Introdução: Os hidroxietilamidos (HES) são considerados expansores plasmáticos efetivos em pacientes submetidos a procedimentos cirúrgicos de grande porte. Entretanto, seu uso clínico é limitado principalmente por sua interferência na hemostasia, representada por alterações da função plaquetária e na coagulação. A extensão dessas alterações está relacionada ao seu ipeso molecular ou à sua substituição molar. Este estudo clínico, foi realizado durante cirurgia de artroplastia de quadril em pacientes adultos para comparar os efeitos do HES (130/0,4) e a solução de Ringer lactato em relação ao sangramento intra-operatório, parâmetros hemodinâmicos, alterações na coagulação, necessidade de transfusões e resultados clínicos. Métodos: Quarenta e oito pacientes candidatos à cirurgia de artroplastia total de quadril sob anestesia subaracnoidea foram distribuídos aleatoriamente em dois grupos 24 pacientes foram selecionados para receber HES (30 ml/kg após anestesia) e 24 pacientes para receber solução de Ringer lactato (30ml/kg). O período de observação teve início após a indução da anestesia e terminou 5 horas após o termino do procedimento cirúrgico. Durante esse período o critério para a infusão de doses adicionais de fluido (10ml/kg de Solução de Ringer lactato para ambos os grupos) foi pressão arterial sistólica inferior a 90 mmHg e/ou um decréscimo de 20% da pressão arterial inicial, frequência cardíaca acima de 100 bpm, e/ou débito urinário menor de 0,4ml/kg/h. Vasopressor foi utilizado nos casos em que a hipotensão persistiu, após a reposição de volume. Transfusão de concentrado de hemácias foi administrada nos pacientes que se mantiveram instáveis hemodinamicamente após bolus adicionais de Ringer lactato ou vasopressor, Parâmetros hemodinâmicos foram mensurados em três períodos da cirurgia; dados bioquímicos foram coletados e testes da coagulação realizados e comparados. Os pacientes foram acompanhados durante sua internação hospitalar. Resultados: Os grupos foram uniformes em relação aos dados demográficos, tipo e duração da cirurgia, assim como a doenças pré-existentes. Não foram observadas diferenças significativas em relação aos parâmetros hemodinâmicos ou temperatura corporal durante o estudo. Os testes de coagulação, função plaquetária, análise de gases sanguíneos e dados bioquímicos mostraramse semelhantes entre os grupos. Perdas sanguíneas foram significativamente maiores no grupo HES (1296x890,p=0,04), necessitou de menos unidades de concentrado de hemácias durante o período observacional (17%versus46%, p=0,029) apresentou menores taxas de infecção (0 versus 4 ,p<0,03), comparado ao grupo Ringer lactato. Conclusões: Em cirurgia de artroplastia total de quadrill, a hemodiluição com hidroxietilamido resultou em maiores taxas de sangramento, menos transfusões sanguíneas e menos infecção pós-operatória. / Introduction: Hydroxyethyl starches (HES) are considered effective plasma expanders in patients undergoing major surgeries. However, the clinical use of HES is limited mainly by their affection of hemostasis, detectable by impaired platelet function and altered coagulation. The extent of such alteration has classically been related to the molecular weight or molar substitution of the used HES solution. This prospective, randomized study was performed during hip arthroplasty in adult patients under spinal anesthesia to compare the effects of HES 130/0.4 with lactateds Ringer solution regarding intraoperative bleeding, hemodynamic parameters, coagulation profile, transfusion requirements and clinical outcomes. Methods: Forty eight patients scheduled to hip arthroplasty after spinal anesthesia were randomized in two groups 24 patients were allocated to receive HES 130/0.4 (30 ml/Kg just after anesthesia) and 24 patients were signaled to receive lactateds Ringer solution (30 ml/Kg). The observational period started after the induction of anesthesia and finished 5 hours after the end of the surgery. During this period, the triggers for infusion of additional boluses of fluids (10 ml/Kg of lactateds Ringer for both groups) were a systolic blood pressure lower than 90 mmHg and/or a decrease of 20% from baseline, a heart rate higher than 100 bpm, and/or a urine output lower than 0.4 ml.Kg-1.h-1. Vasopressors were used if there was persistent hypotension despite of fluid reposition. Red blood cell transfusion was administered if patient remained unstable despite of additional boluses of Ringer or vasopressors, according to the preestablished triggers. Hemodynamic measurements were done in three periods of the surgery, biochemical parameters were analyzed and coagulation tests were performed and compared between groups. After surgery, patients were followed during the hospital stay. Results: The groups were well matched regarding demographic data, type of surgery, and duration of surgery, as well as preexisting diseases. No significant differences in hemodynamic or body temperature were seen during the study. Coagulation variables, platelet function, gases analysis and biochemical parameters were not different between groups. Blood losses were significantly higher in HES 130/0.4 group comparing to Ringers group (1296 x 890 ml, p= 0.046). Despite of that, HES group required less units of blood in the observational period comparing to Ringer group (17% versus 46%, p=0.029). HES group presented lower infection rate compared to Ringer group (0 versus 4 cases, p=0.03). Conclusions: During hip arthroplasty, hemodilution with hydroxyethyl starch 130/0.4 resulted in higher rates of bleeding. However, patients treated with hydroxyethyl starch required less transfusion and presented lower rate of infection.

A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / avaliar a perda sanguÃnea em partos vaginais induzidos pelo misoprostol e em cesÃreas com tentativa prÃvia de induÃÃo do parto pelo misoprostol, atravÃs da dosagem de hemoglobina prà e pÃs-parto; avaliar a perda sanguÃnea em partos vaginais espontÃneos e cesÃreas eletivas, atravÃs da dosagem de hemoglobina prà e pÃs-parto; comparar a perda sanguÃnea, avaliada pela dosagem de hemoglobina prà e pÃs-parto, entre partos induzidos e partos nÃo induzidos. Sujeitos e mÃtodos: realizou-se estudo na Maternidade-Escola Assis Chateaubriand da Universidade Federal do CearÃ, em 101 gestantes com indicaÃÃo para induÃÃo do trabalho de parto, que foram avaliadas pela dosagem de hemoglobina prà e pÃs-parto para estimativa da perda sanguÃnea no parto. As pacientes foram submetidas à ultrassonografia obstÃtrica transabdominal para avaliaÃÃo da estÃtica e peso fetais e Ãndice de lÃquido amniÃtico, e à cardiotocografia basal para avaliaÃÃo da vitalidade fetal. Procedeu-se à induÃÃo do trabalho de parto com misoprostol 25mcg, via vaginal. Os comprimidos foram administrados a cada 6 horas, em um nÃmero mÃximo de seis. O grupo controle foi composto por 30 pacientes que entraram em trabalho de parto espontaneamente e por 30 pacientes que se submeteram à cesÃrea eletivamente. A anÃlise estatÃstica foi realizada com o programa SPSS 10.0 (SPSS Co, Chicago, IL, USA). Os dados foram descritos atravÃs de mÃdias, desvios-padrÃo, medianas, mÃnimos, mÃximos, freqÃÃncias absolutas (n) e relativas (%). Os testes utilizados foram os de comparaÃÃo de mÃdias: T de Student pareado; de medianas: Mann-Whitney, Qui-quadrado ou Exato de Fisher; e o Coeficiente de correlaÃÃo de Spearman. O estudo da hemoglobina, antes e depois do parto foi avaliado atravÃs de ANOVA para medidas repetidas, onde foram verificados o efeito do tempo (prà e pÃs-parto) e o efeito do grupo (com e sem uso do misoprostol). Resultados: foram observadas diferenÃas significativas no tempo, em ambos os tipos de partos (p<0.0001), mas nÃo entre os grupos (p > 0.05). Portanto, existem diferenÃas significativas entre os nÃveis hemoglobina prà e pÃs-parto (p < 0.0001), porÃm as diferenÃas sÃo proporcionais em ambos os grupos, ou seja, a diferenÃa ocorre tanto no grupo que fez uso do misoprostol quanto no grupo que nÃo fez uso do misoprostol (a diminuiÃÃo foi a mesma em ambos os grupos), tanto na cesÃrea (p=0.6845) quanto no parto normal (p=0.2694). ConclusÃes: a induÃÃo do parto com misoprostol nÃo altera a perda sanguÃnea durante o parto, tanto nos partos vaginais induzidos, quanto nas cesÃreas com tentativa prÃvia de induÃÃo, quando comparada respectivamente com a perda sanguÃnea em partos vaginais espontÃneos e cesÃreas eletivas. / ABSTRACT Objectives: to evaluate the blood loss in induced vaginal delivery by misoprostol and caesarians section with induction attempt, through the hemoglobin blood levels pre and post delivery. To evaluate the blood loss in spontaneous vaginal deliveries and elective caesarians through the hemoglobin blood levels pre and post delivery. To compare the blood loss, evaluated by the hemoglobin blood levels pre and post delivery between induced and non induced deliveries. Subjects and methods: this study included 101 pregnant women admitted to the Assis Chateaubriand Maternity School of the Federal University of Cearà which met the criteria for induced delivery labor. Patients were submitted to transabdominal obstetric ultrassound for evaluation of the static and fetal weight and amniotic liquid index, and basal cardiotocography in order to evaluate fetal vitality. Procedures were taken for induced labor delivery with misoprostol 25mcg, by vaginal rout. The pills were administered each 6 hours in a maximum number of six. The control group was formed by 30 patients that initiated labor spontaneously and 30 patients that achieved caesarians electively. The statistical analysis was done with the program SPSS10.0 (SPSS Co, Chicago, IL, USA). The data were described through the medium, standard deviation, median, minimum, maximum, absolute (n) and relative (%) frequencies. The tests used for comparison of media: T of student; of median: Mann-Whitney, Qui-square or Exact of Fisher; and the Coefficient of Spearman correlation. The evaluation of hemoglobin levels before and after delivery was analyzed through the ANOVA test for repeated values, taking in account the effect of time (pre and post delivery) and the effect of the group (with and without the use of misoprostol). Results: there was a statistically significant difference between time in both types of delivery (p<0.0001).There were no statistical significance between the groups (p>0.05). Additionally, there was a similar pattern of decrease in hemoglobin blood levels pre and post labor in both groups evaluated, in the caesarian delivery (p=0.6845) and normal delivery as well (p=0.2694). Conclusions: labor induction with misoprostol does not modify the blood loss during induced vaginal deliveries and caesarians section with induction attempt, when compared to, respectively, the blood loss in spontaneous vaginal deliveries and in elective caesarians.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-16T17:25:01Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Rodrigo Alves Moreira - 2014.pdf: 6048324 bytes, checksum: 289d0c70e7ed1db107b03924d35a81de (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-16T17:37:28Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Rodrigo Alves Moreira - 2014.pdf: 6048324 bytes, checksum: 289d0c70e7ed1db107b03924d35a81de (MD5) / Made available in DSpace on 2015-01-16T17:37:28Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Rodrigo Alves Moreira - 2014.pdf: 6048324 bytes, checksum: 289d0c70e7ed1db107b03924d35a81de (MD5) Previous issue date: 2014-06-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although the mechanism of action at the molecular level is poorly understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently bound to membrane proteins showed that the MT causes a large increase in the molecular dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs) prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL), our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed that the MT concentration that changes the erythrocyte membrane fluidity to an extent that is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies performed with the same spin labels in the membrane of Leishmania (L.) amazonensis (promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic effects on the parasites were also evaluated to investigate the relationships between the cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed that the minimum concentration of MT required to cause a change in the parasite membrane occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus, there is a correlation between the cytotoxicity and changes in the membrane. Although these III membrane alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼ 70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture. / A leishmaniose cut ˆanea ´e uma doenc¸a tropical negligenciada que afetamilh˜oes de pessoas em todo mundo, representando um grave problema de sa´ude p´ublica. A miltefosina (MT) ´e um alquilfosfolip´ıdio aprovado inicialmente para tratamento de cˆancer metast ´atico e tamb´em foi licenciada para o tratamento da leishmaniose visceral, embora seu mecanismo de ac¸ ˜ao a n´ıvel molecular permanec¸a pouco compreendido. A espectroscopia de ressonˆancia paramagn´etica eletr ˆonica (RPE) do marcador de spin lip´ıdico an´alogo do ´acido este´arico (5-DSA) e do marcador de spin derivado do maleimido (6-MSL) ligado covalentemente `as prote´ınas de membrana demonstraram que a MT provoca um grande aumento na dinˆamica molecular das membranas de eritr ´ocito (ghosts) e membranas resistentes `a extrac¸ ˜ao por detergente (DRMs) preparadas a partir de membranas de eritr ´ocito. A t ´ecnica tamb´em demonstrou que em ves´ıculas formadas com lip´ıdios constituintes de rafts, 20 mol% de esfingomielina pode ser substitu´ıdo por 20 mol% de MT, sem qualquer alterac¸ ˜ao em termos de dinˆamica molecular. Al ´em disso, o efeito da MT em DRMs foi mais pronunciado do que em ghosts de eritr ´ocito, consistente com a hip´otese da MT ser um modulador de raft. Na concentrac¸ ˜ao de MT presente no plasma sangu´ıneo durante o tratamento de leishmaniose (31-52 μg/mL), as nossas medidas realizadas diretamente no sangue total indicaram um n´ıvel de 2-5% de hem´olise e tamb´em mostrou que a concentrac¸ ˜ao de MT capaz de alterar a fluidez da membrana de eritr ´ocito a n´ıvel detect ´avel pela espectroscopia de RPE causou cerca de 46% de hem´olise. Posteriormente, estudos de RPE realizados com os mesmos marcadores de spin na membrana de Leishmania (L.) amazonensis (forma promastigota) demonstraram alterac¸ ˜oes similares `as encontradas nas membranas de eritr ´ocito. Os efeitos citot ´oxicos sobre os parasitas tamb´em foram avaliados para investigar as relac¸ ˜oes entre os potenciais citot ´oxicos da MT e sua capacidade de alterar I a fluidez da membrana. Os dados de RPE demonstraram que a concentrac¸ ˜ao m´ınima necess´aria de MT para causar alterac¸ ˜ao na membrana do parasito ocorreu pr ´oximo dos valores de concentrac¸ ˜ao que inibe 50% do crescimento celular (IC50), existindo, portanto, uma correlac¸ ˜ao entre as alterac¸ ˜oes na membrana e a citotoxicidade. Embora o marcador de spin lip´ıdico tamb´em tenha detectado alterac¸ ˜oes na fluidez da membrana, os experimentos de RPE indicaram que a MT atua predominantemente no componente prot ´eico da membrana. A lise celular tamb´em foi detectada atrav´es da an´alise dos sobrenadantes das amostras centrifugadas onde foram encontrados fragmentos de membranas marcadas e tamb´em prote´ınas citoplasm´aticas. Usando um m´etodo para a r ´apida incorporac¸ ˜ao da MT na membrana, os efeitos de citotoxidade foram medidos imediatamente ap´os o tratamento e na mesma faixa de concentrac¸ ˜oes de MT que causam a inibic¸ ˜ao do crescimento celular. A citotoxicidade, estimada atrav´es da contagem microsc´opica das c´elulas vivas e mortas, indicou ∼ 70% de morte celular na concentrac¸ ˜ao de MT em que a espectroscopia de RPE detectou uma alterac¸ ˜ao significativa na dinˆamica da membrana. Ap´os o primeiro impacto no n´umero de parasitas vi ´aveis, o processo de morte e crescimento celular continuou durante as primeiras quatro horas de incubac¸ ˜ao. Os espectros de RPE de prote´ınas de membrana marcadas com o 6-MSL foram consistentes com conformac¸ ˜oes de prote´ına mais expandidas e expostas ao solvente, sugerindo uma ac¸ ˜ao t´ıpica dos detergentes. Nossa interpretac¸ ˜ao ´e que a MT pode formar estruturas semelhantes das micelas ao redor das cadeias polipept´ıdicas, expandindo as prote´ınas e que as prote´ınas com maior hidrofobicidade poderiam induzir a penetrac¸ ˜ao de grupos hidrof´ılicos da MT no interior da membrana, causando sua ruptura e consequentemente a morte da c´elula.

Pour assurer ses besoins de croissance, multiplication, et survie, Plasmodium modifie sa cellule hôte, l'érythrocyte, après l'invasion. Le parasite met en place ainsi un système d'échanges (import/export) avec sa cellule hôte et le milieu extérieur. Nous avons identifié dans la base de données de Plasmodium berghei, le parasite de rongeurs, une famille de gènes, sep, correspondant à la famille etramp chez Plasmodium falciparum. Cette famille de gènes code pour des petites protéines exportées, et conservées dans tout le genre Plasmodium. Les protéines SEP (13?16 kDa) contiennent en N-terminal un peptide signal prédit, un domaine hydrophobe interne, et elles diffèrent au niveau des régions C-terminal et 3' UTR. Toutefois, les protéines SEP sont exprimées à différents moments du cycle de Plasmodium. Durant le cycle érythrocytaire, PbSEP1 et PbSEP3 sont exprimées à partir du stade trophozoïte, et la même quantité de protéine est détectée au stade schizonte et gamétocyte, pendant que PbSEP3 est hautement détectée dans les trophozoïtes mûrs et les gamétocytes. Chez le moustique, PbSEP1 et PbSEP3 sont détectées seulement chez les ookinètes, alors que PbSEP2 est très abondante dans les ookinètes, oocystes, et sporozoïtes des glandes salivaires. Les protéines SEP ont également des localisations différentes. Dans l'érythrocyte, PbSEP1 est localisée dans la membrane de la vacuole parasitophore, alors que PbSEP2 et PbSEP3 sont exportées au-delà de cette vacuole, et sont ainsi localisées dans la cellule hôte, en association avec des structures vésiculaires. Dans cette étude, nous avons identifié les signaux d'adressage des protéines SEP dans la vacuole parasitophore et dans la cellule hôte, chez Plasmodium berghei. L'autre partie du travail, effectuée à l'Université de Montpellier II, a consisté à étudier la localisation de deux protéines du squelette sous- membranaire de l'érythrocyte, la dématine, et l'adducine, durant le développement intra-érythrocytaire de Plasmodium falciparum. Le but de cette étude étant d'identifier un mécanisme potentiel d'internalisation des composants du squelette sous-membranaire de l'érythrocyte dans le parasite. Des études d'immuno-localisation ont montré que la dématine et l'adducine sont internalisées à partir du stade trophozoïte, et sont localisées probablement à la vacuole parasitophore (membrane et/ou lumière). Cette internalisation a été confirmée par des études de fractionnement cellulaire et d'accessibilité à la protéinase K, montrant que la dématine est totalement internalisée, alors l'adducine ne l'est que partiellement, suggérant une localisation de la protéine à la périphérie du parasite. / Plasmodium endurance depends on the ability of the parasite to reorganize the cytosol of the erythrocyte, a terminally differentiated cell, and remodel its skeleton membrane immediately after invasion. In this way the parasite can organize the import/export of the molecules necessary to its survival. The comprehension of cellular trafficking mechanisms which occur during Plasmodium infection is a very important step and fundamental contribute to understand the biology of the malaria parasite.We identified in database of the rodent malaria parasite Plasmodium berghei the gene family sep, corresponding to etramp in P. falciparum, encoding small exported proteins conserved in the genus Plasmodium. SEP proteins (13?16 kDa) contain a predicted signal peptide at the NH2-terminus, an internal hydrophobic region while they differ in their C-terminal region; the genes share the upstream regulative region while differ in the 3' UTR. Despite this, we showed that SEPs have a different timing of expression and a different localization: in the erythrocytic cycle PbSEP1 and PbSEP3 start to be expressed at trophozoite and the same amount of protein is detected also in schizonts and gametocytes, while PbSEP2 is highly detected in mature trophozoites and even more in gametocytes. In mosquitoes stages PbSEP1 and PbSEP3 are expressed only in ookinetes, while PbSEP2 is very abundant in ookinetes, oocysts and in sporozoites of the salivary glands. SEPs also have a different localization in the iRBC: PbSEP1 is targeted to the membrane of the parasitophorous vacuole, while PbSEP2 and 3 are exported beyond the parasite membrane and translocated to the host cell compartment in association with vesicle-like structures. In this study we identified the specific signals necessary for the correct timing of expression and to direct SEP proteins to the vacuolar membrane and to the host cell compartments.The second part of the work was carried out in Montpellier II University and aims to identify the localization of two RBC membrane skeleton components, dematin and adducin, during Plasmodium falciparum infection. Our purpose is to recognize a possible mechanism of internalization of host cytoskeleton components to the parasite compartments. In fact, IFA experiments carried on iRBCs showed that dematin and adducin start to be internalized at trophozoite stage and localize at the periphery of the parasite, most probably at the parasitophoruos vacuole (PV) membrane/lumen. Dematin and adducin internalization during Plasmodium infection is also demonstrated by subcellular fractionation and proteinase K assay: while dematin is fully internalized, adducin is partially protected and suggesting a localization of the protein at the periphery of the parasite where it can be exposed to PK degradation.

<p>Despite previously accepted dogma, several recently published studies in humans and mice have shown that mature red blood cells (RBCs) contain a pool of microRNAs. Their role is currently uncertain; however, it has been suggested that microRNAs may play a role in cellular communications as they can be transferred from the RBC to endothelial cells or other cells. This thesis investigated the set of differentially abundant microRNAs found in mature RBCs of felines with oxidative stress, using diabetes mellitus as an oxidant stressor. We postulated that individual microRNAs identified in this study, might be valuable targets for future studies, investigating the role specific microRNAs play in the development or progression of diabetes and in the oxidative damage inflicted on the red cell and other cells by this disease.</p><p>The first specific objective of this thesis was to document oxidative stress in diabetic cats. In the absence of validated assays to document the presence of oxidative protein damage in felines, we first evaluated the performance of a commonly used colorimetric assay for measurement of protein carbonyls (PC) in serum and plasma. Although within run variation was acceptable and performed well over a wide range of PC content values, there were severe limitations related to excessive between run-variation, hemoglobin interference, and difficulty of assay performance. Therefore, we developed and validated a new method, using a fluorescent probe. This new assay had good within and between-run variations, a broad analytical range, and was easy and rapid to run. Hemoglobin and triglyceride only affected the results when present at moderate to higher levels. To further evaluate their redox status, free-radical production and oxidative stress were measured in diabetic and healthy, control cats. The presence of oxidative stress was assessed by measurement of the resulting damage to biomolecules, and detection of antioxidant levels. Our data indicated the presence of protein and membrane lipid oxidation in diabetic individuals and suggest that the redox status of the mature RBC was shifted toward an oxidation state.</p><p>In the final chapter of this thesis, we document the presence of an abundant and diverse set of microRNAs in the mature erythrocytes of healthy and diabetic cats. While their function in the mature erythrocyte remains unknown, a difference was found in the microRNA expression patterns of diabetic and healthy cats. Our data uncovered severe bias in the microRNA sequencing such that the expression levels of some microRNAs appeared to be artificially increased and other diminished. The library construction kit used, appeared to be the cause of this bias. Among the 899 erythrocyte microRNAs sequenced, 12 differentially abundant microRNAs were identified in diabetic cats, however only 6 were differentially abundant by RT-qPCR. Let-7b, miR-1692, miR-339, miR-486 and a feline specific microRNA were increased in mature RBCs of diabetic cats, while miR-451 was decreased.</p><p><a></a><a></a>In conclusion, we have shown that diabetic cats have evidence of significant systemic protein and lipid oxidation as well as erythrocytic oxidative stress. The new, fluorescent PC content assay developed and validated herein could serve as useful tool to better understand the role and consequence of oxidative stress in feline diabetes or other diseases and to monitor antioxidant treatment. Further, this test could be readily adapted for use in other domestic species. Additionally, we have shown that a set of erythrocytic microRNAs are differently abundant in diabetic in comparison to healthy cats. The significance of such changes is currently uncertain. It could represent adaptation of erythroid precursors to changes in their environment during erythropoiesis and as such, these microRNAs may be useful biomarkers for altered hematopoiesis. If microRNAs play a role in communication between circulating mature RBCs and cells in their surroundings such as endothelial cells, the possibility that changes in their expression in this host cells may result in pathology is an intriguing possibility that need to be further explored.</p>

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Chronic HIV-1 infection is characterized by extensive inflammation/immune activation and also by anaemia. Macrophages and neutrophils produce reactive oxygen species (ROS) which can cause damage to surrounding cells, including erythrocytes. Damaged erythrocytes may die by apoptosis (erythroptosis) or be tagged for clearance by monocytes/ macrophages. In this study we investigated HIV-1-associated anaemia and erythroptosis in asymptomatic, untreated HIV-1 infected individuals and how it relates to oxidative stress and immune activation. / AFRIKAANSE OPSOMMING: Chroniese MIV-1 infeksie word gekenmerk deur uitgebreide inflammasie/immuun aktivering en ook deur anemie. Makrofage en neutrofiele produseer reaktiewe suurstof spesies (ROS), wat kan skade aan omliggende selle, insluitend rooibloedselle veroorsaak. Beskadigde rooibloedselle kan sterf deur apoptose (erythroptosis) of gemerk vir klaring deur monosiete/makrofage. In hierdie studie het ons ondersoek MIV-1-verwante bloedarmoede en erythroptosis in asimptomatiese, onbehandelde MIV-1 besmette individue en hoe dit verband hou met oksidatiewe stres en immuun aktivering. / The Poliomyelitis Research Foundation (PRF

L’objectif de cette étude est de déterminer l’importance clinique de l’antigène érythrocytaire canin Dal, en investiguant sa prévalence, son mode d’héritabilité et son immunogénicité. Un total de 1230 chiens a été recruté à travers l’Amérique du Nord et typés pour le Dal en utilisant des allo-anticorps polyclonaux et une technique sur colonne de gel. Des individus Dal-négatifs ont été identifiés chez les Dalmatiens (n=15/128), Doberman Pinschers (n=183/432), Shih Tzus (n=12/21), chiens de races croisées (3/122), Beagles (2/100), Lhasa Apso (1/3) et Bichon Frisé (1/6). Six donneurs de sang Dal-négatifs ont été identifiés, dont 5 Doberman Pinschers (1/228 donneurs d’une autre race). Tous les autres chiens testés étaient Dal-positifs (n=418). La rareté du sang Dal-négatif place les chiens Dal-négatifs à risque d’incompatibilité transfusionnelle lors de transfusions multiples. Cette étude est la première à identifier des individus Dal-négatifs chez des chiens de race autre que Dalmatien et à établir le mode d’héritabilité du Dal, soit autosomal dominant. Par la suite, 2 Beagles Dal-négatifs ont été sensibilisés spécifiquement pour le Dal en recevant une transfusion Dal-positif. Suivant la sensibilisation, des allo-anticorps anti-Dal ont été détectés à partir du 4ième jour post-transfusion et sont demeurés détectables jusqu’à 2 ans post-transfusion. Les titres d’agglutination maximaux (1:64 et 1:1024) ont été atteints 2 et 1 mois post-transfusion chez le chien #1 et le chien #2, respectivement. Cette étude a confirmé l’immunogénicité du Dal tout en ayant généré une quantité considérable d’allo-anticorps anti-Dal permettant de futurs typages sanguins Dal. / The purpose of this study was to determine the clinical importance of the Dal canine erythrocyte antigen by investigating its prevalence, its mode of inheritance and its immunogenicity. A total of 1230 dogs recruited from North America were blood typed for Dal applying a gel column technique using polyclonal canine anti-Dal sera. Dal-negative dogs were identified mostly in Dalmatians (15/128), Dobermans Pinschers (183/432) and Shih Tzus (12/21), and sporadically in mixed breed dogs (3/122), Beagles (2/100), Lhasa Apso (1/6) and Bichon Frise (1/3). All other dogs tested were Dal-positive (n= 418). Six Dal-negative blood donors were found, including 5 Doberman Pinschers (1/228 non-Dalmatian and non-Doberman Pinscher blood donors). The scarcity of Dal-negative blood donors puts Dal-negative patient at higher risk of transfusion incompatibility if requiring multiple blood transfusions. This study was the first to identify Dal-negative dogs in other breeds than Dalmatians and to establish an autosomal dominant mode of inheritance of the Dal-positive phenotype. Secondly, 2 Dal-negative healthy research Beagles were sensitized specifically for Dal with a Dal-positive packed red blood cell transfusion. Following sensitization, anti-Dal alloantibodies were detected as early as 4 days post-transfusion and remained detectable 2 years post-transfusion, with maximum agglutination titers (1:1024 and 1:64) reached respectively 1 and 2 months posttransfusion in dog #2 and dog #1. Our study confirmed the immunogenicity of the Dal and allowed banking of a considerable amount of polyclonal antisera for further Dal blood typing.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.