Orientadora: Marileda Bonafim Carvalho / Banca: Julieta Rodini Engracia Moraes / Banca: Aguemi Kohayagawa / Resumo: Para este estudo foram avaliados 20 cães adultos sadios (10 machos e 10 fêmeas) e 24 cães adultos (16 machos e 8 fêmeas) com insuficiência renal crônica (IRC), com o objetivo de estudar a proteinúria. Foram empregadas técnicas para determinação quantitativa de proteína da urina e para a separação de suas frações por meio de SDS-PAGE. As quantidades de proteínas presentes na urina de cães sadios foram pequenas, mas o estudo dos perfis eletroforéticos mostrou haver diferenças entre machos e fêmeas. Apesar da ampla variação das concentrações de proteínas entre os animais, o grupo dos portadores de IRC apresentou proteinúria intensa. Os resultados dos portadores de IRC foram agrupados de acordo com o sexo ou em função do tipo de lesão renal predominante, para comparação dos eletroforetogramas. As análises revelaram que os perfis eletroforéticos seguem um padrão para os machos e outro pra as fêmeas. Ainda, o padrão o perfil eletroforético observado quando há predomínio de lesões glomerulares difere do observado quando há predomínio de lesões túbulo-intersticiais. / Abstract: This trial was conducted in 20 adult health dogs (10 male and 10 female) and in 24 adult dogs (16 male and 8 female) in chronic renal failure (CRF), in order to study urinary protein loss. Determination of total urinary proteins and their separation with SDS-PAGE were employed. The results show that CRF dogs had higher proteinuria when compared to controls and the amount of detected protein had large difference among them. In the CRF there are different patterns of protein bands occurrence and distribution according to the patient sex and predominant renal lesion. The difference between protein bands occurrence of male and female already exists in normal dogs. / Mestre

Made available in DSpace on 2015-04-09T12:28:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-08Bitstream added on 2015-04-09T12:48:01Z : No. of bitstreams: 1 000814587.pdf: 233099 bytes, checksum: 86c73283cfc6d5c2954be91ce17d3f29 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Endotoxins are often found in high concentration in the peritoneal fluid from horses with acute gastrointestinal disease and blood of septicemic foals. The conditions mentioned earlier can lead to inflammatory processes and cause changes in the concentrations of acute phase proteins. The objectives of this study were to determine by means of polyacrylamide gel electrophoresis (SDS-PAGE), the concentrations of acute phase proteins (transferrin, ceruloplasmin, albumin, haptoglobin and acid glycoprotein) in serum and peritoneal fluid of horses submitted to experimental endotoxemia and evaluate the possible effects of the administration of lidocaine on the production of these proteins after intraperitoneal injection of endotoxin in these animals. To this end, 12 animals were distributed in two groups, control which was treated with 0.9% NaCl solution and the experimental which was treated with continuous infusion for 6 hours of 2% lidocaine solution. It is concluded that in this experimental model the intraperitoneal administration of endotoxin caused an increase in the concentrations of peritoneal transferrin, ceruloplasmin and haptoglobin in the control group, of albumin in both groups, and the acid glycoprotein in the group treated with infusion of lidocaine. However, the absence of changes in the concentrations of serum ceruloplasmin, serum albumin, haptoglobin and acidic glycoprotein indicate that the amount of the circulatory endotoxin was not enough to alter serum concentrations of these proteins. The continuous infusion of lidocaine interfered in peritoneal acute phase proteins values and had an anti-endotoxemic effect / FAPESP: 2009/15683-0

Doctor of Philosophy / Department of Plant Pathology / Anna E. Whitfield / The Bunyaviridae is a family of animal and plant viruses that pose a threat to human, animal, and plant health worldwide. In nature, the dissemination of these viruses is dependent on arthropod vectors (genera Orthobunyavirus, Nairovirus, Phlebovirus, and Tospovirus) or rodent vectors (genus Hantavirus). The genus Tospovirus is the only one within this virus family that is composed of plant-infecting viruses transmitted by thrips. Tomato spotted wilt virus (TSWV), the type species of the Tospovirus genus, is one of the ten most devastating plant viruses known. It is most efficiently transmitted by the western flower thrips, Frankliniella occidentalis Pergande, in a persistant propagative manner. The insect molecules associated with virus infection and transmission by the thrips vector remain unidentified to date. The aim of this work was to identify F. occidentalis larval thrips proteins that are differentially expressed during TSWV infection of the insect vector and those that directly interact with TSWV. To achieve these goals, I used two-dimensional (2-D) gel electrophoresis and mass spectrometry coupled with Mascot searches. I identified 26 protein spots that displayed differential abundances in response to TSWV infection, which contained 37 proteins. Sixty two percent of these proteins were down-regulated by the viral infection demonstrating a complex response. Moreover, 8 and 11 protein spots that directly interacted with purified TSWV virions and a TSWV glycoprotein (GN), respectively, were identified in overlay assays of larval thrips proteins resolved by 2-D gel electrophoresis. A total of five proteins were identified from these spots. These interacting proteins might play roles in attachment and entry, endocytosis/exocytosis, and escape from different tissues for transmission to occur. Injection of double-stranded RNA (dsRNA) into adult female thrips triggered an RNAi response that resulted in 23% reduction of the target gene transcript level. This significant reduction resulted in increased mortality and decreased fertility compared to insects injected with control dsRNA or water and non-injected insects as well. The work presented here provides new insights on the molecular basis of this virus-vector interaction and describes new tools to conduct functional genomic assays to study gene function and design control strategies of F. occidentalis.

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-01-16Bitstream added on 2014-06-13T19:15:42Z : No. of bitstreams: 1 toledo_egh_me_jabo.pdf: 1480955 bytes, checksum: 264147d52e4fdcb0c91216e8ebe11eca (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Para este estudo foram avaliados 20 cães adultos sadios (10 machos e 10 fêmeas) e 24 cães adultos (16 machos e 8 fêmeas) com insuficiência renal crônica (IRC), com o objetivo de estudar a proteinúria. Foram empregadas técnicas para determinação quantitativa de proteína da urina e para a separação de suas frações por meio de SDS-PAGE. As quantidades de proteínas presentes na urina de cães sadios foram pequenas, mas o estudo dos perfis eletroforéticos mostrou haver diferenças entre machos e fêmeas. Apesar da ampla variação das concentrações de proteínas entre os animais, o grupo dos portadores de IRC apresentou proteinúria intensa. Os resultados dos portadores de IRC foram agrupados de acordo com o sexo ou em função do tipo de lesão renal predominante, para comparação dos eletroforetogramas. As análises revelaram que os perfis eletroforéticos seguem um padrão para os machos e outro pra as fêmeas. Ainda, o padrão o perfil eletroforético observado quando há predomínio de lesões glomerulares difere do observado quando há predomínio de lesões túbulo-intersticiais. / This trial was conducted in 20 adult health dogs (10 male and 10 female) and in 24 adult dogs (16 male and 8 female) in chronic renal failure (CRF), in order to study urinary protein loss. Determination of total urinary proteins and their separation with SDS-PAGE were employed. The results show that CRF dogs had higher proteinuria when compared to controls and the amount of detected protein had large difference among them. In the CRF there are different patterns of protein bands occurrence and distribution according to the patient sex and predominant renal lesion. The difference between protein bands occurrence of male and female already exists in normal dogs.

Resumo:As endotoxinas são frequentemente detectadas em alta concentração no líquido peritoneal de equinos com doença gastrintestinal aguda e no sangue de potros septicêmicos. As condições citadas anteriormente podem levar a processos inflamatórios e provocar alterações nas concentrações de proteínas de fase aguda. Os objetivos deste estudo foram determinar, por meio de eletroforese em gel de poliacrilamida (SDS-PAGE), as concentrações de proteínas de fase aguda (transferrina, ceruloplasmina, albumina, haptoglobina e glicoproteína ácida) no soro e no líquido peritoneal de equinos submetidos à endotoxemia experimetal, e avaliar os possíveis efeitos da administração da lidocaína sobre a produção destas proteínas depois da injeção intraperitoneal de endotoxina nestes animais. Para tanto, foram utilizados 12 animais distribuídos em dois grupos, o controle, tratado com solução de NaCl a 0,9%, e o experimental, tratado com infusão contínua de solução de lidocaína a 2% por 6 horas. Conclui-se que neste modelo experimental a administração intraperitoneal de endotoxina causou o aumento nas concentrações peritoneais de transferrina, ceruloplasmina e haptoglobina no grupo controle, da albumina em ambos os grupos, e da glicoproteína ácida no grupo tratado com infusão de lidocaína. Porém a ausência de alterações nas concentrações séricas de ceruloplasmina, albumina, haptoglobina e glicoproteína ácida indicam que a quantidade de endotoxina que chegou à corrente circulatória não foi suficiente para alterar as concentrações séricas destas proteínas. A infusão contínua de lidocaína interferiu nos valores peritoneais das proteínas mensuradas, tendo efeito anti-endotoxêmico / Abstract:Endotoxins are often found in high concentration in the peritoneal fluid from horses with acute gastrointestinal disease and blood of septicemic foals. The conditions mentioned earlier can lead to inflammatory processes and cause changes in the concentrations of acute phase proteins. The objectives of this study were to determine by means of polyacrylamide gel electrophoresis (SDS-PAGE), the concentrations of acute phase proteins (transferrin, ceruloplasmin, albumin, haptoglobin and acid glycoprotein) in serum and peritoneal fluid of horses submitted to experimental endotoxemia and evaluate the possible effects of the administration of lidocaine on the production of these proteins after intraperitoneal injection of endotoxin in these animals. To this end, 12 animals were distributed in two groups, control which was treated with 0.9% NaCl solution and the experimental which was treated with continuous infusion for 6 hours of 2% lidocaine solution. It is concluded that in this experimental model the intraperitoneal administration of endotoxin caused an increase in the concentrations of peritoneal transferrin, ceruloplasmin and haptoglobin in the control group, of albumin in both groups, and the acid glycoprotein in the group treated with infusion of lidocaine. However, the absence of changes in the concentrations of serum ceruloplasmin, serum albumin, haptoglobin and acidic glycoprotein indicate that the amount of the circulatory endotoxin was not enough to alter serum concentrations of these proteins. The continuous infusion of lidocaine interfered in peritoneal acute phase proteins values and had an anti-endotoxemic effect / Orientador: Francisco Leydson Formiga Feitosa / Banca: Marcos Pinheiro Franque / Banca: Taciana Rabelo Ramalho Ramos / Banca: Paulo Sérgio Patto dos Santos / Banca: Juliana Regina Peiró / Doutor

Orientador: Marco Aurelio Zezzi Arruda / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T13:14:37Z (GMT). No. of bitstreams: 1 Silva_MarceloAnselmoOseasda_M.pdf: 1369513 bytes, checksum: 35e33836144e52420b1164f512aa97e9 (MD5) Previous issue date: 2008 / Resumo: Nesta dissertação foi estudado o comportamento de partição da albumina, proteína presente em elevada concentração no plasma sangüíneo e que interfere na determinação em diversas técnicas analíticas. No estudo efetuado foram avaliados diferentes sistemas aquosos de duas fases, explorando um fenômeno denominado ponto nuvem. Tais sistemas empregam surfactantes, sob condições experimentais específicas, obtendo-se a formação de duas fases imiscíveis: uma rica e outra pobre em tensoativo. Observou-se que devido às características hidrofílicas da albumina, bem como à dimensão dos agregados formados, sua extração para fase rica em tensoativo apresenta valores de coeficiente de partição que não ultrapassam 0,66 o que representa uma eficiência de extração de ca.40%. A extração da proteína apresentou-se viável mediante a utilização de uma mistura composta por um tensoativo não-iônico (Triton® X-114), que possibilita a separação de fases em temperatura biocompatível, na presença de outro tensoativo iônico (dodecil sulfato de sódio - SDS), que atua favorecendo as interações eletrostáticas entre os agregados e as moléculas protéicas, desde que se trabalhe em meio com pH abaixo de 5,0. Análises de dicroísmo circular complementaram o estudo e fornecerem evidências de que a aplicação do sistema baseado na mistura composta por Triton® X-114 e SDS causava desnaturação parcial da proteína, o que não inviabilizou sua aplicação para extração da mesma em uma amostra real. A eficiência deste sistema foi, então, avaliada para a remoção de albumina em plasma sangüíneo. Um coeficiente de partição de 1,1 foi obtido, indicando que ca.51% das proteínas encontravam-se na fase rica em tensoativo. As amostras submetidas ao procedimento de extração também foram avaliadas frente à técnica de eletroforese em gel, sendo que a fase pobre em sufactante apresentou um perfil eletroforético mais detalhado quando comparada a uma amostra que não foi submetida ao procedimento proposto. Já na fase rica em surfactante, foi observada a presença majoritária de albumina e, em menor concentração, outras proteínas de grande abundância no plasma tais como imunoglobulina G e transferrina. Por fim, o método apresentou desempenho semelhante ao de sistemas disponíveis comercialmente para remoção de albumina, tal como o sistema da Millipore®, apresentado neste trabalho / Abstract: In this work the partition behavior of albumin was studied, which is found at high concentrations in blood plasma, which interferes in many analytical techniques determinations. The present study evaluated different aqueous two-phase systems, exploiting a phenomenon called of cloud point. These systems employ surfactants under specific experimental conditions, enabling formation of two immiscible phases: one rich and another poor in surfactant. Due to the hydrophilic characteristcs of albumin, as well as its aggregate dimensions, its extration to the surfactant rich phase presented partition coefficients lower than 0.66, representing and extraction efficiency of ca. 40%. Protein extraction was feasible by applying a mixture comprised of a nonionic surfactant (Triton® X-114), which allowed the phase separation at biocompatible temperatures, and an ionic one (sodium dodecylsufate - SDS), wich promotes eletrostatic interactions between aggregates and protein molecules, since the extraction procedure is carried out at pH 5.0. Circular dichroism analysis complemented the study and it showed that a system based on a Triton® X-114 and SDS mixture causes partial protein denaturation, but its application for a real sample is feasible. The efficiency of this was evaluated for albumin removal from blood plasma. A partition coefficient of 1.1 was obtained, indicating that ca. 51% of proteins were contained in the surfactant rich phase. Albumin depleted samples were submitted to gel eletrophoresis and the surfactant poor phase presented a more detailed gel electrophoresis profile, when compared with a crude sample. The surfactant rich phase reveled that albumin is the predominant protein present, but it is possible to find other highly concentrated plasmatic proteins including immunoglobulin G and transferrin. Finally, the method presented similar performance when compared with commercially available systems for albumin removal, such as the Millipore® system, wich was also evaluated in this work / Mestrado / Quimica Analitica / Mestre em Química

Tese(doutorado) - Universidade Federal do Rio Grande, Programa de Pós–Graduação em Oceanografia Biológica, Instituto de Oceanografia, 2013. / Submitted by Cristiane Gomides (cristiane_gomides@hotmail.com) on 2013-10-15T11:55:34Z No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) / Approved for entry into archive by Sabrina Andrade (sabrinabeatriz@ibest.com.br) on 2013-10-17T17:34:15Z (GMT) No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) / Made available in DSpace on 2013-10-17T17:34:15Z (GMT). No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) Previous issue date: 2013 / A composição (CCB) e a atividade (perfil fisiológico - PFCB) da comunidade bacteriana foram investigadas no estuário da Lagoa dos Patos e região costeira adjacente através de amostragem tipo-Lagrangiana, Euleriana e ao longo de um transecto para responder três perguntas: i) existe um padrão de recorrência (estabilidade) da CCB e PFCB em diferentes faixas de salinidade? ii) a CCB e PFCB respondem a diferentes escalas temporais e espaciais, inclusive a fenômenos climáticos globais que afetam a hidrodinâmica do estuário, tais como o “El Niño Southern Oscillation” (ENSO) ? iii) que fatores, além da salinidade, afetam a CCB e a PFCB? A CCB e a PFCB estiveram associadas à hidrodinâmica do estuário, tanto em escalas curtas (entrada de cunha salina), médias (sazonal) e largas (ENSO). Isto porquê a hidrodinâmica condiciona a variabilidade da salinidade e secundariamente o seston, nutrientes inorgânicos dissolvidos e substratos orgânicos, que afetam as bactérias. A CCB foi primariamente estruturada pela salinidade, seguindo o padrão normalmente encontrado na literatura para estuários, com comunidades características de água salgada e doce. Já a PFCB teve maior influência da quantidade de nutrientes e substratos e de maneira indireta da salinidade. De maneira geral, a atividade bacteriana foi menor em águas salgadas mais oligotróficas. Entretanto, grande atividade bacteriana foi observada em água salgada rica em nutrientes que penetrava no estuário. A maior concentração de nutrientes na água salgada pode ter sido resultado de ressuspensão de sedimento, ou ingresso de água costeira previamente enriquecida com água estuarina. / The composition (BCC) and activity (community level physiological profiles, CLPP) of the bacterial community were investigated in the Patos Lagoon estuary and adjacent coastal region through Lagrangian-like, Eulerian and transect samplings in order to answer three questions: i) is there a pattern of recurrence (stability) of the BCC and CLPP in different salinity ranges? ii) does the BCC and CLPP respond to different temporal and spatial scales, including global climate phenomena that affect the estuary hydrodynamics like El Niño Southern Oscillation (ENSO)? iii) which factors apart from salinity affect the BCC and CLPP? The BCC and CLPP were associated to the estuary hydrodynamics, at short- (salt wedge entrance), meso- (seasonal) and largescales (ENSO). This is because the hydrodynamics conditions the variability of salinity and secondarily the seston, dissolved inorganic nutrients and organic substrates, which in turn affect bacteria. The BCC was primarily structured by salinity, following the pattern commonly found in the literature for estuaries, with characteristic fresh- and saltwater communities. The CLPP had higher influence of the amount of nutrients and substrates and indirectly of salinity. In general the bacterial activity was lower in oligotrophic, saltier waters. However, high bacterial activity was observed in nutrient-rich saltwater that entered the estuary. The higher concentration of nutrients in saltwater is likely the result of ressuspension of the sediment or ingress of coastal water previously enriched with estuarine water.

Orientador: Marco Aurélio Zezzi Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T09:34:49Z (GMT). No. of bitstreams: 1 Barbosa_HerbertdeSousa_D.pdf: 1927584 bytes, checksum: 9e2393e5f41dc205496fac407f5f93ea (MD5) Previous issue date: 2011 / Resumo: Este trabalho de pesquisa consiste na comparação entre dois tipos de sementes de soja [Glycine max (L.) Merrill], transgênica e não-transgênica, em termos proteômicos e metaloproteômicos, de modo a avaliar possíveis diferenças existentes entre as amostras (Ex. diferença de expressão protéica, concentração das espécies metálicas e não-metálicas ligadas às biomoléculas). Para isso, inicialmente, as biomoléculas foram extraídas usando um tampão extrator específico e, após o tratamento adequado da amostra, separadas por meio da técnica de eletroforese bidimensional em gel de poliacrilamida (2D-PAGE) e caracterizadas por meio de técnicas de espectrometria de massas (MALDI-QTOF e ESI-QTOF) e identificadas por meio de buscas em banco de dados relacionados utilizando o programa Mascot, a partir dos dados gerados pelo espectrômetro de massas. Sendo assim, foram identificadas um total de 192 proteínas, sendo 13 proteínas na faixa de pH de 3 a 10 e 179 na faixa de pH de 4 a 7, sendo que esta última faixa de pH mostrou melhor resolução dinâmica na separação das proteínas. A distribuição funcional das proteínas identificadas mostrou que a maior parte (49%) destas estão envolvidas em armazenamento de nutrientes e atividade proteolítica, citando como exemplo deste grupo as proteínas glicina e b-conglicinina e suas subunidades. Para uma avaliação proteômica comparativa, com o intuito de se avaliar possíveis diferenças quanto à expressão das proteínas separadas entre as amostras avaliadas, foi utilizada a técnica de eletroforese em gel diferencial bidimensional (2D-DIGE), a partir de dados como intensidade e volume dos spots protéicos. Sendo assim, foram selecionadas um total de 04 proteínas diferenciais entre as amostras, sendo que todas foram identificadas via espectrometria de massas. Para uma avaliação metaloproteômica comparativa, um estudo para identificação de íons metálicos e semimetálicos livres ou ligados a proteínas foi realizado. Para isso, foi feita uma varredura semi-quantitativa de espécies metálicas e semimetálicas presentes nos extratos protéicos das sementes de soja transgênica e não-transgênica usando a técnica de ICP-MS, sendo que os íons Na, Mg, Si, K, Ti, Mn, Zn e Rb foram mais detectados na soja não-transgênica. Já o íon Cu foi mais detectado nas sementes de soja transgênica. Estes elementos diferenciais entre as amostras foram quantificados via ICP-MS. Além disso, os spots diferenciais obtidos com a técnica de 2D-DIGE foram avaliados quanto a presença de cobre, já que este elemento mostrou diferenças em concentrações em proteínas de soja, conforme trabalhos recentemente publicados na literatura pelo nosso grupo de pesquisa / Abstract: The research compairs two types of soybean [Glycine max (L.) Merrill], transgenic and non-transgenic, in terms of proteomics and metalloproteomics in order to evaluate possible differences among the samples (E.g. difference in protein expression, concentration of metal species and non-metal bounded to biomolecules). Therefore, initially, the biomolecules were extracted using a specific extration buffer and, after proper treatment of the sample, separated by the technique of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and characterized by mass spectrometry techniques (MALDI-QTOF and ESI-QTOF) and identified through searches related database using the Mascot program, from the data generated by mass spectrometer. Thus, we identified a total of 192 proteins, 13 proteins in the pH range 3 to 10 and 179 at pH 4-7, and this latest range of pH showed a better dynamic resolution in the separation of proteins. The functional distribution of identified proteins showed that most (49%) of these are involved in nutrient storage and proteolytic activity, citing as an example of this group glycinin and b-conglycinin and their subunits. For an evaluation of proteomics in order to evaluate possible differences in protein expression between the samples, the differential gel electrophoresis (2D-DIGE) technique was used based on data such as intensity and volume of proteins. Therefore, we selected a total of 04 differential proteins between samples, all of them were identified by mass spectrometry. For a comparative assessment metalloproteomics, a study for identification of metal ions and semimetal free or bound to proteins was performed. In this way, a semi-quantitative sweeping of semi-metal and metal species present in protein extracts of transgenic and non-transgenic soybean seeds was carried out using ICP-MS, and the ions Na, Mg, Si, K, Ti, Mn, Zn and Rb were detected in seeds of non-transgenic soybean. The ion Cu was detected in transgenic soybeans seeds. These differentials elements between the samples were quantified via ICP-MS. Moreover, the differential spots obtained with the 2D-DIGE technique were evaluated according to the presence of copper, since this element showed differences in concentrations in soybean proteins, as recently published in the literature by our research group / Doutorado / Quimica Analitica / Doutor em Ciências

Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures. Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy. Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family. Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae. The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site. The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies. My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

>Magister Scientiae - MSc / The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. / The National Research Foundation and the Norwegian Research Council