Dissertations / Theses: 'Hemolys'

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Sarras, Marcella. "Påverkan av hemolys vid analys av neuronspecifikt enolas på Cobas." Thesis, Malmö universitet, Fakulteten för hälsa och samhälle (HS), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-44406.

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Neuronspecifikt enolas (NSE) är en viktig biomarkör för att diagnostisera t.ex. neuroendokrina tumörer, särskilt småcellig lungcancer (SCLC) och neuroblastom. NSE används även som en del i utredning av hjärnskada vid hjärtstopp. Eftersom NSE finns i höga koncentrationer i erytrocyter, kan hemolys i blodprovet orsaka falskt förhöjda NSE-nivåer i serum utan hjärnskada. Syftet med studien var att utvärdera hur hemolys påverkar NSE-analysen på Cobas, ett helautomatiserat analysinstrument. Mätning av NSE-koncentration utfördes på Cobas 8000 från Roche Elecsys, baserad på immunokemisk sandwich-metod med ElectroChemi-LuminiscenceImmunoassay (ECLI) detektionsteknik. För att studera hemolysens inverkan, tillverkades hemolysat från 20 patientprover. Dessa hemolysat tillsattes till poolat serum, med NSE-nivåer inom referensintervallet (< 17 µg/L). Även graden av hemolys bestämdes på Cobas 8000. Resultatet visade ett linjärt samband mellan de uppmäta hemolysindex (HI) värden och S-NSE värden. Variationen i NSE-tillskott på individnivå undersöktes och resulterade i slutsatsen att varje hemolysenhet motsvarar ett NSE-tillskott på 0,33 ± 0,07 µg/L som frigörs från erytrocyter. Ett förslag för att lösa problemet med hemolys vid analys av S-NSE är att använda en kompenserande faktor för att korrigera NSE-koncentrationen. Kompensering kan utföras med hjälp av det erhållna sambandet i studien (1 HI = 0,33 ± 0,07 µg/L NSE-tillskott) genom att subtrahera tillskottet från den uppmätta NSE-koncentrationen.
Neuron Specific Enolase (NSE) is an important biomarker for diagnosing e.g. neuroendocrine tumors, especially small cell lung cancer (SCLC) and neuroblastoma. NSE is also used as a part of the investigation of brain damage in cardiac arrest. Because NSE is present in high concentrations in erythrocytes, hemolysis in the blood sample can cause falsely elevated NSE levels in serum without brain damage. The purpose of this study was to evaluate how hemolysis affects NSE analysis on Cobas, a fully automated analytical instrument. Measurement of NSE concentration was performed on Cobas 8000 from Roche Elecsys, based on immunochemical sandwich method with ElectroChemi-Luminescence Immunoassay (ECLI) detection technique. To study the effect of hemolysis, hemolysates were prepared from 20 patient samples. These hemolysates were added to pooled serum, with NSE levels within the reference range (<17 μg/L). The degree of hemolysis was also determined on Cobas 8000. The result showed a linear relationship between the measured hemolysis index (HI) values and S-NSE values. The variation in NSE contribution at the individual level was examined with the result that each hemolysis unit corresponds to an NSE contribution of 0.33 ± 0.07 µg/L, which is released from erythrocytes. A suggestion to solve the problem of hemolysis relating to NSE analysis is to use a compensatory factor to correct the NSE concentration. Compensation can be performed by using the relationship obtained in the study (1 HI = 0.33 ± 0.07 µg/L NSE contribution) and subtracting the contribution from the measured NSE concentration.

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Saleh, Amal. "Interferens genom hemolys som påverkar analys av S- bilirubin på Olympus AU2700." Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-30444.

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Felaktig provtagning kan orsaka hemolys som kan påverka mätresultatet av S-bilirubin koncentrationen vid användning av diazometoder. Bilirubin är ett gulfärgat pigment som erhålls när hemolglobin bryts ner. Vätebindningar som finns i bilirubinstrukturen stabiliserar molekylen och avgör om den är löslig eller inte löslig i vatten. Bilirubin konjugeras i levern och blir vattenlösligt, därefter utsöndras bilirubin via gallan och tarmen. Hyperbilirubinemi uppstår vid ökad nedbrytning av erytrocyter eller vid leverskada. Ökad bilirubinkoncentration i blodet sker ofta hos nyfödda och diffunderar till kroppens celler och orsakar icterus. Studien gjordes med syftet att studera hur olika grad av hemolys påverkar analys av S-bilirubin-koncentrationen. S-bilirubinkoncentration analyserades kolormetrisk i Olympus AU2700. Diazoreaktionen sker mellan bilirubin och diazotiserad sulfanilsyra som bildar en färgad produkt, azobilirubin. Azobilirubin analyseras genom absorbansmätning vid 540 nm. Absorbansen är proportionell mot totala bilirubinkoncentrationen. En hemolysat-spädningslösning gjordes och användes som en spädningsslösning till 16 stycken olika serumprover för att få olika hemolysgrader 0 g/L, 0,5 g/L, 1 g/L, 3 g/L respektive 5 g/L. Resultatet visade att hemolys sänkte S-bilirubinkoncentrationen vid mätning i instrumentet som använder diazimetoder. Vid högre grad av hemolys som tillsattes till S-bilirubinkoncentrationen desto lägre blev S-bilirubinkoncentrationen som analyserades i proverna. Alla prover som hade låg S-bilirubinkoncentration hade större avvikelser jämfört med de prover som hade hög S-bilirubinkoncentration. Vid tillsats av hemolys med 0,5 g/L till prover med S-bilirubinkoncentrationen under 20 µmol/L låg avvikelsen mellan 12,00 % och 16,00 % medan vid tillsats av samma hemolysgrad till S-bilirubinkoncentrationen över 20 µmol/L låg avvikelsen mellan 0,67 % och 4,00 %. Slutsatsen är att patientprover som har S-bilirubinkoncentrationer lägre än 20 µmol/L och med hemolys inte kan mätas, prover från patienter med hyperbilirubinemi och hemolysgrad lägre än 1 g/L kan analyseras i de fall de är svåra att ta om t.ex på nyfödda.

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Frohm, Hanna. "Titerbestämning av anti-A och anti-B i trombocytenheter för transfusion över ABO gränsen : utvärdering av rutinanalys och utveckling av en screeningmetod." Thesis, Högskolan Kristianstad, Fakulteten för naturvetenskap, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:hkr:diva-18623.

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Trombocyter är suspenderade i plasma som innehåller antikroppar mot de blodgruppsantigen som saknas på erytrocyterna. För att minimera risken för en hemolytisk reaktion bestäms titern av anti-A och anti-B. Gelkortsteknik används för att detektera antikropp-antigensreaktioner och baseras på agglutinationer i en gel. Syftet med studien var att undersöka titern av anti-A och anti-B i trombocytenheter, samt att utvärdera en rutinanalys och utveckla en screeningmetod. I studien analyserades enheter av blodgrupp O och A. De kontrollerades mot anti-A och/eller anti-B både för IgG och IgM antikroppar. En screeningmetod utvecklades för att kunna screena O-enheterna och en gräns på 1:100 respektive 1:250 undersöktes. Resultatet kunde påvisa en stor skillnad i titer mellan O-och A-enheter. Titern skiljer sig signifikant beroende på om titern bestäms i plasma eller från den färdiga (utspädda) enheten. En screeningmetod på 1:100 påvisade att 86 % av enheterna hade bedömts som hög titer och en screeningmetod på 1:250 visade att andelen sjönk till 31 %. Geltekniken är en känslig metod och är beroende av kompetent personal vid avläsning. En del studier visar liknande resultat men andelen enheter med hög titer varierar och likaså metoder och titergräns. Detta påvisar svårigheterna i att bestämma en kritisk titer och att förutse risker hos patienten. Andra faktorer tros också kunna påverka riskerna. Införande av en screeningmetod på 1:250 kan öka antalet enheter som kan transfunderas över ABO-barriären.
Platelets are suspended in plasma containing antibodies to the blood group antigen missing on the erythrocytes. To minimize the risk of hemolytic reaction, the titrers of anti-A and anti-B are determined. The gel test is used to detect antibody-and antigen responses and is based on agglutinations in gel. The purpose was to investigate the titers of anti-A and/or anti-B in platelets. A routine analysis was evaluated and a screening method was implemented. In the study, units of blood group O and A were analyzed. They were checked against anti-A and anti-B for both IgG and IgM antibodies. A screening method was developed to screen the O-units and a limit of 1:100 and 1:250 was used. The results showed great difference in titers between O and A units. The titers differ significantly depending on whether the titers are determined in plasma or from the finished (diluted) unit. A screening method at 1:100 showed that 86 % of the units was rated as high titer while a screening method of 1:250 showed that this was reduced to 31 %. Gel technology is a sensitive method and is dependent on competent staff when reading the agglutinations. Some studies show similar results, but the proportion of high titer units, methods and critical titers varies. It proves the difficulty in determining a critical titer and predicting risks for the patient. Other factors are also believed to influence the risks. Implementation of a 1:250 screening method is believed to increase the number of units that can be transfused over the ABO barrier.

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Chornogorets, Valeriya, and Валерія Чорногорець. "Study of genetic features of blood groups heredity according to the AB0 system and rhesus factor." Thesis, National Aviation University, 2021. https://er.nau.edu.ua/handle/NAU/50772.

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1. Рагимов А.А., Дашкова Н.Г. Основы трансфузионной иммунологии. М., 2004. С. 68. 2. Blood group terminology 2004: from the International Society of Blood Transfusion committee on terminology for red cell surface antigens / G. Daniels et al. Vox Sang. 2004. № 87. Р. 304-316. 3. Yamamoto F., Yamamoto M. Molecular genetic basis of porcine histo-blood group ABO system. Blood. 2001. Vol. 97, № 10. P. 3308–3310. 4. Лавряшина М.Б., Толочко Т.А., Волков А.Н. Аллоантигены крови человека: учеб. пособ. Кемерово: Кузбассвузиздат, 2006. 100 с. 5. Практическая трансфузиология / под ред. Г.И. Козинца. М.: Практическая медицина, 2005. 544 с.
The work is devoted to the problem of blood transfusion (namely, the study of the most common blood group and rhesus factor in countries), because this issue has become acute around the world. Today, blood transfusions are an acute social problem. Two classifications of human blood groups are of the greatest importance for clinical practice: the AB0 system and the rhesus system, due to the fact that these systems have the greatest antigenic power. Each human-to-human blood transfusion must take into account the compatibility of these two systems, because in the case of a human transfusion of another (incompatible) blood group, agglutination (gluing) and hemolysis (destruction) of erythrocytes occur, which can lead to death. It is also desirable to separate plasma and blood cells.
Робота присвячена проблемі переливання крові (а саме вивченню найпоширенішої групи крові та резус-фактора в країнах), оскільки ця проблема загострилася у всьому світі. Сьогодні переливання крові є гострою соціальною проблемою. Дві класифікації груп крові людини мають найбільше значення для клінічної практики: система AB0 та резус-система через те, що ці системи мають найбільшу антигенну силу. При кожному переливанні крові людині необхідно враховувати сумісність цих двох систем, оскільки у випадку переливання людиною іншої (несумісної) групи крові відбувається аглютинація (склеювання) та гемоліз (руйнування) еритроцитів, що може привести до смерті. Також бажано відокремити плазму та клітини крові.

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Menegazzo, Ana Barbara Bordignon Rodrigues. "Determinação do valor da heptoglobina sérica para diagnóstico de hemólise na síndrome HELLP /." Botucatu, 2014. http://hdl.handle.net/11449/113896.

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Orientador: Joelcio Francisco Abbade
Banca: José Carlos Peraçoli
Banca: Francisco Lázaro Pereira de Souza
Resumo: INTRODUÇÃO: A síndrome HELLP é uma complicação severa da pré-eclâmpsia, caracterizada por hemólise, elevação das enzimas hepáticas e trombocitopenia. Apesar de haver padronização dos valores laboratoriais que definem a síndrome HELLP, ainda existe dificuldade para a caracterização da hemólise. OBJETIVO: Avaliar o valor de haptoglobina que determina a hemólise nas pacientes com síndrome HELLP. MÉTODOS: estudo transversal e prospectivo de gestantes e puérperas com de pré-eclâmpsia. Exame laboratorial avaliado: dosagem sérica de haptoglobina. Construção da curva ROC para determinar o valor de corte da haptoglobina para diagnóstico de hemólise na síndrome HELLP. RESULTADOS: O valor da haptoglobina para diagnóstico de hemólise em pacientes com síndrome HELLP foi de 0,26g/L. DISCUSSÃO: A melhor correlação observada foi a haptoglobina com a DHL, indicando que este é o melhor marcador de hemólise intravascular para o diagnóstico da síndrome HELLP. CONCLUSÃO: A dosagem sérica da haptoglobina nos casos de pré-eclâmpsia deve fazer parte dos exames de rotina para diagnóstico de hemólise intravascular da síndrome HELLP
Abstract: CONTEXT: HELLP syndrome is a severe complication of pre-eclampsia, caracterized by hemolysis, elevated liver enzymes and low platelet count. Although there are standardized laboratory values that define the HELLP syndrome, the difficulty still exists for the characterization of hemolysis. PURPOSE: to evaluate the haptoglobin value to diagnose the hemolysis in HELLP syndrome. METHODS: transversal and prospective study of pregnant and postdelivery women with pre-eclampsia. Laboratory tests evaluated: serum haptoglobin. ROC curve to determine the cutoff value of haptoglobin in the diagnosis of hemolysis in HELLP syndrome. RESULTS: The haptoglobin value for hemolysis diagnosis in HELLP syndrome was 0.26 g / L. DISCUSSION: The best correlation was with haptoglobin and DHL, indicating that this is the best marker of intravascular hemolysis for the diagnosis of HELLP syndrome. CONCLUSION: Serum haptoglobin in cases of pre-eclampsia should be part of routine tests for diagnosis of intravascular hemolysis in HELLP syndrome
Mestre

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Baltyde, Kizzy-Clara. "Implication de la voie adénosine/adénosine récepteur A2B dans les mécanismes physiopathologiques de deux manifestations drépanocytaires : l'hémolyse et le priapisme." Thesis, Antilles, 2016. http://www.theses.fr/2016ANTI0039/document.

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La drépanocytose résulte d’une mutation du gène β-globine entrainant la synthèse d’une hémoglobine anormale, l’HBS, qui polymérise en condition désoxygénée. Le globule rouge deviendra plus rigide et plus fragile, donnant lieu a deux conséquences majeures : l’hémolyse accrue et l’occlusion vasculaire.Récemment, un nouvel acteur moléculaire, l’adénosine, a été identifie. Ce nucléoside présente des effets bénéfiques sur les atteintes pulmonaires en inhibant l’activation des cellules « tueur naturel t inductibles », mais aussi délétères en favorisant la polymérisation de l’HBS et la survenue du priapisme, une des complications drépanocytaires.Les travaux menés ont pour objectifs d’étudier les effets délétères potentiels de l’adénosine, à travers l’étude de l’implication des enzymes de la voie métabolique de l’adénosine dans l’hémolyse ainsi que dans la survenue du priapisme chez des patients drépanocytaires. Pour ce faire, une cohorte composée d’adultes et d’enfants SS a été étudiée. Les résultats obtenus ont permis de préciser le rôle du métabolisme de l’adénosine dans les mécanismes physiopathologiques de la drépanocytose. Nos résultats n’ont pas permis de mettre en évidence de différence d’expression (ARN, protéine) des enzymes du métabolisme de l’adénosine entre les patients présentant des antécédents priapiques et ceux indemnes de cette complication. Néanmoins, nos recherches ont permis d’identifier l’adénylate cyclasse 6 comme gène modulateur de l’hémolyse et d’apporter de nouveaux éléments en accord avec la classification du priapisme comme appartenant au sous-phénotype hyperhémolytique à travers notamment l’étude des caractéristiques hemorhéologiques
Sickle-cell disease is caused by a mutation in the β-globin gene leading to an abnormal hemoglobin, hbs. This change allows polymerization of HBS when deoxygenated. Erythrocytes become more rigid and fragile, leading to the two major manifestations of the disease: hemolysis and vaso-occlusion.Recently, adenosine has been identified as a new molecular actor of this disease. This nucleoside may have beneficial effects by preventing inkt cells activation and pulmonary inflammation. But it may also exhibit deleterious effects by activing a signalling pathway leading to erythrocyte sickling and the occurrence of priapism, a sickle cell disease complication.The purpose of our work, based on the potential deleterious effects induced by adenosine was to precise the involvement of adenosine metabolic pathway enzymes in hemolysis and the occurrence of priapism. Two cohorts of children and adult ss patients and men ss have been studied respectively.Our results had clarified the role of metabolism of adenosine in the pathophysiological mechanisms of sickle cell disease. Our results did not allow detecting evidence of differential expression (rna, protein levels) of adenosine metabolism enzymes between ss adult patients exhibiting priapic events and those who had never experienced this complication. Nevertheless, our work has led to the identification of adenylate cyclase as modifier gene of hemolysis and has bring new elements on the priapism classification to the hyper hemolytic sub-phenotype with the description of the hemorheological features associated with this complication

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Menegazzo, Ana Barbara Bordignon Rodrigues [UNESP]. "Determinação do valor da heptoglobina sérica para diagnóstico de hemólise na síndrome HELLP." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/113896.

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Introdução: A síndrome HELLP é uma complicação severa da pré-eclâmpsia, caracterizada por hemólise, elevação das enzimas hepáticas e trombocitopenia. Apesar de haver padronização dos valores laboratoriais que definem a síndrome HELLP, ainda existe dificuldade para a caracterização da hemólise. OBJETIVO: Avaliar o valor de haptoglobina que determina a hemólise nas pacientes com síndrome HELLP. MÉTODOS: estudo transversal e prospectivo de gestantes e puérperas com de pré-eclâmpsia. Exame laboratorial avaliado: dosagem sérica de haptoglobina. Construção da curva ROC para determinar o valor de corte da haptoglobina para diagnóstico de hemólise na síndrome HELLP. RESULTADOS: O valor da haptoglobina para diagnóstico de hemólise em pacientes com síndrome HELLP foi de 0,26g/L. DISCUSSÃO: A melhor correlação observada foi a haptoglobina com a DHL, indicando que este é o melhor marcador de hemólise intravascular para o diagnóstico da síndrome HELLP. CONCLUSÃO: A dosagem sérica da haptoglobina nos casos de pré-eclâmpsia deve fazer parte dos exames de rotina para diagnóstico de hemólise intravascular da síndrome HELLP
Context: HELLP syndrome is a severe complication of pre-eclampsia, caracterized by hemolysis, elevated liver enzymes and low platelet count. Although there are standardized laboratory values that define the HELLP syndrome, the difficulty still exists for the characterization of hemolysis. PURPOSE: to evaluate the haptoglobin value to diagnose the hemolysis in HELLP syndrome. METHODS: transversal and prospective study of pregnant and postdelivery women with pre-eclampsia. Laboratory tests evaluated: serum haptoglobin. ROC curve to determine the cutoff value of haptoglobin in the diagnosis of hemolysis in HELLP syndrome. RESULTS: The haptoglobin value for hemolysis diagnosis in HELLP syndrome was 0.26 g / L. DISCUSSION: The best correlation was with haptoglobin and DHL, indicating that this is the best marker of intravascular hemolysis for the diagnosis of HELLP syndrome. CONCLUSION: Serum haptoglobin in cases of pre-eclampsia should be part of routine tests for diagnosis of intravascular hemolysis in HELLP syndrome

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Thome, Jacqueline Darc Silva. "Citotoxinas e hemolisinas produzidas por Campylobacter jejuni isolados de diferentes origens." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317312.

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Orientador: Tomomassa Yano
Dissertação (mestrado) - Universidade Estadual de Campinas,. Instituto de Biologia
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Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular

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Coussios, Constantin-Cassios. "Monitoring of hemolysis by acoustic scattering." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620292.

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Russell, Laura René. "Treponema hyodysenteriae: growth and production of hemolysin." Thesis, Virginia Tech, 1988. http://hdl.handle.net/10919/45180.

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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

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Archibong, Edikan. "Optofluidic Spectroscopy Platform for Detection of Hemolysis." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5902.

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In the United States alone, hundreds of millions of blood tests are performed annually, and a significant number of those tests are compromised due to hemolysis: e.g., 31% compromised in emergency rooms (inpatient) and 10% at blood banks, clinics, and other outpatient venues. Currently there is no way to reliably detect hemolysis without plasma separation. As a result, significant delays ensue, potentially negatively affecting patient diagnosis and treatment. In addition to in vitro hemolysis, which compromises the quality of blood tests, hemolysis can also occur in vivo. The in vivo occurrence of hemolysis is an indication of life-threatening complications. Being able to detect early signs of in vivo hemolysis would significantly improve outcomes for many patients, including pregnant women affected by HELLP (Hemolysis, Elevated Liver Enzymes, Low Platelet counts) syndrome. Therefore, there is a critical need to be able to detect hemolysis near the patient, immediately following the collecting of blood sample. The goal of this research is to provide an alternative to the traditional testing of blood samples, which requires large volumes of blood, centrifugation, and bulky instrumentation. The proposed alternative hemolysis detection system is a simple miniature setup that produces test results in minutes. This miniature, near-patient sensor would improve patients’ diagnosis, treatments, general satisfaction, and overall experience. The potential reduction of healthcare costs associated with hemolysis would be another significant benefit. The technology demonstrated in this dissertation is based on a novel combination of microfluidics, spectroscopy, and optical-fiber sensing. The microfluidics provide the capability to handle small volumes of liquid and to filter particles from solution. Novel membrane fabrication and modular integration provides the means to characterize and culture the captured particles. Spectroscopy and optical fibers provide the means to characterize the filtrate. These capabilities can be used for not only the detection of hemolysis but also other biomedical applications. . The first step in detecting hemolysis is to separate blood cells and other unwanted particulates from the plasma needed for optical analysis of concentration of hemoglobin. To that end, we focused initially on the problem of particle separation—specifically, within a microfabricated chamber with a custom-designed transparent membrane. To create a miniature microfluidic system capable of processing microliter blood samples, microelectromechanical systems (MEMS) fabrication techniques were required. The fabrication process included steps such as low-stress vapor deposition, photolithography, plasma, and wet etching. The resulting microdevice proved capable of filtering a variety of biological test fluids, including human lung fibroblast cancer cells from medium. The transparent membrane also allows for spectroscopic studies in broader applications, such as spectroscopic analysis or culturing of the cells retained on the filter. These capabilities were demonstrated using microbeads and cancer cells in solution. Optical techniques are used to analyze the separated blood plasma for concentration of hemoglobin. To integrate spectroscopic capabilities with the above microfluidics system, an optical fiber–based miniature probe was attached to the microfabricated chamber. As proof of concept, this system was tested in an application that required the measurement of physiologically relevant concentrations of cobalamin (vitamin B12). This application was used to address human error in drug administration showing measurements of cobalamin concentration as an example drug that can be monitored. The clinical means range of concentrations is from 1 µg/ml to 1000 µg/ml. The achieved results showed measurements of concentrations between 1 µg /mL to 5 mg/mL to monitor the physiological range and potential overdose in microliter of volume. This device has potential for numerous applications, ranging from single cell spectroscopy to measurements of glucose concentrations. This integrated system was then applied to the detection of hemolysis. The complete system conducts optofluidic spectroscopy with the optical fiber probe connected to the microfabricated chamber, which locally filters out blood cells, and reliably determine amount of free hemoglobin with the need for centrifuging. The utility of the device was demonstrated by its accurate measurement of hemoglobin concentrations in blood plasma. Finally, to apply the concept of the detection system to clinical condition with a reliable, and low-cost system, especially useful for developing countries, a smartphone-based technology, is proposed. This technology delivers ultra-fast results for the detection of early signs of HELLP syndrome and preeclampsia with the goal to decrease mortality and morbidity. The smartphone-based diagnostics is low cost, high speed of operation together with high accuracy. Detection of 1 mg/dL of free hemoglobin was achieved which is comparable to gold standard assay which are time consuming, difficult to operate and expensive. This technology, in summary, integrates microfluidics with microfiltration and spectroscopic technology to conveniently separate and characterize blood plasma. The device can also provide important information about other complex biological samples. These measurements require only very small sample volumes.

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Hyland, Caroline Mair Clark. "Activation and membrane insertion of Escherichia coli hemolysin." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620193.

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Gomez, Lopez Arley. "Phopholipase c and hemolysis in non-tuberculous mycobacteria." Doctoral thesis, Universite Libre de Bruxelles, 2000. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211690.

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14

DRAI, LAURENT. "Formes compliquees de l'hepatite a." Aix-Marseille 2, 1992. http://www.theses.fr/1992AIX20206.

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15

Krzyzaniak, Joseph Frances 1968. "A new in vitro method for evaluating intravascular hemolysis." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/288713.

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The primary focus of this research is to develop an in vitro method that uses an appropriate formulation:blood ratio and contact time to evaluate the degree of hemolysis occurring after an intravenous injection. The effects of both the formulation composition and the formulation:blood contact time on hemolysis are given in Chapters I-IV. Since hemolysis is shown to increase as either the formulation:blood ratio and/or the contact time increases for various pharmaceutical vehicles, a small formulation:blood ratio and short contact time must be selected to determine the degree of hemolysis occurring as the formulation is rapidly diluted by the blood after an intravenous injection. Using a formulation:blood ratio of 0.1 and a contact time of 1 second, a dynamic method has been developed to evaluate intravascular hemolysis. The ability of this method to accurately evaluate hemolysis occurring after an injection is determined by comparing hemolysis data generated with this dynamic method to in vivo hemolysis data obtained from the literature. The results of this comparison are given in chapter V. The in vitro hemolysis data are shown to be in agreement with in vivo hemolysis data.

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Hammerstein, Anne Friederike. "Single-molecule chemistry studies with engineered alpha-hemolysin pores." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:1dd1f11d-2b20-42e9-9dfc-c30498822b77.

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Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. Chapter 1 provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. Chapter 2 describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. Chapter 3 investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn²⁺ ion. The single channel recordings reveal a two step process, in which the Zn²⁺ ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. Chapter 4 demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. Chapter 5 explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]²⁺ binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.

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Ivica, Josip. "α-Hemolysin nanopore sensing of MicroRNA with electrolyte gradients." Thesis, University of Southampton, 2018. https://eprints.soton.ac.uk/424539/.

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Aberrant microRNA expression profiles have been correlated with a range of complex diseases, including specific types of cancer, hence microRNA species are a promising class of molecular cancer biomarkers. Recently, nanopore technology was proposes as a single-molecule methodology to detect and quantify microRNA molecules without amplification or fluorescent labelling. A duplex of microRNA hybridized with a complementary DNA probe is electrophoretically driven to a nanopore, which can be translocated following duplex unzipping at the channel entrance, which is measured as a transient decrease in nanopore electrical current. Nanopore sensing sensitivity is determined by the occurrence frequency of such resistive current pulses, while the specificity is determined by the probe-analyte interaction. This thesis aims to establish the optimal conditions and limitations of nanopore sensing of cancer-related microRNA species with the biological nanopore α-hemolysin as the sensor element. The fragility of aperture-suspended lipid bilayers is one of the main obstacles for sensing with biological pores, hence we first addressed bilayer stability by laser cutting a thin Teflon film to obtain apertures with a tapered wall profile. Nanopore sensing was then investigated with synthetic miRNA 155, overexpressed in lung cancer, in the presence of a complementary DNA probe. Key parameters of duplex nanopore translocation in conventional symmetrical 1 M KC1 were in agreement with previous work, including a relatively low pulse frequency, allowing quantification of miRNA 155 down to 10 nM. We then systematically investigated the effect of cis/trans KC1 gradients across the nanopore. The resistive pulse frequency increased significantly with the salt gradient, indicative of cation-induced filed enhancement at the pore entrance, but bilayer and pore stability were reduced. At a 0.5 / 4 M gradient, the pulse frequency was ~60 times higher than for symmetrical 1 M KC1 conditions, enabling miRNA quantification down to 100 pM. Additionally, experiments with DNA probes with single and double polynucleotide extensions confirmed the necessity of a double-overhang design under salt gradient conditions, while experiments with NaC1, CsC1 and LiC1 electrolyte gradients suggested that Li addition can extend the duplex unzipping time. Finally, trials were performed with total RNA extracts from clinical samples. Here, bilayer stability was no limitation but pore clogging precluded nanopore sensing, most likely due to longer mRNA species with secondary structure, necessitating further extract processing. Another consideration for nanopore analysis of microRNAs from clinical samples is to minimize the extract resuspension volume, implying the use of miniaturized bilayer recording methodologies.

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Dancho, David M. "Analysis of Polyethylene Glycol in the α-Hemolysin Nanopore." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/483.

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Nanopores have been shown to be a useful analytical tool for single molecule detection. They have been used to study the composition of DNA and other molecules of interest. These pores are usually α-hemolysin which is a toxin from Staphylococcus aureus or more recently nanoscale synthetic solid state pores. Now we are beginning to look at other molecules or proteins by sending them into the nanopores and measuring a characteristic partial current blockade. In this thesis we look at polyethylene glycol (PEG) as it enters and blocks current through a single alpha hemolysin pore. We report the effects of ionic strength, PEG size, and applied voltage on the depth and duration of the current blockades. We also apply autocorrelation analysis on the arrival times of PEG molecules to the pore see if we can identify if the PEG is translocating through the pore or escaping from the same side it enters. This suggests a new approach to current blockade analysis.

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Nguyen, Trong Tin 1979. "Development of blood analogs for flow visualization and hemolysis investigations." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82624.

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The experimental investigation of blood flow using in vitro models is very important to derive relevant and quantitative information in order to understand the biomechanical conditions resulting from blood flow dynamics. Furthermore, such studies can help us analyze and evaluate flow disturbances caused by pathologies and by cardiovascular prostheses such as aortic valves and left ventricular assist devices (LVAD). For example, these disturbances can range from high shear flow regions causing blood hemolysis to low shear regions resulting in plaque deposition. Flow visualization studies represent a family of methods to study fluid flow through cardiovascular hydraulic models using blood analogs. Thus, for these analogs, two of the main fluid mechanical properties to be reproduced are the bulk kinematic viscosity and shear-thinning of blood. In a first approach, a blood model is developed to simultaneously match the kinematic viscosity of blood and the refractive index of the material of the cardiovascular model for flow visualization experiments. In a second approach, the development of a non-Newtonian blood analog is presented to match the shear-thinning behavior of blood. This approach also allows the development of a blood analog with hemolytic properties. (Abstract shortened by UMI.)

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Martin, H. S. C. "Molecular dynamics simulations of nucleotide translocation through α-hemolysin nanopores." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302280/.

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The translocation of polynucleotides through transmembrane protein pores is a fundamental biological process with important medical and biotechnological relevance. The complex translocation process is influenced by a range of factors including the diameter and inner surface of the pore, the secondary structure of the polymer, and the interactions between the polymer and protein. Computer simulations are an invaluable means to investigate microscopic systems and thereby provide a unique, atomistic perspective of important states and processes. This thesis explores how two molecular dynamics methodologies can simulate the translocation of nucleotides through the nanopore α-hemolysin. In the first methodology, non-equilibrium constant velocity-steered simulations are combined with Jarzynski's identity to derive the free energy profiles for the passage of a polynucleotide molecule through the pore. In the second methodology, the free energy profiles are calculated from a biasing force which varies in response to energy barriers encountered during the simulation. Both approaches are used to explain the experimentally observed differences in translocation time through the nanopore between polyadenosine and polydeoxycytidine. In addition to polynucleotides, the study also investigates single nucleotide translocation. Together, the simulations highlight the role of molecular interactions between the nucleic acid molecules and the protein pore. In particular, we find that specific residues of the protein pore dominate the translocation. The unique data set helps assess two methodologies to simulate a system of considerable size and complexity.

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Bailey, Marc J. A. "HlyT, a transcriptional regulator of the Escherichai coli hemolysin operon." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259542.

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22

Zydney, Andrew Lawrence. "Cross-flow membrane plasmapheresis : an analysis of flux and hemolysis." Thesis, Massachusetts Institute of Technology, 1985. http://hdl.handle.net/1721.1/15235.

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23

Choi, Lai-Sheung. "Single-molecule chemistry studied using the protein pore -α-hemolysin." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:d9f32132-a6ae-4d49-9649-7bf9cd4b0dd2.

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Single-molecule detection has provided insights into how molecules behave. Without the averaging effect of ensemble measurements, the stochastic behaviour of single molecules can be observed and intermediate steps in multistep transformations can be clearly detected. The single-molecule reactants range from small molecules (e.g. propene) to proteins of several tens of kDa (e.g. myosin). One single-molecule detection technique is single-channel electrical recording. This approach is based on the measurement of the transmembrane ionic current flowing through a nanoscale transmembrane pore under an applied potential. In this thesis, the protein α-hemolysin was employed as a nanoreactor. α-Hemolysin is a toxin secreted by Staphylococcus aureus. Its transmembrane pore (~100 Å in length and ≥14 Å in diameter) allows ions, water and small molecules to pass through its lumen. Under an applied potential, chemical changes in reactants attached to the internal wall of the pore modulate the flow of ions, leading to changes in the transmembrane ionic current. Analysis of this current provides information about the reaction kinetics and mechanisms. Chapter 1 – Single-Molecule Chemistry and α-Hemolysin is an introductory chapter that is divided into two parts. Section 1.1 provides an overview of the different techniques for the detection of chemical reactions at the single-molecule level. Section 1.2 gives a brief review of the protein pore α-hemolysin, including its structure, properties and various applications. Chapter 2 – S-Nitrosothiol Chemistry applies cysteine-containing α-hemolysins to study the biologically relevant chemistry of S-nitrosothiols (RSNO). RSNO are important molecules involved in cell signalling, which control physiological processes such as vasodilation and bronchodilation. Three reactions, namely transnitrosation (the transfer of the ‘NO’ group from RSNO to a thiol), S-thiolation (the formation of a disulfide from RSNO and thiol) and S-sulfonation (the generation of an S-sulfonate (RSSO₃⁻) from RSNO and sulfite ion), were investigated at the single-molecule level. The pH-dependency of the two competing reactions (transnitrosation and S-thiolation), the lifetime of the proposed transnitrosation intermediate, and nature of the chemical reaction between RSNO and sulfite (a bronchoconstrictor) were determined. Chapter 3 – Silver(I)-thiolate and cadmium(II)-thiolate complexes describes the kinetics of the formation and breakdown of these two metal-thiolate complexes. Ag⁺ and Cd²⁺ are commonly used in probing the membrane topology and gating properties of ion channels using the scanning cysteine accessibility method (SCAM). The binding of two Ag⁺ ions per thiol group and the stepwise build-up and dissociation of Cd²⁺-glutathione complexes were unambiguously characterized. Chapter 4 – Copper(II)-Catalyzed Diels-Alder Reactions reports the attempt to carry out copper(II)-catalyzed Diels-Alder reactions inside an engineered α-hemolysin. An iminodiacetate ligand was covalently attached within the lumen of the α-hemolysin pore. This ligand chelates Cu²⁺ ion, which can bind bidentate dienophiles and activate them towards Diels-Alder reaction with dienes. However, due to the ‘slow’ reaction rate of the Diels-Alder reaction (rate constant ~10⁻¹ M⁻¹s⁻) relative to the time-scale of the single-molecule experiment, we failed to observed chemical conversion at the single-molecule level. Nevertheless, the engineered metal-binding α-hemolysin may be useful for sensing molecules bearing metal-coordinating groups.

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Nieuwoudt, Stephnie. "Preparation, stability and in vitro evaluation of liposomes containing chloroquine / Stephnie Nieuwoudt." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4740.

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Malaria is currently a huge treat worldwide, as far as infections are concerned, and is responsible for thousands of deaths per annum. The dilemma associated with the development of anti–malarial drug resistance over the past few decades should be addressed as a matter of urgency. Novel drug delivery systems should be developed in order to employ new and existing anti–malarial drugs in the treatment and management of malaria. The aim of these delivery systems should include an improvement in the efficacy, specificity, acceptability and therapeutic index of anti–malarial drugs. Previous studies have suggested that liposomes have the ability to encapsulate, protect and to promote the sustained release of anti–malarial drugs. Two liposome formulations, namely liposomes and chloroquine entrapped in liposomes, were formulated during this thesis and evaluated by conducting a stability study and an in vitro study with the main focus on cell viability. The stability study consisted of a series of stability tests regarding the stability of nine liposome and nine chloroquine entrapped in liposome formulations over a period of twelve weeks. The in vitro study included three assays such as a reactive oxygen species assay, a lipid peroxidation assay and a hemolysis assay. The aims of these studies included the manufacturing of liposomes, the incorporation of chloroquine into liposomes, the determination of the stability of the formulations as well as the evaluation of the possible in vitro toxicity of liposomes. Results obtained from these studies revealed that liposomes remained more stable over the stability study period in comparison to chloroquine entrapped in liposomes. The entrapment of chloroquine within liposomes was possible, although the initial entrapment efficiency (%) of 14.55 % was much too low. The production of reactive oxygen species occurred to a small extent in the red blood cells and the infected red blood cells. Equal amounts of reactive oxygen species (%) was observed within both the red blood cells and the infected red blood cells with a maximum value of 23.27 % in the presence of the chloroquine entrapped in liposomes at varying concentrations. Red blood cells experienced the highest degree of lipid peroxidation (%) in the presence of chloroquine, at varying concentrations, entrapped in liposomes. The maximum amount of lipid peroxidation (%) was 79.61 %. No significant degree of hemolysis (%) was observed in the red blood cells neither in the presence of the liposomes nor in the presence of the chloroquine entrapped in liposomes at varying concentrations. It can be concluded that liposomes are a more stable formulation and have less toxic effects on red blood cells and infected red blood cells in comparison to the chloroquine entrapped in liposome formulations. Future studies should investigate the possibility of a more stable and less toxic chloroquine entrapped in liposome formulation.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

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Nakaguchi, Yoshitsugu. "The urease gene cluster of Vibrio parahaemolyticus does not influence the expression of the thermostable direct hemolysin (TDH) gene or the TDH-related hemolysin gene." Kyoto University, 2003. http://hdl.handle.net/2433/148733.

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26

Periodismo, Alumnos de la Escuela de Comunicación y., and Universidad Peruana de Ciencias Aplicadas (UPC). "Hemos Perdido La Fe En Ellas." Universidad Peruana de Ciencias Aplicadas (UPC), 2015. http://hdl.handle.net/10757/345922.

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PARENT, LEPARMENTIER MAUD. "Dosage de l'activite hemolytique du composant c4 du complement humain par methode cinetique : contribution a l'etude des deficits genetiques en c4." Strasbourg 1, 1995. http://www.theses.fr/1995STR15102.

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Peeling, Peter Daniel. "Exercise induced hemolysis, inflammation and hepcidin activity in endurance trained runners." University of Western Australia. School of Sport Science, Exercise and Health, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0122.

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[Truncated abstract] Iron is a trace mineral used by the body in many physiological processes that are essential to athletic performance. Commonly, the body's iron stores are compromised by exercise via several well established mechanisms. One such mechanism is the destruction of red blood cells (hemolysis), in response to the mechanical stress and circulatory strain of exercise. Although it appears that a force-dependent relationship between the heel-strike of the running gait and ground contact exists, the effects of the intensity trained at and the ground surface type trained upon have not been documented. Similarly, the effects of a cumulative training stress (i.e. multiple daily sessions) has not been examined. In addition to hemolysis, exercise also invokes an inflammatory response that results in an up-regulation of the cytokine interleukin-6 (IL-6). This cytokine is the primary mediator of hepcidin expression, a liver-produced hormone that regulates iron metabolism in the gut and in macrophages. The influence of exercise on hepcidin expression is relatively unknown, and as such it is possible that this hormone may be a mitigating factor implicated in athletic-induced iron deficiency. Therefore, the purpose of this thesis was to investigate the effect of different training frequencies, intensities and ground surfaces on the hemolytic response. In addition, the impact of exercise-induced inflammation on hepcidin expression in the 24 h post-exercise was investigated, with the aim of determining whether this hormone may be a potential new mechanism associated with athletic-induced iron deficiency. Finally, an interaction between hemolysis and hepcidin activity was examined to investigate their potential combined effect on iron status in the 24 h post-exercise. ... Venous blood and urine samples were collected pre- and immediately post-exercise, and at 3 and 24 h of recovery. Samples were analysed for circulating levels of IL-6, free Hb, Hp, serum iron, ferritin and urinary hepcidin activity. At the conclusion of both the T1 and T2 interval runs, the free Hb and serum Hp were significantly increased (p<0.05) from pre-exercise levels. Furthermore, a cumulative effect of two running sessions was shown in the T2 trial, via a further significant fall in serum Hp. The IL-6 and hepcidin activity were significantly increased after each running session (p<0.05) with no cumulative effect seen. Serum iron and ferritin were significantly increased post-exercise after each interval run (p<0.05), but were not influenced by the addition of a prior LSD run 12 h earlier. As a result, this investigation showed a cumulative effect of consecutive training sessions on RBC destruction in male athletes. Furthermore, post-exercise increases to serum iron and hepcidin, and their interaction was suggested to have potential implications for an athlete's iron status. Overall, the findings of this thesis show that hemolysis is evident at the conclusion of endurance running, and is influenced by training intensity and frequency. The results enabled a time-line for hepcidin expression post-exercise to be established, and the implications of increases to the activity of this hormone, in association with the hemolytic changes seen with endurance exercise are discussed.

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MacDonald, Leslie Anne. "Antigenic relationship of enterohemorrhagic Escherichia coli hemolysin to other RTX toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ56344.pdf.

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Thompson, James Russell. "Imaging the assembly of the Staphylococcal pore-forming toxin alpha-Hemolysin." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:e320004a-6118-4dac-af2a-eca6e90be7ac.

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Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

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Malheiros, Sonia Valeria Pinheiro. "Contribuição do sistema microssomal hepatico na hemolise induzida por trifluoperazine (TFP)." [s.n.], 1995. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314590.

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Orientador: Nilce Correa Meirelles
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-07-20T00:10:31Z (GMT). No. of bitstreams: 1 Malheiros_SoniaValeriaPinheiro_M.pdf: 2861416 bytes, checksum: de7c425fc1049e8d786b3ab7fff79dfd (MD5) Previous issue date: 1995
Resumo: As drogas fenotiazínicas são amplamente utilizadas como neurolépticos e antipsicóticos. A trifluoperazina (TFP), um derivado fenotiazínico, dependendo da concentração, pode induzir hemólise ou proteger eritrócitos da lise em condição isosmótica. Nesse trabalho verificamos a interferência do sistema microssomal hepático (SMH), que é o principal responsável pela biotransformação de drogas nos organismos vivos, no efeito hemolítico causado pela TFP. O efeito hemolítico da TFP é abolido em presença de microssomas de fígado de rato (SMH), o que se deve em parte à biotransformação dirigida pelas enzimas P-450 microssomais e em parte à partição da droga na membrana microssomal, diminuindo a concentração livre da droga. Foi detectado um sobrenadante pós-microssomal (SPM) que mantinha a mesma característica de proteção hemolítica apresentada pelo SMH. Para essa fração solúvel (SPM) de composição proteica 48% e ribonucleica 24 % foi feita uma caracterização bioquímica, constituída basicamente de: determinação do pH, condutividade, caracterização dos espectros de absorção óptica e de ressonância magnética nuclear de prótons (lH-RMN), além da análise cromatográfica por HPLC e determinação da massa molecular aparente por eletroforese. Para todas as nove frações de SPM obtidas na separação cromatográfica, cujos pesos moleculares aparentes variam de 12 a 160 kDa, foi observada proteção tanto contra hemólise induzi da pela TFP quanto contra a hemólise mecânica
Abstract: Phenothiazine drugs are mainly used as neuroleptics and antipsychotic agents. Trifluoperazine (TFP), a phenothiazine derivative, produces either hemolysis or protection of erythrocytes under isosmotic conditions in a dose-dependent manner. In these work we verify the interference of mouse liver microsomes (MLM), which are the main responsible for drug biotransformation in organisms, in the hemolytic effect caused by TFP. The hemolytic effect induced by TFP is abolished in the presence of MLM which is due, in part, to microsomal cytochrome P-450 driven biotransformation and a TFP partition into the microsomal membrane that decreases the free concentration of the drug. A post microsomal supematant (PMS) composed of proteins (48 %) and ribonucleic acid (24 %) has been found to exhibit the same protection against hemolysis as MLM. In this work, we showed the PMS biochemical characterization that consisted of: pH and conductivity determination, optical and nuclear magnetic resonance (NMR) spectroscopy, besides cromatographyc and electrophoretic analysis. The nine fractions obtained from PMS by cromatographic separation, which molecular weight was between 12 and 160 kDa, protected erythrocytes against mechanical and TFP induced hemolysis
Mestrado
Bioquimica
Mestre em Ciências Biológicas

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Aviat, Florence. "Étude d'une protéine de leptospires : Hemolysis Associated Protein 1 : Hap 1." Nantes, 2006. http://www.theses.fr/2006NANT2012.

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La leptospirose est une zoonose de représentation mondiale concernant la plupart des Mammifères dont l'homme. Hap1 a été purifiée dans une fraction protéique de 32 kDa extraite de leptospires, bactéries responsables de la leptospirose. La protéine Hap1 s'est révélée être associée à une protection hétérologue contre la leptospirose chez des gerbilles. Le gène hap1 a été exprimé par E. Coli afin de produire la protéine recombinante rHap1. Ce travail confirme et prolonge les travaux antérieurs : Hap1 est secrétée par les seuls leptospires pathogènes lors de leur multiplication. Très immunogène et protectrice dans les conditions naturelles, cette protéine sous sa forme recombinante ne permet pas de reproduire les activités protectrices dans les conditions naturelles. L'objectif de ce travail était donc de purifier la ou les formes naturelles de la protéine Hap1 afin d'en définir les présentations structurales expliquant cette contradiction
Leptospirosis is a zoonosis with a worldwide distribution concerning most Mammalians among which humans. Hap1 was purified from an antigenic zone with an apparent molecular mass of 32 kDa extracted from leptospires, bacteria responsible for leptospirosis. Immunization with Hap1 expressed by adenovirus or plasmid induced in gerbils a protection against leptospirosis. Then hap1 gene was expressed by E. Coli to product the recombinant protein rHap. This present work confirms these previous data: Hap1 is secreted during the multiplication of only pathogenic leptospires. Very immunogenic and protective in the natural conditions, this protein in the recombinant form doesn't permit to reproduce protective activity in the natural conditions. So, the purpose of this work was to purify one or many Hap1 naturally forms in order to determine the structural difference to understand this contradiction

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33

Mantri, Shiksha. "Engineered α-hemolysin pores with chemically and genetically-fused functional proteins." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:55450bd3-b93f-410f-b795-0110449c0da9.

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Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane  barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.

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34

Roxström-Lindquist, Katarina. "Innate immunity in insects : function and regulation of hemolin from Hyalophora cecropia /." Stockholm : Univ, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-3.

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35

Preté, Paulo Sérgio Castilho. "Solubilização de membranas eritrocitarias : analise quantitativa do efeito hemolitico induzido por surfatantes." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314156.

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Orientador: Eneida de Paula
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Surfatantes ou detergentes são compostos anfifílicos que, na presença de água, têm a característica de formar agregados micelares. Surfatantes induzem a desestruturação de outros agregados como bicamadas sendo, por isso, usados para ruptura celular ou solubilização de lipídios e proteínas de membrana. A capacidade lítica dos surfatantes resulta de sua estrutura química, que determina o modo de interação dos mesmos com as membranas. Em concentrações mais altas (acima da concentração micelar crítica), os surfatantes desestabilizam as bicamadas lipídicas, levando à formação de micelas-mistas. Ensaios hemolíticos são bons modelos para estudo do efeito lítico de surfatantes em biomembranas. Aplicando em eritrócitos humanos o tratamento quantitativo proposto por Lichtenberg (1985) para estudo da solubilização de bicamadas lipídicas mensurou-se, neste trabalho, as concentrações para início (Csat) e 100% de hemólise (Csol), induzidas por 25 surfatantes clássicos, pertencentes a cinco diferentes famílias. A variação dos valores de Csat, Csol determinada com diferentes hematócritos permitiu o cálculo da constante de ligação surfatante/membrana e da razão surfatante/lipídio de membrana (Re) para início e 100% de hemólise. O parâmetro Re foi usado para classificar os detergentes como fortes, médios ou fracos agentes solubilizantes, com boa correlação com dados da literatura o que nos permitiu propor seu uso para descrever o efeito lítico de surfatantes, como uma alternativa simples e aplicável as membranas biológicas. As transições durante o processo hemolítico foram acompanhadas pela técnica de Ressonância Paramagnética Eletrônica, com uso marcador de spin 5 doxil-estearato (incorporado a 1 mol% nas membranas de eritrócito) e lise induzida pelo surfatante não iônico Triton X100. Concomitante ao aparecimento de hemoglobina e fosfato livres no sobrenadante - indicadores da ruptura da membrana, medidas do parâmetro de ordem daquele marcador de spin permitiram estudar as transições que acontecem durante (membrana:membrana mista) e após (membrana mista:micela mista) a hemólise
Abstract: Surfactants or detergents are amphiphilic compounds that form micellar aggregates in the presence of excess water. Surfactants are able to induce disruption of lamellar aggregates, justifying their use for cell lysis or in the extraction of membrane constituents such as lipids and proteins. The lytic capacity of a given surfactant is determined by its chemical structure, that rules its interaction with the membranes. At high concentration (above the critic micelle concentration), surfactants destabilize lipid bilayer leading to mixed micelle formation. Hemolytic assays are a good model to study the lytic effect of surfactants on biomembranes. In this study we have applied to human erythrocytes the quantitative treatment proposed by Lichtenberg (1985) to describe the solubilization of model lipid membranes. The concentration for onset (Csat) and complete (Csol) hemolysis induced by 25 classic surfactants from five different families were measured. Changes in Csat and Csol values at different hematocrits allowed the determination of the surfactant/membrane lipid molar ratio (Re) for beginning and 100% lysis. The Re arameter was used to classify the surfactants as strong, medium or weak membrane solubilizers. The classification was in good correlation with data in the literature, allowing us to recommend the use of Re parameter to describe the lyric effect of surfactants on biomembranes. The transitions in the hemolytic process were accompanied by Electron Paramagnetic Resonance, using the 5-doxyl-stearate spin-probe (1 mol%, incorporated in the erythrocyte membrane) and the non-ionic surfactant Triton X100. Simultaneously to the appearance of hemoglobin and phosphate released in the supernatant, measurements of the order parameter of the spin probe were used to characterize the transitions that take place during (membrane :mixed membrane) and after (mixed: membrane: mixed micelle) hemolysis
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

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36

Jung, Yunhee. "Remodelling the cavity of a transmembrane pore by genetic engineering." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/3880.

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The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Ã (volume ~32,600 Ã3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

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37

Buzin, Marta Pitali. "Atividades enzimaticas e biologicas do extrato de sacos de veneno da vespa social Polistes lanio lanio." [s.n.], 1999. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313645.

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Orientador: Stephen Hyslo
Texto em portugues e ingles
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Os venenos de insetos da Classe Hymenoptera contêm enzimas, peptídeos, aminas e aminoácidos excitatórios. Neste trabalho, investigamos as atividades enzimáticas e biológicas de um extrato aquoso do saco de veneno (ESV) da vespa social Po/istes lanio lanio. O ESV de P. l. lanio possui baixa atividade fosfodiesterásica, elastásica, fosfatásica (ácida e alcalina) e L-amino-ácido oxidase; moderados níveis de hialuronidase e protease e um alto nível de fosfolipase. A eletroforese (SDS-P AGE) revelou a presença de mais de 10 componentes com pesos moleculares variando de 14.000-100.000. A cromatografia de ESV em Sephacryl S200 HR resultou em sete picos, o primeiro destes contém atividades hemolítica e fosfolipásica. O ESV exibiu uma potente atividade hemolítica direta em eritrócitos humanos, sendo dose (12,5-200 ~g/ml) e tempo (até 5 h) dependente (n=3). Eritrócitos humanos e de cão não foram aglutinados pelo ESV (2,5 mg/ml; n=3). Comparado ao ADP (3 lJM), ESV (50-400 ~g/ml) produziu uma agregação dose¬ dependente fraca em plasma humano rico em plaquetas (n=3). Por outro lado, ESV (400 ~g/ml) não agregou plaquetas humanas lavadas, porém desencadeou agregação por doses não agregantes de trombina (n=3). Em íleo isolado de rato, o ESV produziu contrações dose-dependente (4-16 ~g/ml), as quais não foram afetadas pelo pré-tratamento do tecido com atropina (antagonista de receptor muscarínico, 0,6 ~g/ml) ou ciproheptadina (antagonista de histamina e serotonina, 0,2 ~g/ml), mas foram completamentes abolidas pelo HOE-140 (1 ~g/ml, n=3), antagonista do receptor B2 da bradicinina, sugerindo a presença de cininas no ESV. Estes resultados indicam que as atividades enzimáticas e biológicas do ESV de P. l. lanio são similares às de outras vespas sociais
Abstract: Hymenopteran venoms contain enzymes, peptides, amines and excitatory amino acids. We have examined the enzymatic and biological activities of an aqueous venom sac extract (VSE) ITom the Brazilian social wasp Po/istes lanio lanio. The VSE had low phosphodiesterase, elastase, acid and alkaline phosphatase and L-amino acid oxidase activities, moderate levels of hyaluronidase and protease and high phospholipase activity. SDS-P AGE revealed the presence of several components with molecular weights of 14,000-100,000. Gel filtration ofVSE resulted in seven peaks, the first ofwhich contained the hemolytic and phospholipase activities. The VSE exhibited potent direct hemolytic activity on human erythrocytes which was dose- (12.5-200 ~g!ml) and time- (up to 5 h) dependent. VSE (50-400 ~g!ml) produced weak, dose-dependent aggregation of human platelet-rich plasma and did not aggregate human washed platelets (400 ~g!ml), but potentiated the responses to non-aggregatory doses of thrombin. Dog and human erythrocytes were not agglutinated by VSE (2.5 mg!ml). In the rat isolated ileum, VSE (4-16 ~g!ml) produced contractions which were not prevented by atropine (muscarinic receptor antagonist, 0.6 ~g!ml) or cyproheptadine (histamine and serotonin receptor antagonist, 0.2 ~g!ml), but were abolished by the bradykinin B2 receptor antagonist HOE¬140 (1 ~g/ml), suggesting the presence of kinins. These results indicate that the composition of P. 1. lanio VSE is similar to that of other wasp species
Mestrado
Farmacologia
Mestre em Ciências Médicas

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38

YU, Hai [Verfasser], and Dominique [Akademischer Betreuer] Thevenin. "Flow design optimization of blood pumps considering hemolysis / Hai Yu. Betreuer: Dominique Thévenin." Magdeburg : Universitätsbibliothek, 2015. http://d-nb.info/1072685698/34.

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39

Thom, David Charles. "The hemolysis and cytotoxicity of a zeolite-containing root filling material in vitro." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ63009.pdf.

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40

Assefaw-Redda, Yohannes. "Hemolin expression during Cecropia development and its effect on malaria parasites." Doctoral thesis, Stockholm : Institutionen för genetik, mikrobiologi och toxikologi, Stockholms universitet, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-482.

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41

Bannan, Jason David. "The cloning and characterization of a Bordetella pertussis gene encoding a putative hemolysin." Diss., The University of Arizona, 1992. http://hdl.handle.net/10150/185908.

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Bordetella pertussis, the etiologic agent of whooping cough or pertussis, produces a multitude of virulence factors including a hemolysin. Virulent phase B. pertussis isolates are hemolytic, whereas avirulent isolates are not. Other investigations concerning B. pertussis adenylate cyclase toxin indicate it has hemolytic activity and is a member of the bacterial RTX toxin family. In an attempt to further characterize hemolysis by B. pertussis, a new B. pertussis gene was isolated which conferred a hemolytic phenotype on non-hemolytic E. coli. DNA sequencing of the putative B. pertussis hemolysin gene revealed it encoded a 27 kDa protein similar to HlyX, an FNR-like transcriptional regulator from Actinobacillus pleuropneumonia, which also confers hemolysis upon E. coli. No similarity to bacterial cytolysins was found. The B. pertussis transcriptional regulator-like gene and its encoded protein were named btr and BTR, respectively. BJB1, a BTR deficient B. pertussis strain was constructed. The btr::kan mutation was shown to have no effect on the production, or phenotypic modulation, of hemolysis by B. pertussis. BTR production was not regulated by the BvgA-S two component sensor-regulator. An FNR deficient E. coli, JRG1728 (Δfnr), was transformed with a btr recombinant plasmid pHLY1A. The B. pertussis btr gene complemented the FNR deficient E. coli to grow anaerobically on a non-fermentable carbon source. This suggested that BTR may function as a B. pertussis transcriptional regulator which responds to anoxic conditions.

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42

YU, Hai Verfasser], and Dominique [Akademischer Betreuer] [Thévenin. "Flow design optimization of blood pumps considering hemolysis / Hai Yu. Betreuer: Dominique Thévenin." Magdeburg : Universitätsbibliothek, 2015. http://nbn-resolving.de/urn:nbn:de:gbv:ma9:1-6390.

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43

Brunetta, Denise Menezes. "Prevalence and risk factors for immune hemolysis in patients submitted to liver transplan." Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=18954.

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CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Liver transplant (LT) anemia is multifactorial. Immune hemolysis occurs due to auto-antibodies, drug induced or not, or due to allo-antibodies, formed by transfusion or passenger lymphocyte syndrome (PLS). The aim of this study was to evaluate the prevalence and risk factors for immune hemolysis in LT. Between September 2014 and April 2016, 175 patients submitted to 178 LT were included. Multi-organ recipients were excluded. Samples from before, seven consecutive days and weekly for four weeks were analyzed for complete blood cound, reticulocyte count, lactate dehydrogenase (LDH), indirect bilirrubin (IB) and imummohematological tests. SPSS 24 was used for statistical analysis, p<0.05 was considered significant. The mean age was 52.1 Â 14.6 years old, with 105 male patients (60%). The most frequent causes of cirrhosis were hepatitis C virus (HCV, 59 â 33.7%) and alcohol (44 â 25.1%). Anemia before LT was present in 140 patients (74.2%), with lower hemoglobin (Hb) concentration in those with positive direct antiglobulin test (DAT, p=0.014). Nine patients (5.1%) presented positive antibody screen (AS) before transplant, with 2.3% of clinical significance. This finding was more frequent in RhD negative patients (p=0.017). Positive DAT occurred in 53 patients (30.3%) and was related to high MELD score (p=0,048), HCV (p=0.005) and furosemide use (p=0.001). These patients presented higher levels of IB (p<0.001). Ninety six patients (55%) were transfused in the studied period. One hundred and fourty five patients (87.8%) were still anemic on the fourth week. Twenty two patients (12.5%) presented positive AS after LT, with nine patients (5.7%) presenting clinically significant antibodies. Positive AS occurred more frequently in RhD negative (p=0.021) and in those transfused with red blood cells units (RBCU, p=0.022). Sixteen patients received grafts with minor ABO incompatibility. Post-transplant positive DAT was associated with higher levels of LDH (p=0.006), piperacillin-tazobactam use (p=0.021) and was more frequent in the non identical ABO group (p=0.0038). In this group, five of eleven positive DAT patients presented anti-A (2) or anti-B (3) on the eluate, representing PLS. All PLS patients received liver graft O and were using mycofenolate, tacrolimus and steroids. Four patients presented hemolysis and three were transfused due to PLS. These patients, compared to all the other patients, presented lower Hb concentration (p=0.043) and higher LDH levels (p=0.008) and reticulocyte counts (p=0.008). The presence of auto and allo-antibodies against red blood cell antigens is frequent in LT, but clinical significant hemolysis occurred in only 2.8%. Antibodies are more frequent in patients with higher MELD scores, with HCV, in use of pre-transplant furosemide, in those transfused patients with RBCU, RhD negative and piperacillin-tazobactam use after LT. The only risk factor for PLS is minor ABO mismatch between donor and recipient.
Anemia no transplante hepÃtico (TH) Ã multifatorial. HemÃlise imune ocorre por autoanticorpos, com ou sem relaÃÃo com drogas, ou aloanticorpos, formados por transfusÃo ou sÃndrome do linfÃcito passageiro (SLP). O objetivo deste estudo foi avaliar a prevalÃncia e fatores de risco para hemÃlise imune no TH. Foram incluÃdos, entre setembro de 2014 e abril de 2016,175 pacientes submetidos a 178 TH, sendo excluÃdos transplantes de mÃltiplos ÃrgÃos. Amostras prÃ-TH, de 7 dias consecutivos e semanalmente atÃ 4 semanas foram avaliadas com hemograma, reticulÃcitos, lactato desidrogenase (LDH), bilirrubina indireta (BI) e testes imuno-hematolÃgicos. SPSS 24 foi usado para estatÃstica, com p<0,05 significante. A idade mÃdia foi de 52,1 Â 14,6 anos, com 105 homens (60%). As etiologias mais frequentes da cirrose foram vÃrus da hepatite C (VHC, 59 - 33,7%) e Ãlcool (44 - 25,1%). Anemia prÃ-transplante estava presente em 140 pacientes (74,2%), com menores concentraÃÃes de hemoglobina (Hb) naqueles com teste direto da antiglobulina (TAD) positivo (p=0,014). Nove pacientes (5,1%) apresentaram pesquisa de anticorpos irregulares (PAI) positiva prÃ-TH, sendo 2,3% clinicamente significantes. Esse achado foi mais frequente em RhD negativo (p=0,017). TAD positivo prÃ-TH ocorreu em 53 pacientes (30,3%), com relaÃÃo com escore MELD elevado (p=0,048), VHC (p=0,005) e uso de furosemida (p=0,001). Esses pacientes apresentaram BI mais elevada (p<0,001). Noventa e seis pacientes (55%) receberam hemocomponentes no perÃodo estudo. Cento e quarenta e cinco pacientes (87,8%) ainda estavam anÃmicos na 4a semana. Vinte e dois pacientes (12,5%) apresentaram PAI positiva pÃs-TH, sendo nove pacientes (5,7%) com anticorpos clinicamente significantes. PAI positiva foi mais frequente em RhD negativo (p=0,021) e nos transfundidos com concentrado de hemÃcias (CH - p=0,022). Dezesseis pacientes receberam enxerto ABO nÃo idÃntico. TAD positivo pÃs-TH esteve associado a aumento de LDH (p=0,006), uso de piperacilina-tazobactam (p=0,021) e foi mais frequente no grupo ABO nÃo idÃntico (p=0,0038). Nesse grupo, cinco dos 11 com TAD positivo apresentaram eluato com anti-A (02) ou anti-B (03), configurando SLP. Todos receberam fÃgado O e estavam em uso de micofenolato, tacrolimus e corticoide. Quatro apresentaram hemÃlise e trÃs foram transfundidos pela SLP. Esses pacientes, quando comparados aos demais, apresentaram Hb menor (p=0,043) e LDH (p=0,008) e reticulÃcitos (p=0,008) maiores. A presenÃa de auto e aloanticorpos contra antÃgenos eritrocitÃrios Ã frequente no TH, porÃm hemÃlise clinicamente manifesta ocorreu em apenas 2,8%. A presenÃa de anticorpos Ã mais frequente em pacientes com escore MELD mais elevado, com VHC, que utilizam furosemida prÃ-transplante, naqueles transfundidos com CH, RhD negativo e que utilizam piperacilina-tazobactam pÃs-transplante. O Ãnico fator de risco para o desenvolvimento de SLP encontrado Ã a incompatibilidade ABO menor entre doador e receptor.

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44

Campos, Elisa Regina. "A nanopore-based stochastic detection method: Single molecule characterisation of nanoparticles using ()- hemolysin." Doctoral thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica, 2013. http://hdl.handle.net/10362/12028.

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Dissertation presented to obtain the Ph.D degree in Chemistry.
Single-molecule techniques are revolutionising analytical chemistry methods. By observing one molecule at a time, the specific dynamics of (sub) populations can be revealed, and the averaging of signals from many molecules, typically obtained in bulk analysis techniques, is avoided. Nanopores have emerged as sensors par excellence for the label-free analysis of single molecules. Nanopore applications are expanding, helped by the fact that biological nanopores can be engineered to improve their suitability for a particular application, and also due to the recent advances in nanofabrication techniques used in the production of solid-state nanopores.(...)

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45

Hurt, Nicholas S. "Electronic detection of DNA polymerase complex formation and dissociation using an alpha-hemolysin nanopore /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2009. http://uclibs.org/PID/11984.

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46

Hornblower, Breton. "The alpha-hemolysin nanopore as an analytical device to probe non-covalent molecular interactions /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2006. http://uclibs.org/PID/11984.

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47

Shaver, Caryl Smith 1959. "An in vitro model of arsine induced hemolysis and its application to possible treatments." Thesis, The University of Arizona, 1991. http://hdl.handle.net/10150/277980.

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Arsine gas is a potent hemolytic agent. Early work suggested glutathione depletion preceded, and oxygen required for hemolysis to occur. This study developed an in vitro model of arsine hemolysis, using the solubility of arsine gas in aqueous solutions. A total of 75% of the arsine was taken up into the cells within 5 minutes. Hemolysis occurred after 1-2 hours and reached 40-50%. Glutathione depletion occurred, but only after hemolysis reached its maximum. Increasing intracellular glutathione did not prevent hemolysis. The use of an intracellular chelator, monomethyldimercaptosuccinic acid did not prevent hemolysis. Hemolysis occurred in an oxygen excluding atmosphere but carboxyhemoglobin prevented hemolysis. Glutathione depletion is not a critical first step in arsine induced hemolysis. The interaction of arsine with the heme site of hemoglobin is critical to hemolysis. It is likely that a free radical intermediate of oxygen or arsine is the ultimate hemolytic agent.

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48

Trent, Michael S. "Biochemistry of Hemolysin Toxin Activation by Fatty Acylation: Characterization of an Internal Protein Acyltransferase." Digital Commons @ East Tennessee State University, 1998. https://dc.etsu.edu/etd/2985.

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Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyltransfer was assayed. Acyl-ACP was the obligate acyl donor. Acyltransfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was detected and shown to catalyze the reverse reaction. The reaction mechanism was examined by steady state kinetics, and the nature of inhibitions by reaction products was determined. The kinetic mechanism of the internal protein acylation was compatible with an uni uni iso uni uni ping pong with isomerization of the F form of the enzyme. Clues to the chemical mechanism for the acyltransferase were elucidated by both chemical modification studies and site directed mutagenesis of the enzyme. Chemical modification experiments ruled out any critical cysteines, serines, and lysine residues, but suggested a role for histidine(s) and tyrosine(s) in acyltransferase function. In order to examine the function of specific residues and possibly corroborate the chemical findings, site directed mutagenesis studies of the acyltransferase were employed. Seventeen residues that were conserved among 13 different RTX toxin acyltransferases were individually mutated, and the respective HlyCs expressed, and characterized. Residues that were critical for acyltransferase function included Gly 11, His 23, Tyr 70, and Gly 85. As with chemical modification data, mutagenesis ruled out any conserved, essential, cysteines or serines critical for HlyC acyltransferase activity.

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49

Roxström-Lindquist, Katarina. "Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia." Doctoral thesis, Stockholms universitet, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-3.

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Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

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50

Turri, Rosimary de Jesus Gomes. "Estudo de fatores de virulencia em amostras de Klebsiella pneumoniae e Escherichia coli B - lactamases espectro estendidas." [s.n.], 2003. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314707.

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Orientador: Tomomasa Yano
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A patogênese da K.pneumoniae e Ecoli pode ser diretamente influenciada pelos fatores de virulência, dentre os quais encontram-se polissacarídeos capsulares, lipopolissacarídeos tóxicos, adesinas e sistema de aquisição de ferro. Além desses fatores de virulência, muito pouco é conhecido sobre outros fatores que podem participar da patogênese da K.pneumoniae. O presente trabalho estuda os fatores de virulência de K.pneumoniae e Ecoli isoladas de infecções nosocomiais em amostras ESBL (+) e ESBL (-), tendo como objetivos a comparação do grau de patogenicidade e produção de fatores de virulência por essas amostras in vivo e in vitro; a verificação da influência das condições de cultivo sobre a expressão de citotoxinas; o estudo do padrão de adesão em linhagens celulares; análise da influência da dose subinibitória de antibióticos beta-Iactâmicos sobre a produção de fatores de virulência pelas amostras ESBL (+). K.pneumoniae e Ecoli ESBL (+) apresentaram atividades hemolíticas moduladas pelo meio de cultura e pelas condições de cultivo, frente a sangue de eqüino e carneiro. A dose subinibitória do antibiótico estimulou a produção da hemolisina, sobre hemácias de eqüinos em meio Müller-Hinton apenas na presença do oxigênio. A carência de ferro nos meios induziu a produção de hemolisina, independente da aeração para hemácias eqüinas pelas amostras ESBL (+). O sobrenadante das culturas bacterianas apresentou atividade citotóxica em culturas de células HeLa, Vero e HT-29. Amostras ESBL (+) foram mais resistentes à ação bactericida do soro humano normal Que as ESBL (-). O padrão de adesão predominante entre amostras foi do tipo agregativo. Não foi observada diferença no grau de virulência para camundongos entre amostras ESBL (+) e ESBL(-)
Abstract: Virulence factors, such endotoxin, adhesins, cell envelope and the acquisition of iron system may be associated with K. pneumoniae and E. coli pathogenicity. However others virulence factors are not yet studied for understanding pathogeneses of K. pneumoniae. The aim of the present study is the comparation of potential virulence factors in strains of K. pneumoniae and E.coli ESBL (+) and ESBL (-) isolated from nosocomial infections, such characterization of adhesive properties in eukaryotic cells , influence of sub inhibitory dose of beta-Iactams antibiotics in toxins expression and virulence factors production in ESBL (+) strains. K. pneumonia e and E. coli ESBL (+) showed hemolytic activity towards sheep and horse erythrocytes regulated by culture conditions. The sub inhibitory dose of beta lactams antibiotics increased the hemolysin production in Mueller-Hinton agar with horse erythrocytes in oxygen presence. The iron absence in media culture improved the hemolytic activity expression, with horse erythrocytes in ESBL (+) strains independent of oxygen. The bacterial supematant culture showed cytotoxic activity in HeLa, Vero and HT -29 cell lines. The ESBL (+) strains were more resistant to the bactericidal action of normal human serum than the ESBL (-). The aggregative pattern of adherence was the predominant among E. colí and K. pneumoniae and was not observed virulence differences in mice in ESBL (+) and ESBL (-) strains
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

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