Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-05T14:55:06Z No. of bitstreams: 1 arquivototal.pdf: 6265632 bytes, checksum: b400bacf85cbecabd2bc0a3fd0a7f19c (MD5) / Made available in DSpace on 2017-09-05T14:55:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 6265632 bytes, checksum: b400bacf85cbecabd2bc0a3fd0a7f19c (MD5) Previous issue date: 2017-04-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Snakebite or snakebite is the chart of poisoning from the inoculation of toxins through the snake inoculation apparatus. The objective of this study was to evaluate and in vitro the cytotoxic, oxidative and homeostasis effects (on erythrocytes, hemoglobins and human plasma, respectively) caused by the presence of the crude venoms of Bothrops jararacussu and Bothrops moojeni snakes. The protein profile of each venom was characterized by reverse phase chromatography and SDS-PAGE. The venom compositions of both species showed similar protein profiles. The amounts of the crude venoms from B. jararacussu and B. moojeni induced significant indirect hemolysis in the presence of fatty acid and fatty acid plus calcium chloride. The crude venom of B. jararacussu presented a higher cytotoxic potential. The venoms of B. jararacussu and B. moojeni, showed no anti-hemolytic effect. The venom of B. jararacussu presented a haemagglutinating effect in the analyzed amounts of 100 and 1000μg and did not present an oxidizing effect. The B. moojeni venom presented an oxidizing effect only in the amount of 1000μg. Venoms of both species showed no anti-oxidant effect. The crude venom of B. moojeni had a higher coagulant potential compared to B. jararacussu venom and all plasma clots formed were turbid, rigid and stable. The clot formation time was inversely proportional to the amount of venom evaluated. Therefore, the information obtained from the comparisons of the biological effects caused by the venoms, showed that B. jararacussu showed indirect hemolytic activity, hemagglutinating and coagulant, and B. moojeni presented indirect hemolytic activity, oxidant and coagulant. / Acidente ofídico ou ofidismo é o quadro de envenenamento decorrente da inoculação de toxinas através do aparelho inoculador de serpentes. O objetivo desse estudo foi avaliar e comparar, in vitro, os efeitos citotóxico, oxidativo e na homeostase (sobre eritrócitos, hemoglobinas e plasma humano, respectivamente), causados pela presença das peçonhas brutas das serpentes Bothrops jararacussu e Bothrops moojeni. O perfil proteico de cada peçonha foi caraterizado por cromatografia de fase reversa e SDS-PAGE. As composições das peçonhas de ambas as espécies mostraram perfis proteicos similares. As quantidades das peçonhas brutas de B. jararacussu e B. moojeni induziram hemólise indireta significativa na presença de ácido graxo e de ácido graxo mais cloreto de cálcio. A peçonha bruta de B. jararacussu apresentou maior potencial citotóxico. As peçonhas de B. jararacussu e B. moojeni, não exibiram efeito anti-hemolítico. A peçonha de B. jararacussu apresentou efeito hemaglutinante nas quantidades analisadas de 100 e 1000μg e não apresentou efeito oxidante. A peçonha de B. moojeni apresentou efeito oxidante somente na quantidade de 1000μg. As peçonhas de ambas as espécies não mostraram efeito anti-oxidante. A peçonha de B. moojeni apresentou maior potencial coagulante em relação a peçonha de B. jararacussu e todos coágulos plasmáticos formados eram turvos, rígidos e estáveis. O tempo para formação dos coágulos foi inversamente proporcional à quantidade de peçonha avaliada. Portanto, as informações obtidas das comparações dos efeitos biológicos provocados pelas peçonhas, mostraram que B. jararacussu apresentou atividade hemolítica indireta, hemaglutinante e coagulante, e B. moojeni apresentou atividade hemolítica indireta, oxidante e coagulante.

Orientador: Helia Harumi Sato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T08:10:00Z (GMT). No. of bitstreams: 1 Rodrigues_GiselledeArruda_M.pdf: 925514 bytes, checksum: 300372a4285d7efd417f8a717a850023 (MD5) Previous issue date: 2008 / Resumo: A enzima ß-1,3-glucanase da bactéria Cellulosimicrobium cellulans 191 é capaz de lisar a parede celular de diversas leveduras dentre elas as linhagens de Kluyveromyces sp., produtoras da enzima intracelular ß-galactosidase. Este trabalho visou a produção de ß-1,3-glucanase lítica de Cellulosimicrobium cellulans 191 para obtenção das preparações enzimáticas bruta concentrada e purificada com alta atividade lítica para aplicação na obtenção de protoplastos de leveduras e lise das células de Kluyveromyces marxianus var. bulgaricus para extração de b-galactosidase. A ß-1,3-glucanase extracelular foi produzida pela bactéria Cellulosimicrobium cellulans 191 em meio otimizado composto de 10g/L de parede celular de levedura, 2,0g/L de (NH4)2SO4; 0,2g/L de MgSO4.7H2O em tampão fosfato 0,2M, pH 7,5 como descrito por SCOTT e SCHEKMAN (1980) e modificado por SOARES (2002). Foi obtida maior produção da ß-1,3-glucanase na fermentação do microrganismo em frascos aletados a 30ºC, 200rpm por 24 horas do que em fermentador contendo o mesmo meio a 30ºC, 1,5vvm por 24 horas, obtendo-se 0,724 e 0,351U/mL, respectivamente. Os sobrenadantes dos meios fermentados em frascos agitados e em fermentador de 5L apresentaram atividade de 0,120 e 0,064U/mL de protease, respectivamente. Foi estudada a influência dos compostos sacarose (10mM), lactose (10mM), maltose (10mM), glicerol (10mM), gelatina (125mg/mL), albumina de ovo (125mg/mL), sulfato de amônio (10mM), EDTA (10mM), b-mercaptoetanol (10mM) e cisteína (10mM) na preservação da atividade de ß-1,3-glucanase da preparação enzimática bruta a 45ºC, 50ºC e 55ºC, no entanto, nenhum dos compostos testados aumentou a estabilidade da enzima. Foi testada a concentração da preparação enzimática bruta de ß-1,3-glucanase por liofilização, ultrafiltração, fracionamento com sulfato de amônio, pela remoção de água com carboximetilcelulose (CMC) e precipitação com os solventes orgânicos acetona e etanol. A concentração da preparação enzimática bruta de ß-1,3-glucanase utilizando-se sacos de diálise e CMC em pó para a adsorção da água mostrou-se como o método mais eficiente, sendo que a enzima foi concentrada 6,38 vezes com manutenção de 96% da atividade inicial. A preparação enzimática bruta de ß-1,3-glucanase foi purificada aproximadamente 2 vezes por fracionamento com sulfato de amônio 30% de saturação. A ß-1,3-glucanase da preparação enzimática bruta foi purificada aproximadamente 6,66 vezes através de fracionamento com sulfato de amônio a 30% de saturação, dessalinização em coluna de exclusão Sephadex PD10 e cromatografia em coluna de troca iônica DEAE-Sephacelè, com rendimento de 18%. Dentre as linhagens das leveduras Kluyveromyces marxianus var. bulgaricus, Kluyveromyces marxianus var. lactis, Kluyveromyces drosophilarium, Kluyveromyces marxianus NRRL7571, Kluyveromyces marxianus NRRL1196, a primeira foi selecionada como maior produtora da enzima b-galactosidase. As preparações enzimáticas bruta concentrada e purificada de ß-1,3-glucanase apresentaram capacidade de lisar as células de Kluyveromyces marxianus var. bulgaricus a 37ºC. Para a formação de protoplastos foi utilizado 0,1U de ß-1,3-glucanase por mL de suspensão de levedura equivalente a 1,3mg de massa celular seca, durante 1 hora a 37ºC. A extração da ß-galactosidase de Kluyveromyces marxianus var. bulgaricus foi testada pelos métodos químico (solventes orgânicos), enzimático (ß-1,3-glucanase) e físico (ultrasonicação). O método de pré-tratamento enzimático e posterior aplicação de ultrasonicação apresentou melhores resultados seguido pelos métodos de permeabilização com etanol 50%, b-1,3-glucanase purificada, ultrasonicação, permeabilização com tolueno 20% e ß-1,3-glucanase bruta, obtendo-se, respectivamente, atividades de ß-galactosidade (massa celular seca) iguais a 25,13U/mg/min; 14,00U/mg/min; 13,11U/mg/min; 10,95U/mg/min; 10,75U/mg/min e 7,25U/mg/min utilizando-se lactose como substrato. A b-galactosidase bruta apresentou atividade ótima em pH 7,0 e 32ºC e mostrou-se estável na faixa de 30 a 35ºC após 3 horas em pH 7,0 e na faixa de pH entre 6,5 e 7,5 após 3 horas a 35ºC. A b-galactosidase de Kluyveromyces marxianus var. bulgaricus foi purificada 2,3 vezes após fracionamento com sulfato de amônio a 60% de saturação, diálise e cromatografia em coluna de troca iônica DEAE-Sephacelè, com rendimento de 31%. As preparações enzimáticas bruta e purificada da ß-glucanase lítica da bactéria Cellulosimicrobium cellulans podem ser utilizadas na obtenção de protoplastos de leveduras e extração de enzimas intracelulares de leveduras / Abstract: The enzyme ß-1,3-glucanase from the bacteria Cellulosimicrobium cellulans 191 is capable of lysing the cell wall of various yeasts including strains of Kluyveromyces sp., producers of the intracellular enzyme ß-galactosidase. This work aimed to produce lytic ß-1,3-glucanase from Cellulosimicrobium cellulans 191 in order to obtain crude, concentrated and purified enzyme preparations with high lytic activity, for application in the obtaining of yeast protoplasts and in the lysis of Kluyveromyces marxianus var. bulgaricus to extract ß-galactosidase. Extracellular ß-1,3-glucanase was produced by the bacteria Cellulosimicrobium cellulans 191 in an optimised culture medium composed of 10g/L yeast cell wall, 2.0g/L (NH4)2SO4 and 0.2g/L MgSO4.7H2O in 0.2M phosphate buffer, pH 7.5. The production of ß-1,3-glucanase from the microorganism was greater in flasks with flaps incubated at 30ºC and 200rpm for 24 hours than in a fermenter containing the same medium and incubated at 30ºC and 1.5vvm for 24 hours, obtaining yields of 0.724U/mL and 0.351U/mL, respectively. The supernatants from the media fermented in the shaken flasks and in the 5L fermenter presented protease activities of 0.120U/mL and 0.064U/mL, respectively. The influences of sucrose (10mM), lactose (10mM), maltose (10mM), glycerol (10mM), gelatine (125mg/mL), egg albumin (125mg/mL), ammonium sulphate (10mM), EDTA (10mM), b-mercaptoethanol (10mM) and cysteine (10mM) in the preservation of the ß-1,3-glucanase activity of the crude enzyme preparation at 45ºC, 50ºC and 55ºC, were studied, but none of the compounds tested increased the stability of the enzyme. Concentration of the crude ß-1,3-glucanase preparation was tested using freeze drying, ultrafiltration, ammonium sulphate fractionation, by the removal of water using carboxymethylcellulose (CMC) and by precipitation with the organic solvents acetone and ethanol. Concentration of the crude ß-1,3-glucanase preparation using dialysis bags and powdered CMC to adsorb the water was shown to be the most efficient method, the enzyme being concentrated 6.38 times with 96% maintenance of the initial activity. The crude ß- 1,3- glucanase preparation was purified about two-fold by ammonium sulphate fractionation at 30% saturation. The ß-1,3-glucanase of the crude enzyme preparation was purified about 6.66 times using ammonium sulphate fractionation at 30% saturation, desalting in a Sephadex PD10 exclusion column and ion exchange column chromatography using DEAE-Sephacelè, with an 18% yield. Of the following yeast strains, Kluyveromyces marxianus var. bulgaricus, Kluyveromyces marxianus var. lactis, Kluyveromyces drosophilarium, Kluyveromycesmarxianus NRRL7571 and Kluyveromyces marxianus NRRL1196, the first one was selected as the greatest producer of the enzyme ß-galactosidase. The crude, concentrated and purified ß-1,3-glucanase preparations were capable of lysing the cells of Kluyveromyces marxianus var. bulgaricus at 35ºC. In order to form protoplasts a ß-1,3-glucanase preparation with 0.1U activity was used per mL yeast suspension equivalent to 1.3mg dry cell mass, for 1 hour at 35ºC. Physical (ultrasound), chemical (organic solvents) and enzymatic (ß-1,3-glucanase) methods were tested for their efficiency in extracting ß-galactosidase from Kluyveromyces marxianus var. bulgaricus. The method using an enzymatic pre-treatment followed by the application of ultrasound gave the best results, followed by methods of permeabilization by ethanol 50%, purified ß-1,3-glucanase, ultrasonication, toluene 20% and crude ß-1,3- glucanase, obtaining ß-galactosidase activities (dry cell mass) of, respectively, 25.13U/mg/min, 14.00U/mg/min, 13.11U/mg/min, 10.95U/mg/min, 10.75U/mg/min e 7.25U/mg/min using lactose as the substrate. The crude ß-galactosidase preparation showed optimum activity at pH 7.0 and 32ºC and was stable in the range from 30-35ºC after 3 hours at pH 7.0 and between pH 6.5 and 7.5 after 3 hours at 35ºC. The ß-galactosidase from Kluyveromyces marxianus var. bulgaricus was purified 2.3 fold after ammonium sulphate fractionation at 60% saturation, dialysis and ion exchange column chromatography on DEAE-Sephacelè, with a 31% yield. The crude and purified lytic ß-glucanase preparations of Cellulosimicrobium cellulans bacteria can be used to obtaining yeast protoplasts and extraction of intracellular enzymes of yeasts / Mestrado / Mestre em Ciência de Alimentos

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Motivation In the black population of South Africa, cardiovascular disease (CVD) is rapidly increasing due to urbanisation. Stroke is usually accompanied by a prothrombotic haemostatic profile. Changing lifestyle factors that accompany the urbanisation process could have a negative impact on the haemostatic profile of black South Africans. Elevated levels of pro-coagulant factors, von Willebrand factor (vWF), fibrinogen and fibrin D-dimer have been reported in the black population, which could increase the black population’s susceptibility to CVD. However, low levels of plasminogen activator inhibitor-1 (PAI-1) previously reported in the black population could contribute towards a pro-fibrinolytic state, which may counteract the hypercoagulant state. This may have a beneficial effect on the haemostatic profile of the black population. More investigation into the haemostatic profile of black South Africans is therefore needed to determine if an altered haemostatic profile exists in this group, and if so, to what extent these alterations may relate to cardiovascular dysfunction. This study included markers of both the coagulation (vWF, fibrinogen, fibrin D-dimer) and fibrinolytic (PAI-1, fibrin D-dimer and fibrinolytic potential) systems in an attempt to investigate the haemostatic profile of the black population of South Africa, and for comparison purposes that of the white population as well. The relationship of these markers’ with selected markers of cardiovascular function was also examined to determine if they could possibly contribute to an increase in cardiovascular risk, especially in the black population. Aims The aims of this study were to first compare coagulation and fibrinolysis markers in the black and white populations of South Africa. Furthermore, to determine if associations exist between the selected components of the haemostatic system and markers of cardiovascular function, especially in the black population of South Africa, who tends to be at a higher cardiovascular risk due to altered metabolic and haemostatic profiles. Methodology The Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study was a prospective cohort study that consisted of 409 participants at baseline (2008-2009) that were equally distributed according to both ethnicity (200 black; 209 white) and gender (black, 101 men, 99 women; white, 101 men, 108 women). At follow-up (2011/2012) the cohort totalled 359 participants (170 black, 88 men and 82 women; 189 white, 93 men and 96 women). Data from baseline measurements were used for the first two manuscripts (chapters 2 and 3), while followup data was used for the third manuscript (chapter 4). vWF, fibrinogen, PAI-1, fibrin D-dimer, CLT, serum peroxides, glutathione, glutathione peroxidase and reductase activity were determined, and ambulatory blood pressure and the retinal vessel calibres were measured. The groups were stratified by ethnicity as specified by statistical interaction terms. T-tests and chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between the components of the haemostatic system and cardiovascular function markers. This was followed by multiple linear regression analyses to investigate whether independent associations exist between the variables in both ethnic groups. P-values ≤0.050 were deemed significant. Results and conclusion of each manuscript The first manuscript (chapter 2) compares the haemostatic profiles of the black and white population to determine whether ambulatory blood pressure is related to components of the haemostatic system. The black participants displayed a prothrombotic profile with significantly higher vWF, fibrinogen, PAI-1, fibrin D-dimer and a longer CLT than their white counterparts. Furthermore, partial and multiple linear regression analyses showed a positive association of systolic and diastolic blood pressure with fibrin D-dimer in the black population, while a negative association existed between ambulatory blood pressure and CLT in the white population. These associations suggest that fibrin D-dimer may contribute, at least in part, to the high prevalence of hypertension in the black population. The second manuscript (chapter 3) determined associations between markers of the haemostatic and oxidant-antioxidant systems in the black and white populations. In addition to the prothrombotic profile that exists in the black population, this group also had significantly higher serum peroxides (oxidative stress) and lower glutathione peroxidase activity (antioxidant) levels. Multiple linear regression analyses indicated positive associations between fibrinogen and serum peroxides in both populations. In the white population, an additional positive association was found between serum peroxide and CLT. In the black population, vWF and CLT were negatively associated with GPx activity. The results suggest that there are ethnic-specific relationships between the haemostatic and oxidant-antioxidant systems. The third manuscript (chapter 4) investigated the relationships between the retinal vessel calibres and components of the haemostatic system in the black and white population. The investigation focussed specifically on arteriolar diameters in the lower median, since a narrow arteriolar diameter is known to be associated with elevated blood pressure. In both ethnic groups, a narrower arteriolar calibre was accompanied by narrower venular calibres. Independent positive associations were found between the central retinal vein equivalent (CRVE) and fibrinogen in the black population, as well as vWF and CLT in the white population. In addition, independent negative associations were found between the central retinal artery equivalent and CLT in the black population and with vWF in the white population. The results suggest that haemostatic alterations are linked to early vascular changes that may differ between ethnicities. General conclusion Ethnic-specific relationships between the components of the haemostatic system and measures of cardiovascular function are evident. The prothrombotic profile that is observed in the black population, together with the adverse associations of the haemostatic components with blood pressure, a compromised oxidant-antioxidant profile, and retinal vessel calibres may contribute, at least in part, to the high cardiovascular and cerebrovascular risk evident in this population group. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015

Foot-and-mouth disease virus (FMDV) is a highly infectious virus affecting cloven-hoofed animals. The most prominent of its clinical signs is the development of vesicular lesions on the feet and in or around the mouth, which are a consequence of extensive FMDV-induced epithelial cell death. Currently, there is no certain biological knowledge on why extensive epithelial cell death occurs in some FMDV-infected tissues, but not in others. Using the epithelial tissues of tongue and dorsal soft palate as examples of a tissue where lesions occur and one that does not visibly exhibit FMDV-induced cell death, this work aims to identify the potential drivers of epithelial cell death and survival. A partial differential equation (PDE) model informed by experimental data on epithelial structure, is used to test epithelium thickness and cell layer structure as potential determinants. A second PDE model investigates FMDV-interferon (IFN) dynamics and their impact on the levels of cell death and survival, while an experimental study is undertaken to provide data for model validation. The work carried out casts light on the important role of a variety of factors including FMDV replication, IFN production and release, and IFN antiviral action.

Après déshydration, les boues biologiques présentent en moyenne une siccité de 30%, ce qui pose d'importants problèmes environnementaux. Afin de comprendre les mécanismes physico-chimiques de rétention d'eau dans ces boues, nous les avons modélisées par une souche bactérienne pure présentant la particularité de produire ou non des surstructures, suivant sa température de culture. Les suspensions bactériennes ainsi obtenues ont été floculées par du polyéthylèneimine de différentes masses moléculaires. Des mesures de densité optique, mobilité électrophorétique, conductivité, pH, absorbance, granulométrie, ainsi que des observations en MET et AFM ont été effectuées. Cela nous a permis de caractériser la structure des agrégats ainsi obtenus et d'évaluer les influences relatives de la masse moléculaire du polymère ainsi que de sa concentration et de la présence/absence de surstructures sur l'agrégation de cellules bactériennes. Des mesures d'élasticité, de constante de raideur et de pression de turgescence ont été effectuées par spectroscopie de force, ce qui nous a permis de caractériser les propriétés nanomécaniques des agrégats. Dans un second temps, les suspensions floculées ont été déshydratées par centrifugation, procédé mis en oeuvre dans certaines station d'épuration. Des mesures d'élasticité et de viscosité (gradient de cisaillement infini) ont été effectuées par rhéologie. La corrélation de celles-ci avec des mesures de siccité nous a permis de conclure à un impact important des surstructures, de la masse moléculaire et de la concentration en polymère sur la déshydratation des boues biologiques / After dewatering, a biological sludge still contains an average water content of 70%, thus causing huge environmental problems. To achieve a better understanding of the mechanisms underlying physicochemical interactions inducing water retention in the sludge, the bacterial sludge was modeled by a pure strain producing or not surface appendages, depending on the growth temperature. These bacterial suspensions were flocculated by polyethyleneimine of various molecular weights. Measurements of optical density, electrophoretic mobility, conductivity, pH, absorbance, particle size, as well as AFM and TEM observations were performed. This allowed to characterize aggregates structures and to estimate the influence of molecular weight and concentration of polymer and presence/absence of surface appendages. Measurements of elasticity, spring constant and turgor pressure were carried out by force spectroscopy allowing to characterize nanomechanical properties of aggregates. In a second step, a dewatering of these flocculated suspensions was performed by centrifugation, process used in some wastewater treatment plants. Measurements of elasticity and viscosity were carried out by rheology. Correlations with dryness measurements allowed to conclude that the presence/absence of surface appendages, and also the molecular weight and concentration of the polymer have a significant impact on biological sludge dewatering

Plato’s Socrates is famous for claiming that “I know one thing: That I know nothing” (see e.g. Ap. 21d and Meno 81d). There is one subject that Socrates repeatedly claims to have expertise in, however: ta erôtika (see e.g. Symp. 198d1). Socrates also refers to this expertise as his erôtikê technê (Phdr. 257a7–8), which may be translated as “erotic expertise”. In this dissertation, I investigate Socrates’ erotic expertise: what kind of expertise is it, what is it constituted by, where is it put into practice, and how is it practiced? I argue that the purposes this expertise serve are, to a significant extent, educational in nature. After first having clarified the dissertation’s topic and aim, as well as my methodological approach, I present an initial account of erôs and Socrates’ erotic expertise. While discussing what constitutes Socrates’ erotic expertise, I account for two erotic educational methods: midwifery and matchmaking. I further argue that these methods tend to be accompanied by two psychological techniques, namely charming and shaming. I argue that these methods and techniques are systematically applied by Socrates when he puts his erotic expertise into practice. In the dissertation, three dialogues where Socrates practices his erotic expertise are scrutinized: Lysis, Charmides, and Alcibiades I. I focus on Socrates’ encounters with the eponymous youths of the dialogues, and each dialogue is devoted a chapter of its own. I show how these dialogues are erotically charged, and also how Socrates in these dialogues demonstrates his erotic expertise. I argue that Socrates’ expertise on erôs plays an essential role in his attempts to engage the three youths in the processes of self-cultivation, learning, and the very practice of philosophy. In the final chapter of the dissertation I turn to some questions that arise in light of my readings, and summarize the results of my investigation.

O uso das técnicas de análise multivariada está reservado aos grandes centros de pesquisa, µas grandes empresas e ao ambiente acad^emico. Essas técnicas s~ao muito interessantes porque utilizam simultaneamente todas as variáveis respostas na interpretação teórica do conjunto de dados, levando em conta as correlações existentes entre elas. Uma das principais barreiras para a utilização dessas técnicas é o seu desconhecimento pelos pesquisadores interessados na pesquisa quantitativa. A outra dificuldade é que a grande maioria de softwares que permitem esse tipo de análise (SAS, MINITAB, BMDP, STATISTICA, S-PLUS, SYSTAT, etc.) não são de domínio público. A disseminação do uso das técnicas multivariadas pode melhorar a qualidade das pesquisas, proporcionar uma economia relativa de tempo e de custo, e facilitar a interpretação das estruturas dos dados, diminuindo a perda de informação. Neste trabalho, foram confirmadas algumas vantagens das técnicas multivariadas sobre as univariadas na análise de dados de expe- rimentos agropecuários. As análises foram realizadas com o auxílio do software R, um software aberto, \"amigável\" e gratuito, com inúmeros recursos disponíveis. / The use of the techniques of multivariate analysis is restricted to large centers of research, the higher companies and the academic environment. These techniques are very inte- resting because of the use of all answers variables simultaneously in theoretical interpretation of the data set, considering the correlations between them. One of the main obstacle to the usage of these techniques is that researchers interested in the quantitative research do not know them. The other di±culty is that most of the software that allow this type of analysis (SAS, MINITAB, BMDP, STATISTICA, S-PLUS, SYSTAT etc.) are not in public domain. Publishing the use of Multivariate techniques can improve the quality of the research, decrease the time spend and the cost, and make easy the interpretation of the structures of the data without cause damage of the information. In this report, were con¯rmed some advantages of the multivariate techniques in a univariate analysis for data of agricultural experiments. The analysis were taken with R software, a open software, \"friendly\" and free, with many statistical resources available.

Archaea is one of the three domains of life and studies of archaeal biology are important for understanding of life in extreme environments, fundamental biogeochemical processes, the origin of life, the eukaryotic cell and their own, unique biology. This thesis presents four studies of the archaeal cell, using the extremophilic Sulfolobus and ocean living Nitrosopumilus as model systems. Cell division in crenarchaea is shown to be carried out by a previously unknown system named Cdv (cell division). The system shares homology with the eukaryotic ESCRT-III system which is used for membrane reorganization during vesicle formation, viral release and cytokinesis. Organisms of the phylum Thaumarchaeota also use the Cdv system, despite also carrying genes for the euryarchaeal and bacterial cell division system FtsZ. The thaumarchaeal cell cycle is demonstrated to be dominated by the prereplicative and replicative stage, in contrasts to the crenarchaeal cell cycle where the cell at the majority of the time resides in the postreplicative stage. The replication rate is remarkably low and closer to what is measured for eukaryotes than other archaea. The gene organization of Sulfolobus is significantly associated with the three origins of replication. The surrounding regions are dense with genes of high importance for the organisms such as highly transcribed genes, genes with known function in fundamental cellular processes and conserved archaeal genes. The overall gene density is elevated and transposons are underrepresented. The archaeal virus SIRV2 displays a lytic life style where the host cell at the final stage of infection is disrupted for release of new virus particles. The remarkable pyramid-like structure VAP (virus associated pyramids), that is formed independently of the virus particle, is used for cell lysis. The research presented in this thesis describes unique features of the archaeal cell and influences our understanding of the entire tree of life.