The author's inaugural dissertation, Basel. / "Literatur": p. [7]-8.

Thesis (Ph. D.)--Drexel University, 2005. / Includes abstract and vita. Includes bibliographical references (leaves 102-114).

The author's inaugural dissertation, Basel. / "Literatur": p. [7]-8.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains v, 56 p. : ill. Includes abstract. Includes bibliographical references (p. 30-35).

Thesis (Ph.D.)--Cornell University, August, 2008. / Includes bibliographical references (leaves 117-129).

Thesis (Ph.D.)--University of California, Berkeley, Spring 2005. / Includes bibliographical references (p. 450-468).

Despite progress through expanded use of bed nets and anti-malarial drugs, Plasmodium falciparum (Pf) malaria caused about 200 million cases and 500,000 deaths in 2015. An ideal vaccine would reduce the burden of disease and interrupt transmission. Despite decades of effort, there is no vaccine that can adequately address the global burden of malaria. This thesis focuses on two potential weaknesses in the parasite life-cycle. First, I investigate two vaccination strategies aimed at improving the antibody response to RH5, an essential and conserved protein for erythrocyte invasion. Due to instability of the resultant recombinant vaccine constructs, these efforts have required re-engineering of the vaccine platform, which remains an ongoing effort. Second, the immunogenicity and mechanism of protection of a live-attenuated whole sporozoite vaccine, PfSPZ Vaccine, was assessed. In a study that examined PfSPZ Vaccine at intravenous (IV) doses between 1.35 &tiles; 10⁵ to 4.5 &tiles; 10⁵ PfSPZ, I demonstrate that PfSPZ antibody responses correlated with durable sterile protection against controlled human malaria infection (CHMI). Surprisingly, the pre-vaccine frequency of Vγ9⁺Vδ2⁺ T cells, an innate T cell that recognizes conserved Plasmodium phosphoantigens, also correlated with durable sterile protection. Regarding the mechanism of protection, PfSPZ-specific antibodies as well as CD8 and CD4 T cells in the blood decreased substantially over time, yet sterile protection was maintained. In non-human primates, the CD8 T cell response in the liver at a memory time point was measured to be about 100-fold higher than found in the blood. Collectively, these data suggest that PfSPZ Vaccine confers durable protection in humans by long-lived, tissue-resident CD8 T cells. These findings were extended with a study using 9.0 x 10⁵ PfSPZ, wherein I demonstrate that T cell responses peaked immediately after the first vaccination with minimal T cell activation despite additional immunizations. This suggests that anti-PfSPZ immunity may be limiting the effectiveness of subsequent immunizations. Finally, I examined the T cell response to PfSPZ attenuated by chloroquine (termed PfSPZ-CVac). T cell responses were substantially higher than achieved with comparable PfSPZ Vaccine doses. Additionally, a significantly higher proportion of PfSPZ-specific CD4 T cells were polyfunctional, simultaneously expressing IFN-γ, IL-2, and TNF-α, in subjects that were protected from CHMI. In sum, these studies provide insight into the immunobiology of a protective immune response that may guide future malaria vaccine development efforts.

nÃo hà / A periodontite agressiva representa um tipo de doenÃa periodontal inflamatÃria que, embora rara, geralmente acomete indivÃduos em idade precoce e à caracterizada pela destruiÃÃo rÃpida e debilitante do periodonto de suporte. A patogÃnese desta doenÃa vincula-se a um fator etiolÃgico primÃrio, o biofilme dental e tambÃm à resposta inflamatÃria do hospedeiro susceptÃvel à agressÃo bacteriana. O objetivo deste estudo foi avaliar o efeito do tratamento a curto prazo com etoricoxib como adjuvante à terapia de raspagem e alisamento radicular (RAR) sobre os nÃveis de PGE2 e sobre os parÃmetros clÃnicos e radiogrÃficos em pacientes portadores de periodontite agressiva. Os pacientes foram randomicamente alocados para TESTE e CONTROLE (n=10 em cada grupo) e submetidos ao tratamento com etoricoxib (120 mg/dia) ou placebo durante 7 dias. Profundidade de sondagem (PS), nÃvel de inserÃÃo clÃnica (NIC), recessÃo gengival (RG), Ãndice de placa visÃvel (IP), sangramento à sondagem (SS), mensuraÃÃo da distÃncia linear (DL) e anÃlise dos nÃveis de cinza (NC) foram registrados antes e um mÃs apÃs a instituiÃÃo das terapias. A anÃlise visual por vÃdeo e por negatoscÃpio bem como a anÃlise por subtraÃÃo radiogrÃfica digital foram feitas no inÃcio e ao final do perÃodo experimental. A dosagem de PGE2 no fluido crevicular gengival (FCG) foi avaliada por radioimunoensaio no inÃcio, com 7 dias apÃs o inÃcio dos tratamentos e 30 dias apÃs a finalizaÃÃo dos mesmos. Ao final do perÃodo experimental, nÃo foram observadas diferenÃas estatisticamente significantes entre os grupos em relaÃÃo aos parÃmetros clÃnicos, embora ambos os grupos tenham apresentado melhora significativa em todas as variÃveis avaliadas. Houve um decrÃscimo no NIC de 5,54Â0,47 mm para 3,59Â0,53 mm no grupo TESTE e de 5,92Â1,10 para 3,69Â0,80 mm no grupo CONTROLE. Uma significativa reduÃÃo nos nÃveis de PGE2 foi observada apÃs 7 dias de tratamento. A mensuraÃÃo da DL evidenciou diferenÃa entre os grupos. Em conclusÃo, etoricoxib nÃo foi capaz de promover benefÃcio adicional nos parÃmetros clÃnicos, contudo, promoveu reduÃÃo inicial nos nÃveis de PGE2 e discreta melhora na condiÃÃo Ãssea. / Aggressive periodontitis is an inflammatory type of periodontal disease which, although rare, generally affects individuals at an early age and is characterized by the rapid and debilitating destruction of the support periodontium. The pathogenesis of this pathology is related to primary etiologic factor, dental biofilm, associated to the immunoinflammatory response of susceptible host to this aggression. The purpose of this study was to assess the effect of short duration treatment with etoricoxib as adjuvant therapy to scaling and root planing (SRP) on prostaglandin E2 (PGE2) levels and the clinical and radiographic parameters in aggressive periodontitis. Patients were randomly allocated to TEST or CONTROL (N=10 in each group) and submitted to SRP and treatment with 120 mg/day of etoricoxib or placebo during 7 days. Probing depth (PD), clinical attachment level (CAL), gingival recession (GR), visible plaque index (VPI), bleeding on probing (BOP), measurement of the linear distance (LD) and analysis of the gray levels (GL) were recorded before and one month after the therapies. The visual analysis from video and negatoscope and digital subtraction radiographic was made in beginning and in the final experimental period (30 days). The PGE2 dosage in the gingival crevicular fluid (GCF) was measured by radioimmunoassay at the beginning, and 7 and 30 days afterwards. No significant difference was observed between the groups in the clinical parameters at the end of the experimental period, although both groups presented significant improvement in all the variables examined. There was a decrease in CAL from 5.54Â0.47 mm to 3.59Â0.53 mm in the TEST group and from 5.92Â1.10 to 3.69Â0.80 mm in the CONTROL group. A significant reduction in PGE2 was found after 7 days of treatment. The LD measurement was shown to differ between the groups. In conclusion, etoricoxib did not promote additional improvement in the clinical parameters, however it produced an initial reduction in the PGE2 levels in the GCF, which could be related to the discrete improvement in the bone condition.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / The Systemic Inflammatory Response Syndrome (SIRS) is characterized by an excessive release of inflammatory mediators as a systemic inflammatory response to a serious clinical injuries. The use of glutamine in nutraceutical doses has been studied as a strategy in tissue protection and preservative of tissue metabolic function in stressful situations, helping to improve the immune response of patients. The effects of enteral glutamine supplementation in nutraceutical doses on the inflammatory markers, of glycolytic metabolism, of immune system and of oxidative stress were studied in adult and elderly patients with SIRS in a prospective, clinical, randomized, controlled, double-blind crossover study. Thirty six moderately severe patients admitted to the Intensive Care Unit were selected according to pre-defined criteria, diagnosis of SIRS and the APACHE II score (>10<20), distributed into two groups and submitted to the supplementation with 1 litre of enteral nutrition with addition of 30g of L-glutamine or calcium caseinate or 1 litre of enteral nutrition with addition of 30g of calcium caseinate or L-glutamine for two days, pause for one day only with diet, followed by four days of supplementation. Blood samples were collected before (T0) and after (T1) each supplementation. For evaluation blood parameters (hematocrit, leukocytes, lymphocytes, monocytes, prealbumin, blood urea nitrogen, creatinine, glucose, lactate, C-peptide and insulin), IL-1, IL-6, IL-10 and TNF&#945; were also assayed. Glutathione, TBARS, and glutamine and glutamate amino acids were measured. Six patients died during the study. Thirty patients finished the study, 16 men (53%) and 14 (47%) women, median age 74.4 years (30-92 years) in moderately severe state of health (APACHE II 13.1 - range 10-19). All patients developed SIRS and were given enteral nutrition supplemented with L-glutamine or calcium caseinate, 1464kcal/day (range 792-1914kcal/day). The use of L-glutamine in nutraceutical dose of 30g/day showed no changes in blood parameters. All laboratory parameters remained within normal values except the blood urea [Calcium Caseinate T1=47.0mg/dL (range 34.0-69.0 mg/dL) versus Glutamine T1=50.0mg/dL (36.75-75.0mg/dL); p=0.030]. Creatinine concentrations were not statistically different. There was no statistically significant difference in assessment of inflammatory parameters (IL-1, IL-6, IL-10 E TNF&#945;). Leukocytes count decreased significantly in both groups [Calcium Caseinate T0=13.650 1/mm3 (10.148-18.250 1/mm3) versus T1=11.500 1/mm3 (8.050-29.100 1/mm3); p=0,019] and [Glutamine T0=12.850 1/mm3 (11.155-15.550 1/mm3) versus T1=11.000 1/mm3 (9.200-16.325 1/mm3); p=0.046]. There was increase statistically significant difference in lymphocytes count between groups [Calcium Caseinate T1=1085 1/mm3 (range 805-1363 1/mm3) versus Glutamine T1=1916 1/mm3 (1301-2517 l/mm3); p<0.0001] and Calcium Caseinate group decreases [T0=1288 1/mm3 (range 834-2209 1/mm3) versus T1=1085 1/mm3 (range 805-1363 1/mm3); p=0.0324] and Glutamine group increases [T0=954 1/mm3 (range 785-1442 1/mm3) versus T1=1916 1/mm3 (range 1301-2517 l/mm3); p<0.0001]. Blood concentration of TBARS decreased significantly in both groups [Calcium Caseinate T0=20.56&#61549;mol MDA/ml (range 13.64-20.56&#61549;mol MDA/ml); p=0.001] and [Glutamine T0=17.67 &#61549;mol MDA/ml (range 8.11-34.98 &#61549;mol MDA/ml) versus T1=16.52 &#61549;mol MDA/ml (range 5.41-21.86 &#61549;mol MDA/ml); p=0.020]. The blood concentrations of Gluthatione showed a statistically significant reduction in caseinate group (T0=486.0&#61549;mol/ml (range 486.0Â165.8&#61549;mol/ml versus T1=451.0Â167.4&#61549;mol/ml; p=0.047) and no statistically significant difference in the glutamine group, nor between groups. However, there were no differences between groups. Glutamine and glutamate were not statistically different. Enteral nutrition supplemented with glutamine in nutraceutical doses of 30g/day increase lymphocyte count, helps to reduce lipid peroxidation and maintains the antioxidant glutathione capacity, interfering beneficially modulating the inflammatory response and stress, but present no effect upon cytokines concentrations or glycolytic parameters. / A SÃndrome da Resposta InflamatÃria SistÃmica (SRIS) caracteriza-se por uma liberaÃÃo excessiva de mediadores inflamatÃrios a uma sÃrie de situaÃÃes clÃnicas graves. A utilizaÃÃo da glutamina em doses nutracÃuticas tem sido estudada como uma estratÃgia de proteÃÃo tecidual e metabÃlica em situaÃÃes de estresse, melhorando a resposta imune de pacientes. Os efeitos da nutriÃÃo enteral suplementada com 30g/dia de glutamina sobre os marcadores inflamatÃrios, do metabolismo glicolÃtico, da funÃÃo imune e do estresse oxidativo foram estudados em pacientes adultos e idosos com SRIS. Foi realizado estudo clÃnico prospectivo, randomizado, controlado, duplo-cego, cruzado. Trinta e seis pacientes internados em Unidade de Terapia Intensiva foram selecionados pelos critÃrios do estudo, diagnÃstico da SRIS e score APACHE II (>10<20), distribuÃdos em dois grupos e submetidos à suplementaÃÃo com 1 litro de dieta enteral suplementada com 30g de L-glutamina ou caseinato de cÃlcio ou 1 litro de dieta enteral suplementada com 30g de caseinato de cÃlcio ou L-glutamina por dois dias, intervalo de um dia somente com dieta, perfazendo quatro dias de dieta com suplementaÃÃo. Amostras de sangue foram coletadas antes (T0) e apÃs (T1) cada suplementaÃÃo. Foram realizadas anÃlises do hematÃcrito, leucÃcitos, linfÃcitos, monÃcitos, prÃ-albumina, urÃia, creatinina, glicose, lactato, peptÃdeo-C e insulina, das IL-1, IL-6, IL-10, TNF&#945;, glutationa, TBARS e dos aminoÃcidos glutamina e glutamato. Seis pacientes foram a Ãbito durante o estudo e trinta pacientes concluÃram o estudo, sendo 16(53%) homens e 14(47%) mulheres, mediana de idade 74,4 anos (30-92 anos), moderadamente graves, mediana de APACHE II 13,1 (10-19) e mediana de ingestÃo calÃrica de 1464kcal/dia (792-1914kcal/dia). O uso L-glutamina em dose nutracÃutica de 30g/dia nÃo mostrou alteraÃÃes nos parÃmetros hematolÃgicos. Houve aumento da urÃia [Caseinato T1=47,000mg/dL (34,000-69,000mg/dL) versus Glutamina T1=50,000mg/dL (36,750-75,000mg/dL); p=0,030] na comparaÃÃo intergrupos, mas nÃo houve diferenÃa estatisticamente significante de creatinina em nenhum dos grupos. NÃo houve alteraÃÃo estatisticamente significante nos parÃmetros inflamatÃrios (IL-1, IL-6, IL-10 e TNF&#945;). A contagem de leucÃcitos diminuiu significantemente em ambos os grupos [Caseinato T0=13.650 1/mm3 (10.148-18.250 1/mm3) versus T1=11.500 1/mm3 (8.050-29.100 1/mm3); p=0,019] e [Glutamina T0=12.850 1/mm3 (11.155-15.550 1/mm3) versus T1=11.000 1/mm3 (9.200-16.325 1/mm3); p=0,046]. Houve aumento estatisticamente significante na contagem de linfÃcitos na comparaÃÃo intergrupos [Caseinato T1=1.085 1/mm3 (805-1.363 1/mm3) versus Glutamina T1=1.916 1/mm3 (1.301-2.517 l/mm3); p<0,0001], uma diminuiÃÃo estatisticamente significante no grupo Caseinato [T0=1.288 1/mm3 (834-2.209 1/mm3) versus T1=1.085 1/mm3 (805-1.363 1/mm3); p=0,0324] e aumento no grupo Glutamina [T0=954 1/mm3 (785-1.442 1/mm3) versus T1=1.916 1/mm3 (1.301-2.517 l/mm3); p<0,0001]. Observou-se reduÃÃo estatisticamente significante na dosagem do TBARS na comparaÃÃo intragrupos [Caseinato T0=20,56&#61549;mol MDA/ml (13,64-20,56&#61549;mol MDA/ml) versus T1=15,08 &#61549;mol MDA/ml (13,64-20,56 &#61549;mol MDA/ml); p=0,001] e [Glutamina T0=17,67 &#61549;mol MDA/ml (8,11-34,98 &#61549;mol MDA/ml) versus T1=16,52 &#61549;mol MDA/ml (5,41-21,86 &#61549;mol MDA/ml); p=0,020], mas nÃo houve diferenÃas intergrupos. A concentraÃÃo sanguÃnea de glutationa apresentou uma reduÃÃo estatisticamente significante no grupo Caseinato (T0=486,00&#61549;mol/mlÂ165,80&#61549;mol/ml) versus T1=451,00Â167,40&#61549;mol/ml; p=0,047) e nÃo houve diferenÃa no grupo Glutamina, tampouco entre os grupos. Glutamina e glutamato nÃo demonstraram diferenÃas estatisticamente significantes. Conclui-se que a nutriÃÃo enteral suplementada com glutamina em dose nutracÃutica de 30g/dia em pacientes moderadamente graves promove um aumento dos linfÃcitos, contribui para reduzir a peroxidaÃÃo lipÃdica e mantÃm a capacidade antioxidante da glutationa, interferindo de forma benÃfica na modulaÃÃo da resposta inflamatÃria e do estresse, mas nÃo apresenta nenhum efeito sobre a concentraÃÃo de citocinas ou parÃmetros glicolÃticos.

Orientador: Maria Isabel Rodrigues / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T14:23:26Z (GMT). No. of bitstreams: 1 Burkert_JanainaFernandesdeMedeiros_D.pdf: 9150119 bytes, checksum: 05d911513e3637048890ed7ef1eb3867 (MD5) Previous issue date: 2003 / Resumo: O interesse na produção de lipases microbianas tem aumentado significativamente nas últimas décadas, devido ao seu amplo potencial de aplicações industriais, seja na indústria de alimentos como aditivos (modificação de aromas), química fina (síntese de ésteres), detergentes (hidrólise de gorduras), tratamento de efluentes (decomposição e remoção de substâncias oleosas), couro (remoção de lipídios das peles dos animais), farmacêutica e na área médica (remédios, digestivos e enzimas para diagnósticos). A produção de lipase pode ser influenciada por diferentes variáveis, como o microrganismo produtor da enzima, as fontes de carbono, nitrogênio e lipídio, as condições de aeração e agitação, o tipo do impulsor, e até mesmo a geometria do reator. Na primeira etapa deste trabalho foram realizados testes com diferentes indutores (óleo de soja, óleo de milho, óleo de girassol, óleo de canola e óleo de oliva) para produção de lipase utilizando o microrganismo Geotrichum candidum NRRL- Y552, obtendo como melhor indutor o óleo de soja. Em seguida, um estudo para padronização do inóculo foi realizado possibilitando o início da otimização do meio de cultura, em frascos agitados, obtendo como meio otimizado 3,58% de peptona e 0,64% de óleo de soja, com pH inicial de 7,0 a 30°C e 250 rpm, alcançando 16 U/mL em 48 horas de fermentação. Utilizando as condições de produção otimizadas, a enzima foi caracterizada quanto a pH ótimo, temperatura ótima e de estabilidade, a influência de sais minerais na atividade enzimática e a determinação dos parâmetros cinéticos KM e Vmáx. Seqüencialmente, com o meio de cultura otimizado, foi verificada a influência dos impulsores turbina de Rushton, hélice naval e pás inclinadas na produção da lipase em fermentadores de bancada, atingindo em tomo de 25 g/L de biomassa para todos os impulsores e 19 U/mL em 30 horas, 12 U/roL em 30 horas e 22 U/mL em 54 horas, respectivamente, de atividade lipolítica. Utilizando o agitador tipo pás inclinadas foi investigado o efeito clã agitação e aeração no processo fermentativo, sendo obtida as condições de 300 rpm e 1 vvm a 30°C como ótimas para produção da lipase, alcançando aproximadamente 22 U/mL em 54 horas de processo. Ainda foi investigado o efeito de diferentes taxas de aeração em um reator não convencional "air lift" que permitiu obter cerca de 20 U/mL de atividade lipolítica em 30 horas de fermentação, possibilitando nesta geometria de reator uma produtividade 40% maior em relação aos reatores convencionais. Estes resultados para o processo de produção da lipase foram superiores em relação aos relatados na literatura para o mesmo microrganismo / Abstract: The interest in microbial lipase production increased significantly in the last decades, because of the large potential in industrials applications such as: additives in foods (flavor modification), fine chemicals (synthesis of esters), detergents (hydrolysis of fats), waste water treatment (decomposition and removal of oily substances), leather (removal of lipids of animal skins), pharmaceutical and medical areas (remedies, digestives and enzymes for diagnostics). The lipase production can be influenced by different variables such as the microorganism, the carbon sources, nitrogen and lipid, the aeration and agitation conditions, the impeller type, and also including the geometry of the reactor. In the first stage of this work tests was carried out with different inductors (soy oil, com oil, sunflower oil, canola oil and olive oil) for lipase production by Geotrichum candidum NRRL- Y552, getting as the best inductor the soy oil. After that, a study for standardization of inoculum was carried out, making possible the beginning of the optimization of the culture medium, in shaker-flasks, getting as half optimized 3.58% of peptona and 0.64% of soy oil, with initial pH of 7,0, 30°C and 250 rpm that conditions allow reaching 16 U/mL in 48 hours of fermentation. Using the optimized conditions of production, the enzyme was characterized concerning to optimum pH, optimum temperature and stability temperature, the influence of salts in the enzymatic activity and the determination the kinetic parameters KM and Vmáx, Sequentially, with the optimized medium culture, the influence of the impellers it was verified for Rushton turbine, helix naval and pitched blade up in the production of lipase in fermenter, reaching around 25 g/L of biomass for all impellers and 19 U/mL in 30 hours, 12 U/mlL in 30 hours and 22 U/mlL in 54 hours, respectively. Using the impeller type pitched blade up the effect of the agitation and aeration in the of lipase production was investigated, being the optimum conditions 300 rpm and 1 vvm at 30°C for enzyme production, reaching approximately 21 U/mL in 54 hours of fermentation. The effect of different aeration rates in a not conventional reactor air lift was also investigated, resulting in about 20 U/mL of lipolytic activity ín 30 hours of fermentation, making possible to obtain with this geometry of reactor a larger productivity (40% greater ín comparison to the conventional reactors). These results for the process of lipase production are larger than the reported ones in the literature for the same microorganism / Doutorado / Doutor em Engenharia de Alimentos