously generates 10 output concentrations that cover three orders of magnitude. We show the efficacy of our serial dilution approach by demonstrating an assay for dose-dependent permeabilization of A549 cells in different concentrations of digitonin integrated into a single device. Our demonstration of component miniaturization in conjunction with a high degree of integration illustrates the promise of 3D printing to enable highly functional and compact microfluidic devices for a variety of biomolecular applications.

Philosophiae Doctor - PhD / Wastewater is normally composed of a mixture of pollutants. The type and composition of pollutants in a particular wastewater depend on the source of origin. The source and characteristics of a particular wastewater determine the ideal method of sewage treatment. Specific treatment techniques are effective in the removal of certain types of pollutants and may have no impact on the levels of other types of pollutants. Therefore, a combination of treatments and assessment of the quality of effluent before release into the environment is normally recommended. The assessment of effluent can be achieved by various techniques including chemical analysis and biological assays. Chemical analyses are commonly employed; however, they often pose detection problems and are considered to be uneconomical.

Amifostine (WR-2721, Ethyol®), a phosphorylated thiol, demonstrates the unique ability to protect normal but not tumour tissue from cytotoxic damage induced by radiation therapy and chemotherapy. This study tested the effect of amifostine's active metabolite, the free thiol, WR-1065, on the cytotoxicity of standard anticancer drugs against human A2780 ovarian and MCF7 breast cancer cell lines in vitro, using the well-characterised sulphorhodamine B assay. 50% inhibitory concentration (IC50) values were determined for each of 16 different anticancer drugs in the presence and absence of the highest nontoxic dose of WR-1065 from concentration-response curves constructed in triplicate and based on 18 replicate cell culture plates for each tested drug concentration. Pretreatment with WR-1065 had no statistically significant effect on the IC50 value of any of the 16 drugs tested against either the A2780 or MCF7 human tumour cells. These data expand upon previous reports showing that amifostine does not protect tumours from the cytotoxic effects of anticancer agents. The ability of amifostine to protect against dose-limiting toxicity to a variety of normal tissues without protection of tumour should enhance the efficacy ratio of a wide range of standard anticancer drugs.