CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / A anemia falciforme (AF) Ã uma doenÃa hereditÃria resultante de uma mutaÃÃo pontual (GAG &#61664; GTG) no cÃdon do gene da &#946;S - globina, levando a uma substituiÃÃo de Ãcido glutÃmico por valina na sexta posiÃÃo da cadeia polipeptÃdica, gerando uma hemoglobina (Hb) anormal denominada de HbS, em homozigose. A AF se caracteriza por anemia hemolÃtica crÃnica associada a mÃltiplos eventos tais como processo inflamatÃrio crÃnico, aumento do estresse oxidativo, dano endotelial, diminuiÃÃo da biodisponibilidade do Ãxido nÃtrico (NO), ativaÃÃo da coagulaÃÃo, dentre outros. Os biomarcadores de hemÃlise tais como: contagem de reticulÃcitos, lactato desidrogenase (LDH), Ãcido Ãrico e arginase I sÃo fundamentais na avaliaÃÃo do grau da hemÃlise, principalmente, de natureza intravascular contribuindo com o monitoramento da anemia nesses pacientes. O presente estudo teve como objetivo avaliar o impacto dos haplÃtipos do gene &#946;S sobre os marcadores de hemÃlise em adultos com AF em estado basal, acompanhados no ambulatÃrio do serviÃo de hematologia no Hospital UniversitÃrio Walter CantÃdio (HUWC). Um total de 50 pacientes adultos com AF foi selecionado, com diagnÃstico confirmado por estudo molecular. Os pacientes se encontravam em uso de hidroxiurÃia (HU), dose variando de 500 mg a 1,5 g/kg/dia por no mÃnimo seis meses. Um grupo controle foi elaborado, sendo constituÃdo por 20 indivÃduos supostamente saudÃveis. Foram coletados 10 mL de sangue venoso em tubo de coleta a vÃcuo, contendo o anticoagulante EDTA (etileno-diamino-tetracÃtico), para a realizaÃÃo da contagem de reticulÃcitos e 6 mL de sangue venoso em tubo de coleta a vÃcuo contendo gel separador, sem anticoagulante, para as dosagens sÃricas de LDH , Ãcido Ãrico e arginase I. As variÃveis idade, sexo, dosagem e tempo de uso do medicamento, concentraÃÃo da Hb e da hemoglobina fetal (HbF) foram obtidas nos prontuÃrios mÃdicos no momento da realizaÃÃo do estudo. As anÃlises estatÃsticas foram realizadas no programa GraphPad Prism (versÃo 5.0) e o nÃvel de significÃncia estabelecido foi p < 0,05. Foi verificado aumento significante nos nÃveis de reticulÃcitos, LDH, Ãcido Ãrico e arginase I nos pacientes com AF em relaÃÃo ao grupo controle (p < 0,05). Foi observada diferenÃa significativa na Hb nos grupos Bantu/Benin em relaÃÃo aos demais haplÃtipos do cluster da &#946;s-globina. Os nÃveis de HbF apresentaram uma tendÃncia a aumento no haplÃtipo Benin/Benin em relaÃÃo aos demais. Foi verificada uma tendÃncia no aumento de LDH no genÃtipo Bantu/Bantu em comparaÃÃo com os demais haplÃtipos. Foi observada diferenÃa significativa da arginase I entre os grupos Bantu/Bantu vs Bantu/Benin e Bantu/Bantu vs Benin/Benin. Os resultados do presente estudo reforÃam a hipÃtese de que a arginase I possa ser utilizada como possÃvel indicador de gravidade uma vez que a mesma foi associada ao haplÃtipo Bantu.

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacterium that causes respiratory disease in poultry characterized by rhinitis, tracheitis, and pneumonia with mortality averaging 2-3%. In the Shenandoah Valley of Virginia, the seroprevalence for ORT among turkey flocks as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 70.9% (n=175). Additionally, the seroprevalence for hemorrhagic enteritis virus (vaccine induced), Bordetella avium, and paramyxovirus-1 was 100%, 74.8%, and 6.3% respectively. No significant interactions were detected. The type strain of ORT is characteristically non-hemolytic at least for 96 hours at 37°C on Columbia Blood Agar. In recent years, atypical isolates that rapidly produce hemolysis have been isolated with increasing frequency. A variety of in vitro tests were used to determine differences between representative isolates of the hemolytic (H) and non-hemolytic (NH) phenotypes. Findings suggest that the H isolate contains a 4 kb plasmid similar to that found in Reimerella anatipestifer. No plasmid was found in the NH isolate. Differences in growth characteristics and resistance to tetracyclines were also noted. No differences in proteins, biochemical characteristics or 16S rRNA sequences were found, the latter serving as confirmation that the isolates were both ORT. Embryo inoculation was used to assess virulence. No significant differences were observed and most embryos survived through to the day of hatch (pip) despite the fact that ORT could be re-isolated. In turkey poults however, the H phenotype did appear less virulent. A significant depression in weight gain was noted for birds inoculated intratracheally with the NH isolate at 7 days post-inoculation (dpi). NH inoculates also had significantly higher antibody levels on ELISA at 14 and 21 dpi and histopathological lesion scores for lung at 7, 14, and 21 dpi. The NH isolate could be re-isolated from NH-inoculated poults through 21 dpi; whereas the H isolate could only be re-isolated through 14 dpi. In conclusion, there are numerous differences between the NH and H isolates found in the field with the H isolate appearing less virulent and as such, making it a potential vaccine candidate. The phenotypic difference appears to correlate with this, but may not suffice to explain it. / Ph. D.

Treponema hyodysenteriae is the causative agent of swine dysentery, a mucohemorrhagic disease of the intestines. T. hyodysenteriae requires phospholipids and cholesterol (or cholestanol) for growth, and it produces a a-hemolysin (TH) which is induced (300 fold) by the addition of RNA core to cultures. TH is bound to the RNA core which acts as a carrier or stabilizer. I\lyobjectives were to (i) obtain successful continuous growth of T. hyodysenteriae; (ii) study the production of the hemolysin produced by T. hyodysenteriae; (Ui) study the effects of different oligonucleotide carriers on the induction of hemolysin, (iv) examine various purification procedures for nipid, efficient partial purification of the hemolysin, (v) separate the hemolysin from the RNA core carrier, and (vi) to detennme whether T. hyodysenteriae can use coprostanol, the most common intestinal sterol, to satisfy its sterol requirement. I found that optimal growth of T. hyodysenteriae could be achieved by using BHI- glucose broth supplemented with calf serum (I O~/o). serum replacement (100/0), or phosphatidylcholine liposomes which contained cholesterol or cholestanol. Optimal growth required 1 % 02 and stirring of the culture. l\laximal hemolytic titers were obtained during late-log to early-stationary phase by cultures grown in the presence of RNA core. Hemolysin production was induced as soon as 5 minutes after addition of RNA core to cultures. This production was inhibited by chloramphenicol. Polyguanylic acid and RNase treated RNA core did not significantly increase hemolytic titers of cultures grown in their presence. Partial purification of hemolysin was achieved by acetic acid clarification followed by ammonium sulfate precipitation (65% saturation). With these procedures > 90% of the hemolytic activity was recovered. Although, hydroxylapatite adsorption and polyethyleneimine precipitation completely adsorbed or precipitated hemolytic activity I I was unable to efficiently recover the activity. Partially purified hemolysin was c}10toxic to CHO cells, and caused lysis rather than the rounding effect caused by many cytotoxins. / Master of Science

O avanço da bioengenharia atual tem sido motivado pela crescente necessidade da humanidade em se buscar formas para amenizar o sofrimento, garantir tempo para um tratamento, melhorar a qualidade de vida ou sanar quadros clínicos de pacientes. Assim, a crescente necessidade de órgãos artificiais impulsiona a área de bioengenharia para que seja dado um suporte no desenvolvimento destes mecanismos. Neste contexto se encontra o presente trabalho. Aqui, apresenta-se uma metodologia numérica para que seja aplicada a desenvolvimento e testes de Dispositivos de Assistência Cardíaca, de forma a otimizar o processo de desenvolvimento e estimar os possíveis problemas inerentes ao seu funcionamento. Compondo a metodologia, uma ampla discussão de cada etapa numérica foi elaborada, contribuindo para uma metodologia flexível para uma ampla variedade de aplicações. A hemólise também foi investigada através dos principais modelos da literatura, bem como foram propostos modelos adaptados para tentar estimar a hemólise verificada experimentalmente. Além das metodologias para cada etapa, uma metodologia geral utilizando Sistemas de Referências Múltiplas (SRM) compõe os resultados, para uma sequência de passos numéricos que possa obter resultados satisfatórios em comparação com resultados de bancada por loop test. Outros resultados encontrados e devidamente discutidos foram inerentes a uma densidade de malha a ser utilizada para que ocorra simulações com independência de malha, na qual uma densidade a partir de 318,43 elementos/mm³ para referenciais não-inerciais é proposta. O mapeamento da turbulência por seis modelos que aplicam a média de Reynolds também é discutido, indicando os melhores modelos a serem empregados para um número de Reynolds relativo (Re*) que relaciona a influência dos contornos nos resultados numéricos obtidos. Além disso, uma nova abordagem por tensão fisiológica é proposta para o cálculo da hemólise, sendo comparada aos modelos clássicos adotados. Os resultados para hemólise indicam um bom ajuste para o novo modelo proposto, bem como indica o correto tratamento de unidades para os modelos tradicionais. Por fim, as análises recaem na melhoria do dispositivo e conclui a metodologia numérica a ser empregada, preenchendo lacunas em cada etapa e determinando uma maneira de análise numérica cujos resultados são confiáveis. / The advance of current bioengineering has been driven by the increasing need of humanity to seek ways to alleviate the suffering, ensure time to treatment, improve the quality of life or cure medical conditions of patients. Thus, the growing need for artificial organs boosts bioengineering area to be given a support in the development of these mechanisms. In this context is the present work. Here, we present a numerical methodology to be applied to development and Cardiac Assist Devices tests in order to optimize the development process and estimate the potential problems inherent in their operation. Compounding the methodology, a comprehensive discussion of each numerical step was developed, contributing to a flexible methodology for a wide variety of applications. Hemolysis was also investigated through the main models of literature, and have been proposed models adapted to try to estimate hemolysis verified experimentally. In addition to the methodologies for each step, a general methodology using Multiple Reference Systems (MRF) makes up the results for a sequence of numeric steps you can get satisfactory results compared to bench test results for loop. Other findings were discussed and properly attached to a mesh density being used for simulations occurring independently mesh in which a density from 318.43 elements/mm³ Non-inertial frames is proposed. The mapping of turbulence six models applying Reynolds medium is also discussed, indicating the best designs to be employed for a number of relative Reynolds (Re*) that relates the influence of the contours of numerical results obtained. In addition, a new approach by physiological stress is proposed for the calculation of hemolysis, being compared to classic models adopted. The results for hemolysis indicate agreement with the proposed new model, and indicates the correct treatment units to the traditional models. The final analyses fall into improved device and completes the numerical methodology to be used by filling gaps in each step and determining a way to numerical analysis results which are dependable.

Introdução: Anemias hemolíticas induzidas por fármacos (AHIF) são caracterizadas por uma anemia hemolítica imune após exposição a um fármaco. Testes sorológicos têm o potencial de confirmar a presença de anticorpo contra fármacos, mas a realização destes testes ainda é um desafio. Os anticorpos IgM e IgG envolvidos na AHIF são principalmente de dois tipos: fármaco-dependentes e fámaco-independentes. Os anticorpos fármaco-independentes reagem com os eritrócitos \"in vitro\" sem a presença do fármaco, e os resultados imunohematológicos são idênticos aos encontrados nas anemias hemolíticas autoimunes idiopáticas. Anticorpos fármaco-dependentes resultam em teste da antiglobulina direto (TAD) positivo e eluato negativo, os anticorpos só reagem \"in vitro\" com os eritrócitos na presença do fármaco. Indivíduos saudáveis podem desenvolver anticorpos contra fármacos, mesmo na ausência de anemia hemolítica. Os indivíduos podem ser sensibilizados durante exposição prévia a fármacos, exposição a antibióticos usados em ração animal, tanques de piscicultura, berçários de criação de suínos ou tratamento de mastite em bovinos. Estes indivíduos apresentam um maior risco de desenvolvimento de AHIF grave na ocasião de uma segunda exposição ao fármaco. Objetivos: Padronizar testes imunohematológicos para a detecção de anticorpos dirigidos contra fármaco-dependentes. Materiais e métodos: Amostras de 162 doadores de sangue foram submetidas a pesquisa de anticorpos fármaco-dependentes anti-cefalexina, anti-rifampicina e anti-diclofenaco de sódio. Amostra de um doador de plaquetas colhidas por aférese com TAD positivo foi investigada para detecção de anticorpos fármaco-dependentes anti-valsartana. Amostras de 8 pacientes com suspeita diagnóstica de AHIF foram investigadas para a presença de anticorpos contra os fármacos que foram prescritos ao paciente no período de duas semanas prévias ou até o momento em que a hemólise aguda foi constatada. Anticorpos fármaco-dependentes que reagem na presença do fármaco ligado covalentemente à membrana da hemácia, ou na presença do fármaco em solução, foram investigados em doadores de sangue (técnicas em gel) e em pacientes (técnicas em tubo e em gel). Resultados: Os testes laboratoriais detectaram a presença de anticorpo anti-valsartana no eluato de um doador de plaquetas colhidas por aférese com TAD positivo e a presença de anticorpo anti-ceftazidima no soro de um paciente com suspeita clínica de AHIF. A investigação de anticorpo anti-cefalexina e anti-rifampicina foi negativa em amostras de doadores de sangue. O teste laboratorial para investigação de anticorpo anti-diclofenaco de sódio não foi finalizado devido a presença de hemólise em hemácias sensibilizadas com o fármaco e após a adição do fármaco na solução de hemácias. Conclusão: A detecção de anticorpo anti-valsartana em um doador de plaquetas colhidas por aférese evidenciou a presença de anticorpo anti-fármaco na ausência de hemólise. A detecção de anticorpo anti-ceftazidima em amostra de um paciente com anemia hemolítica confirmou a suspeita clínica de AHIF. Reconhecer os sinais clínicos da AHIF precocemente e a disponibilidade de testes sorológicos é essencial para confirmação diagnóstica, orientação terapêutica e prevenção de uma hemólise potencialmente fatal. / Introduction: Drug-induced immune hemolytic anemia (DIHA) is characterized by immune hemolytic anemia following exposure to a drug. Serological testing has the potential to confirm the presence of anti-drug antibodies, but the testing is still a challenge. The IgM and IgG antibodies involved in DIHA comprise mainly two types: drug-dependent and drug-independent. Drug-independent antibodies react with erythrocytes in vitro without the presence of the drug, and the immunohematologic results are identical to those found in idiopathic autoimmune hemolytic anemias. Drugdependent antibodies induce positive direct antiglobulin tests (DAT) and negative eluates, and in vitro antibody reactions with erythrocytes are only detected in presence of the drug. Healthy individuals can develop antibodies against drugs even in the absence of hemolytic anemia. Individuals may be sensitized during previous exposure to drugs, exposure to antibiotics used in animal feed, fish culture ponds, swine nurseries or mastitis treatment in bovines. These individuals present higher risk of developing severe DIHA on the second exposition. Aims: To standardize immunohematological tests for detection of anti-drug dependent antibodies. Materials and methods: Samples from 162 blood donors were tested for anti-cephalexin, anti-rifampicin and antidiclofenac antibodies. Sample from one platelet apheresis donor with positive DAT was investigated for anti-valsartan drug-dependent antibody. Samples from 8 patients with suspected DIHA were investigated for the presence of antibodies directed against the drugs that were prescribed to the patient in the period of two weeks prior to or until the time when acute hemolysis was detected. Drug-dependent antibodies that react in presence of the drug covalently attached to the erythrocyte membrane, or in presence of the drug in solution were investigated in samples from blood donors (gel techniques) and from patients (tube and gel techniques). Results: Laboratory tests detected antivalsartan antibody in the eluate of an platelet apheresis donor with positive TAD and anti-ceftazidime antibody in the serum of a patient with clinical suspicion of DIHA. Investigation of anti-cephalexin and anti-rifampicin antibody was negative in blood donor samples. The laboratory test for anti-diclofenac antibody was not possible due to the presence of hemolysis in erythrocytes sensitized with the drug and when the drug in solution was added to the erythrocyte. Conclusion: The detection of anti-valsartan antibody in an platelet apheresis donor revealed the presence of an anti-drug antibody in the absence of hemolysis. Detection of anti-ceftazidime antibody in a patient with hemolytic anemia confirmed the clinical suspicion of DIHA. It is essential to recognizing early clinical signs of DIHA and have serological tests available for diagnostic confirmation, therapeutic guidance and prevention of potentially fatal hemolysis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1977. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Vita. / Includes bibliographical references. / Ph.D.

Flavonoids are of great interest for their antioxidant and health-promoting activities. Ascorbate (vitamin C) has antioxidant activities but also sometimes displays pro-oxidant activities <i>in vitro</i> and reportedly <i>in vivo</i>. This research investigated to what extent flavonoids moderate oxidative stress from vitamin C <i>in vivo</i>.<p>Dietary experiments were conducted in two phases using adult male Wistar rats. First, all animals were maintained for two weeks on a control flavonoid-free diet with the dietary requirement (27 IU) of vitamin E/kg diet. In the subsequent four weeks, the animals were treated in four groups (8 rats/group), being fed the following diets: flavonoid-free control (C), ascorbate-supplemented (7.55 mmol/kg diet) (A), flavonoid-supplemented (2.67 mmol/kg diet) (F) and flavonoids (2.67 mmol/kg diet) plus ascorbate (7.55 mmol/kg diet)-supplemented (T). Measurements were done on in vivo biomarkers of oxidative stress, tissue antioxidants and on tissue in vitro susceptibility to oxidative stress.<p>In the combined feeding of ascorbate plus flavonoids, endogenous thiobarbituric acid reactive substances (TBARS) increased in liver by 114%. No effects of dietary ascorbate or flavonoids were seen on endogenous TBARS in brain or heart, or on plasma thiols or erythrocyte fragility.<p><i>In vitro</i>, the susceptibility to TBARS formation of liver homogenate (incubated for 60 min at 37ºC in air) showed a significant 60% increase in ascorbate-fed animals compared to control, but no increase in animals fed ascorbate plus flavonoids, suggesting that the additional feeding of flavonoids helped to prevent the increase produced by ascorbate-feeding. Incubation of liver mitochondria with 300 µM ascorbate in vitro produced a large (2-7 fold) increase in TBARS, but there was no difference among mitochondria from the different feeding groups.<p>The ability of flavonoid-feeding in protecting against oxidative stress from ascorbate in vivo could not be demonstrated in this study, even showing pro-oxidant effects of flavonoids in combination with ascorbate in liver. However, in vitro tests in liver suggest a protective effect of flavonoid-feeding against susceptibility to oxidative stress from ascorbate. Further investigations are needed in order to resolve the differences observed in vitro and in vivo and to determine the endogenous effects of specific flavonoids under ascorbate-induced oxidative stress.

Flavonoids are of great interest for their antioxidant and health-promoting activities. Ascorbate (vitamin C) has antioxidant activities but also sometimes displays pro-oxidant activities <i>in vitro</i> and reportedly <i>in vivo</i>. This research investigated to what extent flavonoids moderate oxidative stress from vitamin C <i>in vivo</i>.<p>Dietary experiments were conducted in two phases using adult male Wistar rats. First, all animals were maintained for two weeks on a control flavonoid-free diet with the dietary requirement (27 IU) of vitamin E/kg diet. In the subsequent four weeks, the animals were treated in four groups (8 rats/group), being fed the following diets: flavonoid-free control (C), ascorbate-supplemented (7.55 mmol/kg diet) (A), flavonoid-supplemented (2.67 mmol/kg diet) (F) and flavonoids (2.67 mmol/kg diet) plus ascorbate (7.55 mmol/kg diet)-supplemented (T). Measurements were done on in vivo biomarkers of oxidative stress, tissue antioxidants and on tissue in vitro susceptibility to oxidative stress.<p>In the combined feeding of ascorbate plus flavonoids, endogenous thiobarbituric acid reactive substances (TBARS) increased in liver by 114%. No effects of dietary ascorbate or flavonoids were seen on endogenous TBARS in brain or heart, or on plasma thiols or erythrocyte fragility.<p><i>In vitro</i>, the susceptibility to TBARS formation of liver homogenate (incubated for 60 min at 37ºC in air) showed a significant 60% increase in ascorbate-fed animals compared to control, but no increase in animals fed ascorbate plus flavonoids, suggesting that the additional feeding of flavonoids helped to prevent the increase produced by ascorbate-feeding. Incubation of liver mitochondria with 300 µM ascorbate in vitro produced a large (2-7 fold) increase in TBARS, but there was no difference among mitochondria from the different feeding groups.<p>The ability of flavonoid-feeding in protecting against oxidative stress from ascorbate in vivo could not be demonstrated in this study, even showing pro-oxidant effects of flavonoids in combination with ascorbate in liver. However, in vitro tests in liver suggest a protective effect of flavonoid-feeding against susceptibility to oxidative stress from ascorbate. Further investigations are needed in order to resolve the differences observed in vitro and in vivo and to determine the endogenous effects of specific flavonoids under ascorbate-induced oxidative stress.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-104).

The conditions by which the production, in liquid media, of hemolytic activity by growing cells of T. hyodysenteriae and T. innocens are described. / From a ribonucleic acid added culture broth of T. hyodysenteriae, two hemolysins are shown in the supernatant. For each of them, a purification scheme allowing the obtention of an homogeneous hemolytic preparation by gel filtration and electrophoresis, is proposed. One hemolysin is associated with ribonucleic acid (HN) while the later is associated with bovine serum albumin (HP). They share some properties: their synthesis is blocked by chloramphenicol, they do not have proteolytic or phospholipasic activities, they are insensitive to oxygen and their activity does not require bivalent cations or is sensitive to EDTA's action; they are lytic to all red blood cell's type tested. They differ by the size of their apparent molecular weight, their mode of action, the stability of their activity to temperature and different pH and, their pattern of sensitivity to proteolytic enzymes. / The isolation of a non-hemolytic mutant strain of T. hyodysenteriae and its use in a ligated ileal loop model in rabbit has shown that there is a relation between the loss of the hemolytic activity and the loss of the enterotoxic activity.