Este trabalho de investigação refere-se ao uso do reator anaeróbio híbrido para tratamento de esgoto sanitário, com configuração baseada no reator anaeróbio de manta de lodo (UASB) com inclusão de: meio suporte sobre as calhas de coleta de gás (denominado reator anaeróbio híbrido - UAHB) e, também, meio suporte na zona de reação (denominado reator anaeróbio híbrido modificado - UAHBmod). Para o desenvolvimento desta pesquisa, no Campus I da USP de São Carlos-SP foram construídos dois reatores experimentais de 18,8 m3 cada: um reator UASB, com função de controle, e um reator UAHB. Primeiramente os reatores foram operados por período de 200 dias, com tempo de detenção hidráulica (TDH) de 6 h. Após serem inoculados, com 80 dias de operação, os reatores atingiram o estado de equilíbrio dinâmico aparente, com geração de alcalinidade, baixa concentração de ácidos voláteis e eficiência de remoção média de DQO, de 84% e 85% e de DBO de 87% e 91%, respectivamente para o UASB e o UAHB. Após esse período, os reatores foram submetidos a aumento da velocidade ascensional (Vasc) (mediante recirculação do efluente) de 0,78 m.h-1; 1,17 m.h-1; 1,56 m.h-1 e de 1,96 m.h-1. O UAHB mostrou ser menos susceptível ao aumento da Vasc do que o UASB. Além da análise da operação dos reatores, foram realizados os ensaios hidrodinâmicos e avaliada a estrutura da comunidade microbiana, por microscopia ótica, epifluorescência e pela técnica do DGGE. Após esse período preliminar, os reatores UAHB e UAHBmod, operados com TDH de 6h e Vasc de 0,78 m.h-1, atingiram o estado de equilíbrio dinâmico aparente, com geração de alcalinidade, baixa concentração de ácidos voláteis e eficiência de remoção média da matéria orgânica, de 71% e 76% em DQO, e de 72% e 87% em DBO, respectivamente para o UAHB e UAHBmod. Após este período, o reator UAHBmod, submetido a Vasc de 1,56 m.h-1, promoveu remoção de 74% de DQO, e de 87% de DBO. / This research refers to the use of a hybrid anaerobic reactor (UAHB) for domestic wastewater treatment. The configuration of this reactor is based on a sludge bed anaerobic reactor (UASB); in the first instance, a media support above the gas collection apparatus (also known as hybrid anaerobic reactor) was provided and later, a media support on the reaction zone (also known as hybrid modified anaerobic reactor - UAHBmod) was provided. Two reactors, with a volume of 18.8 m3, each, were built for this research at Campus I, USP in São Carlos - SP-Brazil. One UASB reactor acted as a control, and the other as a UAHB reactor. In the preliminary essays, the reactors were operated with 6h of hydraulic detention time (HDT) for 200 days. After inoculation, the reactors attained the apparent dynamic equilibrium state after 80 days of operation, with alkalinity generation, low volatile acids concentration and mean organic matter removal of 84% and 85% in terms of COD, and 87% and 91% in terms of BOD, for UASB and UAHB reactors, respectively. After this period, the reactors were submitted to an increasing in up velocity (Vup) of 0.78 m.h-1; 1.17 m.h-1; 1.56 m.h-1 and 1.96 m.h-1. The UAHB reactor showed lesser susceptibility for Vup increase than the UASB reactor. Hydrodynamic tests were also done on the reactors, in addition to routine operational analysis. The structure of the microbial community was evaluated by optical and epifluorescence microscopy, and the DGGE technique. After this step, the UAHB and the UAHBmod reactors were operated out 6h of HDT and Vup of 0.78 m.h-1. The reactors attained the apparent dynamic equilibrium state with alkalinity generation, low volatile acids concentration and mean organic matter removal of 71% and 76% in terms of COD, and 72% and 87% in terms of BOD for the UASB and UAHBmod reactors, respectively. After this period, the UAHBmod reactor was subjected to a Vup of 1.56 m.h-1 and achieved removal efficiencies of 74% COD and 87% BOD.

Made available in DSpace on 2014-07-29T15:26:25Z (GMT). No. of bitstreams: 1 AlessandraMarques2009.pdf: 692808 bytes, checksum: 9a7b1bde8039039ba579f8e25c59ead5 (MD5) Previous issue date: 2009-12-03 / In recent years the number of infections caused by microbacteria non-tuberculous mycobacteria (NTM) has increased mainly due to opportunistic infections in individuals imunocompormetidos and improvement of farming techniques and identification of MTN. Mycobacterium massilienese is an emerging body associated with wound infections, abscesses and pneumonia. An outbreak of infection after videoscopy occurred between 2005 and 2007 in seven hospitals in Goiânia-GO, in central Brazil. The objective of this study was to identify NTM isolated from patients with infection after arthroscopy and lararoscopia by PCR followed by analysis of fragment length polymorphism restrção (PRA-hsp65), compared by gel electrophoresis pulsed-field gel (PFGE), sequencing of the partial rpoB gene and determination of antimicrobial susceptibility in vitro. NTM were recovered from samples (exudate abscess subcutâneio) of 18 patients involved in the outbreak. In the period leading up to this study there was no reported case of infection after videoscopy caused by MTN in Goiania. The 18 isolates were identified as M, massiliene and genotyped as a single clone, indicating that they had a common origin, suggesting a common source of infection for the patients involved in the outbreak. The epidemic isolates were susceptible to amikacin (MIC90 4 micrograms / ml) and clarithromycin (MIC90 <1 ug / ml), but resistance to ciprofloxacin (MIC90 <128g/ml), tobramycin (MIC90 32 micrograms / ml) and intermediate susceptibility to cefoxitin (MIC90 64 ug / ml). In conclusion this study demonstrated the clonality of strains of M. massiliense involved in infections after procedures videoscopes and that they are susceptible to drugs indicated for the treatment / Durante os últimosd anos o número de infecções causadas por microbactérias não-tuberculosas (MNT) tem aumentado principalmente devido às infecções oportunistas em indivíduos imunocompormetidos e ao aprimoramento das técnicas de cultura e identificação das MTN. Mycobacterium massilienese é um organismo emergente, associado a infecções de feridas, formação de abscessos e pneumonias. Um surto de infecção após videoscopias ocorreu entre 2005 e 2007 em sete hospitais privados de Goiânia-GO, na região central do Brasil. O objetivo deste estudo foi identificar MNT isoladas de amostras de pacientes com infecção após artroscopia e lararoscopia por PCR seguida de análise de polimorfismo de fragmentos de restrção (PRA-hsp65), comparação por eletroforese em gel em campo pulsado (PFGE), sequenciamento parcial do gene rpoB e determinação da suscetibilidade antimicrobiana in vitro. MNT foram recuperadas das amostras (exsudato de abscesso subcutâneio) de 18 pacientes envolvidos no surto. No período antecedente a esse estudo não houve nenhum relato de caso de infecção após videoscopias causada por MTN em Goiânia. Os 18 isolados foram identificados como M, massiliene e genotipados como um único clone, indicando que tiveram uma origem em comum, o que sugere uma fonte comum de infecção para os pacientes envolvidos no surto. Os isolados epidêmicos apresentaram sensibilidade a amicacina (CIM90 4 ug/ml) e claritromicina (CIM90 < 1 ug/ml), porém resistência a ciprofloxacina (CIM90 < 128g/ml), tobramicina (CIM90 32 ug/ml), e sensibilidade intermediária a cefoxitina (CIM90 64 ug/ml). Em conclusão este estudo evidenciou a clonalidade de cepas de M. massiliense envolvidas em infecções após procedimentos de videoscopia e que as mesmas são suscetíveis às drogas indicadas para o tratamento

A bactéria Xylella fastidiosa 9a5c é o agente causal da clorose variegada dos citros (CVC) e responsável por grandes perdas econômicas na citricultura. O genoma de X. fastidiosa 9a5c foi totalmente seqüenciado e a sua análise permitiu identificar vários genes possivelmente envolvidos na patogenicidade/virulência da bactéria. Como os sintomas da CVC desenvolvemse um longo tempo após a infecção da planta pela bactéria e a severidade da doença têm sido associada à altas temperaturas, é possível que a expressão de fatores de patogenicidade/virulência seja dependente de densidade celular e/ou estresses térmicos. Desta forma, o crescimento in vitro da X. fastidiosa foi monitorado através da absorbância de suspensões bacterianas à 600nm (A600), número de unidades formadoras de colônias (UFC) e viabilidade celular, durante 16 dias de cultivo em meio PW modificado líquido, à 28 e 32ºC. Proteínas extracelulares foram extraídas e analisadas através da eletroforese bidimensional em gel de poliacrilamida (2D-PAGE). Bioensaios foram utilizados para verificar a produção de moléculas sinais por X. fastidiosa. Os resultados obtidos mostraram que o crescimento de X. fastidiosa à 32ºC, com base na A600, não diferiu do crescimento à 28ºC. No entanto, com base no número de UFCs e viabilidade celular, o crescimento de X. fastidiosa diferiu em função da temperatura, tempos de incubação e a interação entre esses fatores. X. fastidiosa produziu maior número de proteínas extracelulares à 32 do que à 28ºC, mostrando que a secreção de proteínas em X. fastidiosa é regulada pela temperatura de incubação. Várias proteínas extracelulares produzidas por X. fastidiosa à 28 e 32ºC são moduladas durante o crescimento da bactéria. A maior parte das proteínas extracelulares produzidas por X. fastidiosa são proteínas ácidas e de massa molecular aparente entre 20-60 kDa. X. fastidiosa não sintetizou moléculas de lactonas de homoserina aciladas (LHAs) reconhecidas pelo sistema repórter utilizado, mas sintetizou uma molécula extracelular em meio de cultivo, semelhante ao DSF produzido por X. campestris pv. campestris, capaz de restaurar a atividade da endoglucanase através do sistema repórter utilizado. A concentração desta molécula em meio PW modificado foi dependente da densidade de células de X. fastidiosa no meio. / The bacterium Xylella fastidiosa is the causal agent of the citrus variegated chlorosis (CVC) and is responsible for significant economic losses in citriculture. The genome of X. fastidiosa has been completely sequenced and revealed several genes probably involved in pathogenicity/virulence. Since CVC symptoms are develop a long time after infection of plant by the bacterium and the severity of the disease has been associated with high temperatures, it’s possible that the expression of pathogenicity/virulence factors is dependent on cellular density and/or temperature stresses. Thus, the growth of X. fastidiosa in modified liquid PW medium was measured based on the absorbance of suspensions at (A600), number of colony-forming units (CFU) and cellular viability, during 16 days at 28 and 32ºC. Extracellular proteins were extracted and analysed by two-dimensional gel electrophoresis (2D-PAGE). Bioassays were used to determine whether X. fastidiosa produces signal molecules involved in quorum perception. The results showed that temperatures of 28 and 32ºC did not affect the growth of the bacterium, based on A600. Temperatures of 28 and 32ºC, incubation times and the interaction of both factors affected bacterial growth based on CFU numbers and the cellular viability. X. fastidiosa produced higher number of extracellular proteins at 32 than at 28ºC, showing that protein secretion is dependent on growth temperature. Several extracellular proteins produced by X. fastidiosa at 28 and 32ºC were modulated the bacterial growth. Most of the extracellular proteins produced by X. fastidiosa were acidic with apparent molecular mass within 20-60 kDa. X. fastidiosa did not synthesize N-acyl homoserine lactone (AHL) recognizes by the reporter system used. However, it synthesized an extracellular molecule in modified PW medium, similar to DSF produced by X. campestris pv. campestris, which is able to restore endoglucanase activity by the reporter system. The concentration of this extracellular molecule produced by X. fastidiosa was dependent on cellular density.

Philosophiae Doctor - PhD / Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate ((PSNP-OSO3) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 −) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10−8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were <100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 μA/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 μM NO2 −, were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 −; respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors. / South Africa

O trabalho teve por objetivo avaliar fatores associados à ensilagem da cana-deaçúcar, com destaque para a aplicação de aditivos químicos e microbianos sobre a dinâmica fermentativa, composição bromatológica, atividade da álcool desidrogenase e desenvolvimento e diversidade da micloflora em silagens de cana-de-açúcar. No ensaio conduzido durante 110 dias o delineamento experimental adotado foi o inteiramente ao acaso, com 4 tratamentos, 2 repetições, e seis épocas de abertura (1, 3, 7, 15, 35, 110 dias). Os tratamentos foram: uréia 1% MV e os inoculantes microbianos Lactobacillus buchneri (3,65x105 ufc/g da MV) e a combinação de bactérias Pedioccocus pentosassus e Lactobacillus buchneri (1x106 ufc/g MV). As maiores variações na composição bromatológica e perdas de MS, das silagens controle ocorreram dos 7 aos 15 dias, estabilizando após esse período. As regressões ajustadas para perdas de MS e carboidratos solúveis foram bem similares e de forma contrária ao acúmulo de FDN. As perdas por gases alcançaram valores de 28,27%, de carboidratos solúveis em apenas 2,98% e FDN em torno de 67,77% da MS. Os aumentos nos teores de etanol e perda na digestibilidade nas silagens controle se extenderam até o 35º dia, com valores máximos de etanol de 12,23%. Foi possível relacionar etanol com a digestibilidade mostrando que cada 1% de aumento nos teores de etanol, 2 unidades de digestibilidade foram perdidas. Os aditivos uréia e o aditivo Lactobacillus buchneri mais Pediococcus foram eficazes em diminuir a produção de etanol (2,75 e 1,30 vs 8,27% no tratamento controle), em diminuir perdas de MS em 47 e 60%, e de carboidratos soluveis em 22 e 56% em relação à silagem controle, respectivamente. As silagens aditivadas com uréia obtiveram maiores valores de pH e maiores valores de ácido lático em relação às silagen controle. As silagens aditivadas com L. buchneri apenas foram as de maiores produções de etanol, acima da silagem controle (11.53 vs 8.27%), além de grandes perdas de matéria seca e baixa digestibilidade pelo acúmulo de FDN, comparáveis às silagens controle. A diferença entre aditivos na composição químico-bromatológica e perdas ocorreu após 7 dias de fermentação. Os dados apresentados pelos aditivos uréia e L. buchneri mais Pediococcus foram ajustados em curvas simples, através de modelos lineares, para descrever e predizer as variações durante a ensilagem. Os tratamentos controle e a aditivação com L. buchneri apenas, pelas altas taxas fermentativas, observaram melhor ajuste dos dados em polinômios de segundo e terceiro grau. Apesar dos altos teores de ácido acético em todas as silagens, principalmente nas silagens aditivadas com a combinação de bactérias, não foram verificadas efeitos deste sobre a população de leveduras. Os teores obtidos de ácido lático e ácido propiônico e a relação entre esses ácidos e o ácido acético, durante a fermentação, conseguiu explicar parte do sucesso dos tratamentos uréia e L. buchneri mais Pediococcus na redução da atividade da enzima álcool desidrogenase e na producão de etanol. A análise de grupamentos hieráquicos mostrou que os aditivos alteraram a diversidade bacteriana durante a ensilagem. / The present trial aimed to study the ensiling associated factors of sugarcane focusing on chemical and microbial additives on fermentation, chemical composition, enzymatic activity of alcohol dehydrogenase and the microflora development and diversity in sugarcane silages. A complete randomized design was set to a 110-d trial with 4 treatments, two replications within 6 opening dates (1, 3, 7, 15, 35, 110-d). Treatments were described as follows: urea 1% (wet basis), Lactobacillus buchneri (3.65x105 cfu/g of forage), a combination of Pediococus pentosassus and Lactobacillus buchneri (1x106). Major variation observed on the chemical composition and the DM losses in sugarcane silages without additives took place from the day 7 through the day 15. Losses of DM and soluble carbohydrates showed similar trend and in opposition to the NDF increase. Gases losses averaged 28.27%, while the soluble carbohydrates and NDF contents reached respectively, 2.98% and 67.77% when fermentation was stabilized. Conversely, ethanol and the digestibility were changed across the storage period up to the day 35, with ethanol content increasing to 12.23%. 1% of ethanol increase was associated with 2 percentage units of digestibility decrease. Both urea and the combination of microorganisms were effective in decrease the ethanol content (2.75, 1.30 vs 8.27% - without additives), decrease DM losses (47 and 60%) and reduce soluble carbohydrates losses (22 and 56%) when compared to the control treatment. The urea treated silages showed higher pH and lactic acid values. The L. buchneri treatment led to higher ethanol content (11.23 vs 8.27%) compared to the control, resulting in low DM recovery rate, higher losses and decreased digestibility as well as the silages without additives. The major changes on the chemical composition were noticed after the day 7 of fermentation. For the addition of urea and the combination of microorganisms L. buchneri and Pediococcus the variation was better described by linear equations whereas quadratic and cubic effects were more suitable for fitting the data from the control and the L. buchneri added silages. Even tough all silages has shown high acetic acid contents, mainly the combination of lactic bacteria, no significant effects were observed upon the yeast counts. However, the levels of lactic acid and propionic acid and the ratio of both over the acetic acid content were related to the decrease on the activity of the alcohol dehydrogenase enzyme and, furthermore, on the ethanol content of the silages. The cluster analysis based on molecular evaluation demonstrated a change promoted over the bacterial population mediated by the additives applied during the ensiling of sugarcane.

A bactéria Xylella fastidiosa 9a5c é o agente causal da clorose variegada dos citros (CVC) e responsável por grandes perdas econômicas na citricultura. O genoma de X. fastidiosa 9a5c foi totalmente seqüenciado e a sua análise permitiu identificar vários genes possivelmente envolvidos na patogenicidade/virulência da bactéria. Como os sintomas da CVC desenvolvemse um longo tempo após a infecção da planta pela bactéria e a severidade da doença têm sido associada à altas temperaturas, é possível que a expressão de fatores de patogenicidade/virulência seja dependente de densidade celular e/ou estresses térmicos. Desta forma, o crescimento in vitro da X. fastidiosa foi monitorado através da absorbância de suspensões bacterianas à 600nm (A600), número de unidades formadoras de colônias (UFC) e viabilidade celular, durante 16 dias de cultivo em meio PW modificado líquido, à 28 e 32ºC. Proteínas extracelulares foram extraídas e analisadas através da eletroforese bidimensional em gel de poliacrilamida (2D-PAGE). Bioensaios foram utilizados para verificar a produção de moléculas sinais por X. fastidiosa. Os resultados obtidos mostraram que o crescimento de X. fastidiosa à 32ºC, com base na A600, não diferiu do crescimento à 28ºC. No entanto, com base no número de UFCs e viabilidade celular, o crescimento de X. fastidiosa diferiu em função da temperatura, tempos de incubação e a interação entre esses fatores. X. fastidiosa produziu maior número de proteínas extracelulares à 32 do que à 28ºC, mostrando que a secreção de proteínas em X. fastidiosa é regulada pela temperatura de incubação. Várias proteínas extracelulares produzidas por X. fastidiosa à 28 e 32ºC são moduladas durante o crescimento da bactéria. A maior parte das proteínas extracelulares produzidas por X. fastidiosa são proteínas ácidas e de massa molecular aparente entre 20-60 kDa. X. fastidiosa não sintetizou moléculas de lactonas de homoserina aciladas (LHAs) reconhecidas pelo sistema repórter utilizado, mas sintetizou uma molécula extracelular em meio de cultivo, semelhante ao DSF produzido por X. campestris pv. campestris, capaz de restaurar a atividade da endoglucanase através do sistema repórter utilizado. A concentração desta molécula em meio PW modificado foi dependente da densidade de células de X. fastidiosa no meio. / The bacterium Xylella fastidiosa is the causal agent of the citrus variegated chlorosis (CVC) and is responsible for significant economic losses in citriculture. The genome of X. fastidiosa has been completely sequenced and revealed several genes probably involved in pathogenicity/virulence. Since CVC symptoms are develop a long time after infection of plant by the bacterium and the severity of the disease has been associated with high temperatures, it’s possible that the expression of pathogenicity/virulence factors is dependent on cellular density and/or temperature stresses. Thus, the growth of X. fastidiosa in modified liquid PW medium was measured based on the absorbance of suspensions at (A600), number of colony-forming units (CFU) and cellular viability, during 16 days at 28 and 32ºC. Extracellular proteins were extracted and analysed by two-dimensional gel electrophoresis (2D-PAGE). Bioassays were used to determine whether X. fastidiosa produces signal molecules involved in quorum perception. The results showed that temperatures of 28 and 32ºC did not affect the growth of the bacterium, based on A600. Temperatures of 28 and 32ºC, incubation times and the interaction of both factors affected bacterial growth based on CFU numbers and the cellular viability. X. fastidiosa produced higher number of extracellular proteins at 32 than at 28ºC, showing that protein secretion is dependent on growth temperature. Several extracellular proteins produced by X. fastidiosa at 28 and 32ºC were modulated the bacterial growth. Most of the extracellular proteins produced by X. fastidiosa were acidic with apparent molecular mass within 20-60 kDa. X. fastidiosa did not synthesize N-acyl homoserine lactone (AHL) recognizes by the reporter system used. However, it synthesized an extracellular molecule in modified PW medium, similar to DSF produced by X. campestris pv. campestris, which is able to restore endoglucanase activity by the reporter system. The concentration of this extracellular molecule produced by X. fastidiosa was dependent on cellular density.

Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein O-GlcNAc levels. As a first step in elucidating the role of O-GlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modified proteins that decrease during oocyte development. Stage I oocytes due to their high amounts of these proteins, were used as starting material for purification. Multiple affinity and specific antibody based purification technique were initially used in an attempt to enrich the O-GlcNAc proteins. Due to the unique properties of the proteins ultimately identified, these techniques were unable to provide sufficient material for sequencing. However, differential centrifugation coupled with 2D-gel electrophoresis was highly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10. Coomassie stained bands from 2D-analysis were trypsin digested, and peptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpreted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA binding protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A, Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogenic oocytes (stages I-III) is involved in tRNA storage and possess low tRNA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA storage ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in order to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation. This study is the first to characterize these oocyte specific proteins as O-GlcNAc modified proteins. Overall, the presence of several O-GlcNAc proteins in oocytes, the reduction in their levels/ O-GlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in developing oocyte indicates O-GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O-GlcNAc role in their function is a worthwhile pursuit.

Ph.D. / Molecular Microbiology and Immunology / Both poxviruses and γ2-herpesviruses share the K3-family of viral immune evasion proteins. These proteins are characterized by an amino-terminal RING-CH domain followed by two transmembrane domains. We analyzed several human homologues of the K3-family termed membrane-associated RING-CH (MARCH) proteins. All MARCH proteins localized to subcellular membranes while several reduced surface levels of known K3-family substrates. Thus, MARCH proteins appear to be structurally and functionally homologous to viral K3 proteins. One of the major challenges in determining the function of this family is the identification of their physiological substrates. To overcome this we created a quantitative proteomics approach which can be used to identify novel substrates for both the K3- and MARCH-families. Using stable isotope labeling by amino acids in cell culture, we compared the proteome of plasma membrane, golgi, and endoplasmic reticulum membranes in the presence and absence of K5 and MARCH-VIII. Quantitative mass spectrometric protein identification from these fractions revealed that CD316 (bone marrow stromal antigen 2), CD166 (activated leukocyte cell adhesion molecule) and syntaxin-4 were consistently underrepresented in the plasma membrane of K5 expressing cells, while CD44, CD81 (TAPA-1) and B-cell receptor-associated protein 31kDa (Bap31) were consistently underrepresented in the plasma membrane of MARCH-VIII expressing cells. Furthermore, downregulation of each of these proteins was independently confirmed. Our results both identify and characterize a novel family of human ubiquitin ligase enzymes and elucidate a novel technique which can analyze this family and be easily adapted to the analysis of other cellular enzymes viral immune modulators.

<p>Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3 &minus / ) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate (PSNP-OSO3 &minus / ) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 &minus / ) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10&minus / 8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were &lt / 100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 &mu / A/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 &mu / M NO2 &minus / , were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 &minus / , respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors.</p>

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.