Com este estudo objetivou-se avaliar os níveis de estresse e a qualidade de carne de cordeiros mestiços Santa Inês x Dorper, submetidos a transporte de percurso curto (duas horas) e longo (seis horas) e em dois períodos de espera pré-abate (12 ou 24 horas). Foram utilizados 32 cordeiros com 127 ± 7 dias de idade e 30,4 ± 2,1 kg de peso vivo. Antes de serem submetidos aos períodos de transporte, os animais estavam alocados em baias coletivas e receberam ração concentrada (farelo de soja e milho, calcário calcítico e núcleo com monensina), bagaço úmido de citros e capim Napier (Pennisetum purpureum) e água ad libitum. Foram realizadas avaliações de reatividade dos animais no momento do embarque, desembarque e durante a contenção dos animais para as colheitas de sangue, urina e temperatura ocular por termografia infravermelho. Durante o período de espera pré-abate, realizou-se a colheita de dados comportamentais dos animais. Foram avaliadas as concentrações de cortisol no soro, na urina e as concentrações de haptoglobina no soro nos períodos que antecederam a insensibilização, assim como no momento do abate. Os animais apresentaram baixa reatividade durante todos os manejos. Houve diferença significativa no comportamento dos cordeiros durante os períodos de espera (P < 0,05) que durante as 12 horas apresentaram frequência de comportamentos que indicaram bem-estar favorável, enquanto que o período de transporte não afetou (P > 0,05). Os níveis de cortisol no soro mantiveram-se semelhantes da saída dos animais para o transporte até o final do período de espera (P > 0,05), enquanto houve oscilação dessas concentrações no cortisol na urina (P < 0,05), com pico no desembarque dos animais de duas horas de transporte e diminuição ao final do período de descanso. Os níveis de haptoglobina mantiveram-se semelhantes da colheita realizada antes do transporte, no embarque e no desembarque (P > 0,05) e diminuíram no final do período de espera pré-abate (P < 0,05). A temperatura ocular elevou-se no embarque e no desembarque dos animais, com diminuição da temperatura ao final do período de espera (P < 0,05). No momento do abate, foi observado aumento das concentrações de haptoglobina (P < 0,05), enquanto não houve alteração nas concentrações de cortisol no soro (P > 0,05). Animais que permaneceram por 24 horas de espera pré-abate apresentaram maior força de cisalhamento e menor luminosidade (L*) e intensidade de amarelo (b*). As variáveis comportamentais foram pouco afetadas pelos períodos de transporte e de espera pré-abate, porém o período de 12 horas de espera favoreceu a qualidade da carne / This study aimed to evaluate the stress levels and the meat quality from crossbred Santa Inês x Dorper lambs transported in short route (two hours) and long route (six hours) and maintained in two lairage periods (12 or 24 hours). Thirty-two crossbred Dorper x Santa Inês lambs with 127 ± 7 days of age and 30.4 ± 2.1 kg of body weight were used. Before the transport, the animals were allocated in collective pens and received concentrated feed (soy and corn meal, limestone and nucleus with monensin), wet bagasse citrus, Napier grass (Pennisetum purpureum) and water ad libitum. Reactivity evaluations were performed at loading, unloading and restraint of animals during the blood and urine collection and eye temperature by infrared thermography. During the pre-slaughter period (lairage), behavioral data were collected. Cortisol and haptoglobin serum concentrations and urine cortisol concentrations were measured in periods prior to stunning and at bleeding. The animals had low reactivity for all managements. The lairage periods changed the behavior of lambs (P < 0.05) with animals which have remained for 12 hours showed frequency of behaviors that indicate favorable welfare. The transport period did not affect (P > 0.05) the behavior of animals. The serum cortisol levels remained similar of the output for transport to the end of the lairage period (P > 0.05), while there was oscillation these concentrations in urine (p < 0.05) with peak on unloading in animals transported for two hours and a decrease at the end of the lairage period. The haptoglobin levels were similar between the collection performed before transportation, loading and unloading (P > 0.05) and decreased at the end of pre slaughter lairage (P <0.05). Ocular temperature rose at loading and unloading, with decreasing at the end of lairage period (P <0.05). At slaughter was observed increase of haptoglobin concentrations (P < 0.05), while there was no change in cortisol concentrations in serum (P> 0.05). Animals that remained for 24 hours lairaged before slaughter had higher shear force and lower luminosity (L *) and yellow intensity (b *). Behavioral variables were little affected by transport and lairage periods, but 12 hours of lairage favored the quality of meat

O objetivo deste trabalho foi avaliar o efeito de um preparado de anticorpos policlonais (PAP) contra bactérias ruminais específicas, Streptococcus bovis e Fusobacterium necrophorum, em parâmetros ruminais da fermentação, em vacas canuladas, adaptadas ou não a uma dieta de alta proporção de carboidratos prontamente fermentescíveis, após indução à acidose. O delineamento experimental utilizado foi o quadrado latino 3X3 replicado em arranjo fatorial de tratamentos 3X2, sendo 2 aditivos alimentares (PAP na apresentação em pó - PAPP e PAP na apresentação líquida - PAPL) mais um grupo controle (CON) e dois manejos de adaptação à dieta, resultando em seis tratamentos. O primeiro quadrado latino foi submetido a um protocolo de adaptação à dieta do tipo gradual ou step-up: dos dias D0 a D4 os animais receberam 100% de forragem; do D5 ao D9, 30% de concentrados e do D10 ao D14, 60% de concentrados. O segundo quadrado latino recebeu 100% de forragem do D0 ao D14 (sem adaptação). Nos D15 e D16, todos os animais receberam dieta com 80% de concentrados. Para as análises foram coletadas amostras de líquido ruminal a cada 3 horas a partir da 0h antes da alimentação até as 36h (D15 e D16) durante o desafio com uma dieta de 80% de concentrados. Os dados foram analisados pelo procedimento Mixed do SAS com nível de significância de 0,05. Foi observada interação entre tempo e adaptação (P<0,05) para pH ruminal com diferença entre método de adaptação nas 0, 3, 6, 9, 12 e 36 horas pós alimentação, quando o grupo não adaptado teve valores maiores que o grupo adaptado, sendo que na hora 24 ocorreu o contrário. Para a concentração de ácidos graxos de cadeia curta (AGCC), nas horas 0, 3, 6, 9 e 36 pós alimentação o grupo adaptado obteve maiores valores comparado ao grupo não adaptado. Para proporção molar de acetato, a 0 hora o grupo sem adaptação obteve valores maiores comparado ao grupo adaptado. Já nas horas 24, 27 e 30 o grupo com adaptação que obteve maiores valores. Para a proporção molar de propionato o grupo sem adaptação teve valores mais altos em comparação ao outro grupo das 3 às 36 horas pós alimentação. Quanto à proporção acetato:propionato (Ac:Pr) às 6, 12, 24, 27, 30 e 36 horas pós alimentação, o grupo de animais adaptados teve valores mais altos que o grupo não adaptado. Na proporção molar de butirato, o grupo de animais adaptados obteve maiores valores nas horas 0, 3, 6, 9, 12, 33 e 36. Para os valores de nitrogênio amoniacal (N-NH3), às 6 horas pós alimentação, o grupo não adaptado obteve maiores valores que o grupo adaptado (26,1 vs. 19,3, respectivamente). Nas horas 9, 30, 33 e 36 ocorreu o contrário. Observou-se também interação entre tempo e aditivo (P=0,0430) para a proporção molar de butirato. Porém, quando a análise foi realizada por tempo, nenhum efeito foi observado. Para os valores relativos de protozoários mensurados (Dasytricha, Isotricha, Epidinium, Diplodinium e Entodinium) apenas o Entodinium apresentou efeito de adaptação (P<0,0236) tendo sua proporção maior no grupo adaptado. Os valores de haptoglobina também não foram influenciados nem por aditivo nem por adaptação. O preparado de anticorpos policlonais não foi tão eficaz quanto a adaptação gradual à dieta de alto concentrado para controlar alterações dos parâmetros ruminais. / The objective of this trial was to evaluate the effects of polyclonal antibodies preparation (PAP) against specific rumen bacteria Streptococcus bovis and Fusobacterium necrophorum on rumen fermentation parameters in ruminally cannulated cows adapted or not to highly fermentable carbohydrates diets (HFC) after an acidosis challenge. The experimental design was two 3X3 Latin squares in a factorial arrangement of treatments 3X2 regarding two feed additives (PAP in powder presentation - PAPP and PAP in liquid presentation - PAPL) plus control group (CON) and two managements of diets adaptation, resulting in six treatments. The first Latin square had a step-up diet adaptation: from D0 to D4 100% forage; D5 to D9 30% of concentrates and D10 to D14 60% of concentrates. The second Latin square received 100% forage from D0 to D14. On D15 and D16, all animals received a diet with 80% of concentrates. For analysis, rumen fluid was sampled at 0 and every 3 h posfeeding totaling 36 h (D15 and D16) of challenge with a diet with 80% of concentrates. Data were analyzed by MIXED procedure with a significance level of 0.05. An interaction between time and adaptation (P<0,05) was observed for ruminal pH. At 0, 3, 6, 9, 12 and 36 h postfeeding, the non-adapted group had higher values compared to the adapted group and at 24 h postfeeding, the inverse was observed. For total short-chain fatty acids concentration, at 0, 3, 6, 9 and 36 h postfeeding, the adapted group had higher values compared to non-adapted group. For molar proportion of acetate at 0h postfeeding, the non-adapted group had higher values than the adapted group, and at 24, 27 and 30h, the adapted group had greater values than the non-adapted group. For molar proportion of propionate the non-adapted group had greater values compared to the adapted group from 3 to 36h postfeeding. For acetate:propionate (Ac:Pr) ratio at 6, 12, 24, 27, 30 and 36 h postfeeding, the adapted group had greater values compared to the nonadapted group. For butyrate molar proportion at 0, 3, 6, 9, 12, 33 and 36h postfeeding the adapted group had greater values than the non-adapted group. For ammonia nitrogen (NH3- N) concentration at 6h, the non-adapted group had greater values than the adapted group (26.1 vs. 19.3, respectively), however at 9, 30, 33 and 36h postfeeding, the adapted group had higher values compared to the non-adapted group. It was also observed an interaction between time and additive (P=0.0430) for butyrate molar proportion, but when the analysis was performed by time no effect was observed. For the relative values of protozoa measured (Dasytricha, Isotricha, Epidinium, Diplodinium and Entodinium) only Entodinium presented adaptation effect (P<0.0236) with a higher proportion in the adapted group. Haptoglobin values was also not influenced (P>0.05) by additive or adaptation effect. Polyclonal antibodies preparation was not as effective as the gradual adaptation to the diet high concentrate to control changes of ruminal parameters.

Orientador: José Eduadro Tanus dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T14:18:38Z (GMT). No. of bitstreams: 1 Sertorio_JonasTadeuCau_D.pdf: 15317741 bytes, checksum: 8f9e412dcb5f88fa28c13ad829f48faa (MD5) Previous issue date: 2012 / Resumo: O óxido nítrico (NO) exerce um importante papel durante as alterações hemodinâmicas que normalmente acompanham uma gravidez sadia (GS). Entretanto, em mulheres com pré-eclâmpsia (PE), uma síndrome caracterizada por hipertensão e proteinúria, a vasodilatação da circulação sistêmica materna está comprometida. Isso possivelmente ocorre devido a uma menor biodisponibilidade de NO, a qual resulta de uma menor produção e uma maior degradação do NO. Como a hemoglobina livre no plasma sequestra o NO pela rápida reação de dioxigenação, o que pode comprometer a eficiência do processo de vasodilatação mediado pela via do NO/guanilato ciclase, formulamos a hipótese de que pacientes com PE, quando comparadas com grávidas sadias, apresentariam concentrações aumentadas de hemoglobina livre no plasma, o que resultaria em maior degradação (consumo) do NO e menor biodisponibilidade de NO. Além disso, também formulamos a hipótese de que os diferentes fenótipos da haptoglobina (Hp) podem modular a biodisponibilidade de NO ao influenciar o consumo de NO na PE. A haptoglobina é uma proteína polimórfica (Hp1-1, Hp2-1 e Hp2-2) que se liga à hemoglobina para formar um complexo (Hb-Hp) que é removido da circulação, prevenindo o consumo de NO e o estresse oxidativo induzido pela hemoglobina. Os produtos do gene da haptoglobina apresentam diferentes propriedades bioquímicas e biofísicas, o que pode influenciar a taxa de eliminação (clearance) do complexo Hb-Hp. Para avaliar a biodisponibilidade de NO, foram analisadas as concentrações de nitrito no plasma e no sangue total em um analisador de NO. O consumo de NO foi determinado no plasma através do método de quimiluminescência. As concentrações de hemoglobina plasmática e haptoglobina foram analisadas com kits de imunoensaio enzimático (ELISA). Os genótipos da haptoglobina foram determinados pela Reação em cadeia da polimerase em tempo real (RT-PCR). Encontramos concentrações reduzidas de nitrito no plasma e no sangue total de grávidas com PE comparado com grávidas sadias. As amostras de plasma de grávidas com PE consumiram mais NO e apresentaram maiores concentrações de hemoglobina livre do que as amostras de grávidas sadias. Além disso, verificamos que há correlação positiva entre as concentrações de hemoglobina plasmática e o consumo de NO, correlação negativa entre o consumo de NO e as concentrações de nitrito plasmático e no sangue total e, por fim, correlação negativa entre as concentrações de hemoglobina livre plasmática e as concentrações de nitrito no plasma e no sangue total. Esses dados sugerem que níveis aumentados de Hb livre resultam em consumo de NO aumentado e baixo biodisponibilidade de NO em pacientes com PE, quando comparados com grávidas sadias. Não encontramos diferenças nas frequências dos genótipos da haptoglobina entre grávidas com PE e GS. Em grávidas sadias, os genótipos da haptoglobina não apresentaram efeitos nos níveis de hemoglobina livre, no consumo de NO e nas concentrações de nitrito plasmático. Entretanto, pacientes com PE e genótipos Hp2-1 e Hp2-2 apresentaram níveis aumentados de hemoglobina livre (heme), consumo de NO elevado e concentrações reduzidas de nitrito quando comparados com pacientes com PE e genótipo Hp1-1. Esses achados indicam que apesar de o genótipo da haptoglobina não afetar o risco de se desenvolver PE, o genótipo Hp1-1 possivelmente exerce um papel de proteção na PE ao reduzir o sequestro de NO, enquanto que os genótipos Hp2-1 e Hp2-2 podem agravar a PE ao reduzir a biodisponibilidade de NO / Abstract: Nitric oxide (NO) plays an important role in the hemodynamic changes found during normal pregnancy (NP). However, preeclampsia (PE), a syndrome characterized by hypertension and proteinuria, is associated with impaired vasodilatation of the maternal systemic circulation, possibly due to decreased NO bioavailability resulting of lower NO production and increased NO degradation. Since cell-free hemoglobin scavenges NO through a high speed dioxygenation reaction, thus compromising the efficiency of the NO/soluble guanylyl cyclase pathway to elicit vasodilatory activity, we hypothesized that higher plasma hemoglobin concentrations exist in patients with preeclampsia compared with normal pregnant women, and the higher plasma hemoglobin concentrations could lead to increased NO consumption and lower NO bioavailability in preeclamptic patients. Moreover, we hypothesized that haptoglobin (Hp) phenotypes might modulate NO bioavailability by influencing NO consumption in preeclampsia. Haptoglobin is a polymorphic protein (Hp1-1, Hp2-1 and Hp2- 2) that binds hemoglobin to form a complex that is removed from circulation, thus preventing Hb-driven oxidative stress and NO scavenging. Hp protein products differ in biochemical and biophysical properties, which reflects on the Hb-Hp complex clearance rate. To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. A NO consumption assay was used to measure NO consumption. Plasma hemoglobin and haptoglobin concentrations were assessed with a commercial immunoassay (ELISA). Haptoglobin genotypes were determined using Real Time Polymerase Chain Reaction. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients compared with normal pregnant women. Plasma samples from preeclamptic women consumed more NO and had higher plasma Hb levels than those found in normal pregnant women. We found significant positive correlations between plasma Hb and plasma NO consumption, negative correlations between plasma NO consumption and whole blood and plasma nitrite concentrations, and negative correlations between plasma Hb and whole blood and plasma nitrite concentrations. These findings suggest that increased plasma Hb levels lead to xiv increased NO consumption and lower NO bioavailability in preeclamptic patients compared with healthy pregnant women. Furthermore, we found no differences in Hp genotype frequencies between preeclamptic and NP groups. Haptoglobin genotypes had no effects on plasma heme levels, NO consumption and plasma nitrite in normal pregnant women. However, in preeclampsia, Hp2-1 and Hp2-2 were associated with higher plasma heme levels, increased NO consumption, and lower plasma nitrite compared with Hp1-1. These findings indicate that although haptoglobin genotype does not affect the risk of preeclampsia, Hp1-1 genotype may exert a protective role in preeclampsia by reducing NO scavenging, whereas Hp2-1 and Hp2-2 may further aggravate preeclampsia by reducing NO bioavailability / Doutorado / Farmacologia / Doutor em Farmacologia

In couples diagnosed with unexplained infertility, the woman has normal ovulatory cycles and no organic pathology, and the man has normal semen. The period during which the implantation of an embryo can occur is defined as the window of implantation (WOI). For infertile women, disturbances in the endometrium maturation are hypothesized to impair the uterine receptivity for embryo implantaition. However, there are still no methods found to predict endometrial receptivity. Hemoglobin related-proteins hypothesized to be involved in embryo implantation have been found in the endometrium in both fertile and infertile women. The aim of this study was to determine whether there were differences in the expression of hemoglobin-related proteins, hemoglobin-α, cytoglobin, fetal hemoglobin and haptoglobin in the endometrium of fertile and infertile women. Immunohistchemical staining was used. Haptoglobin showed negative expression in glands, luminal epithelium and strong expression in stroma for both gruops. The expression of the hemoglobin-related proteins, hemoglobin-α, cytoglobin, fetal hemoglobin intensity were stronger in fertile than infertile women. More studies are required to find other factors that may have an effect on fertility.

Orientador: Mateus José Rodrigues Paranhos da Costa / Resumo: Objetivou-se avaliar a eficácia do uso do colostro comercial em pó como substituto do colostro de vaca na primeira mamada de bezerras. Foram utilizadas 31 bezerras mestiças Holandês (H) x Gir (G), provenientes dos grupos genéticos 3/4HG, 5/8HG, 7/8HG e LA, divididas em dois grupos experimentais: G1 (n=15) 2L de colostro de vaca e G2 (n=16) 470g de colostro comercial em pó (SCCL, Saskatoon, SK, Canadá) diluídos em 1,5L de água morna, ambos fornecidos nas primeiras três horas de vida, e posteriormente recebendo mais 2L de colostro de vaca em até 12 horas após o nascimento. As concentrações de IgG presentes (p=0,006) foram maiores no colostro comercial em pó, enquanto as médias da β- caseína (p<0,0001), haptoglobina (p<0,0001) e α1-glicoproteína ácida (p=0,002) foram maiores nos colostros de vaca. As concentrações séricas de PT (G1 = 7,70 ± 1,00g/dL e G2 = 6,73 ± 0,65g/dL; p=0,003) e IgG (G1 = 2110,25 ± 595,03mg/dL e G2 = 1567,6 ± 418,25mg/dL; p=0,004) demonstraram diferenças estatísticas entre os grupos de manejo, com maiores médias para o G1. Houve diferenças significativas para a concentração de albumina sérica (p=0,005), com maiores médias para o G1 (4660,7 ± 384,61mg/dL). Não ocorreu diferença significativa para a haptoglobina sérica (p=0,29), porém suas concentrações médias se apresentaram bem mais altas em ambos os grupos de manejo (G1 = 33,86 ± 4,90 e G2 = 29,31 ± 5,63mg/dL). As enfermidades com maior ocorrência foram diarreia e pneumonia, sendo registrados também casos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was value the effectiveness in commercial colostrum powder using as a cow colostrum substitute in the first calf suckling. Twenty-one Holstein (H) x Gir (G) crossbred calves from 3/4HG, 5/8HG, 7/8HG and LA genetic groups were divided into two experimental groups: G1 (n = 15) 2L of cow colostrum and G2 (n = 16) 470g of commercial powdered colostrum (SCCL, Saskatoon, SK, Canada) diluted in 1.5L of warm water, both supplied within the first three hours of life, and subsequently receiving an additional 2L of cow colostrum in up to 12 hours after birth. Present IgG concentrations (p = 0.006) were higher in commercial powdered colostrum, while mean β-casein (p < 0.0001), haptoglobin (p <0.0001) and α1-acid glycoprotein (p = 0.002) were higher in cow colostrums. Serum concentrations of PT (G1 = 7.70 ± 1.00 g/dL and G2 = 6.73 ± 0.65 g/dL; p = 0.003) and IgG (G1 = 2110.25 ± 595.03 mg/dL and G2 = 1567.6 ± 418.25 mg/dL; p = 0.004) demonstrated statistical differences between management groups, with higher means for G1. There were significant differences in serum albumin concentration (p = 0.005), with higher means for G1 (4660.7 ± 384.61mg / dL). There was no significant difference for serum haptoglobin (p = 0.29), but its mean concentrations were much higher in both management groups (G1 = 33.86 ± 4.90 and G2 = 29.31 ± 5.63 mg/dL). The most common diseases were diarrhea and pneumonia, with cases of parasitic sadness and omphalitis, and two deaths in G2. Althou... (Complete abstract click electronic access below) / Mestre

No description available.

Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar.<br /> Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. <br /> Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet.<br /> Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid Fructosyl-Valin-Histidin-Leucin-Threonin-Prolin (glkPP) als Kompetitor und Affinitätsligand immobilisiert und so eine regenerierfähige Oberfläche geschaffen. Beim Sandwich-Immunoassay wurde im ersten Schritt Gesamt-Hämoglobin an die mit Haptoglobin (Hp) modifizierte Festphase angereichert und im zweiten Schritt der gebundene HbA1c-Anteil nachgewiesen. <br /> Für die Konstruktion des HbA1c-Immunosensors wurden Affinitätsmatrizen durch Modifizierung von Cellulose-Dialysemembranen mit glkPP bzw. Hp hergestellt. Grundlegend studiert wurde die Aktivierung der Cellulose-Membranen mit 1,1'-Carbonyldiimidazol (CDI) und 1-Cyano-4-dimethylaminopyridintetrafluoroborat (CDAP) als Aktivierungsagenzien. Eine gerichtete Immobilisierung der Liganden wurde realisiert, indem glkPP über dessen C-Terminus (einzige Carboxylatgruppe) und Hp über dessen periodat-oxidiertem Kohlenhydratrest an die amino- oder hydrazidfunktionalisierte Membranen kovalent gekoppelt wurden. <br /> Mit dem Einsatz der glkPP- und Hp-modifizierten Membranen in der elektrochemischen Messzelle war erstmalig der biosensorische Nachweis von HbA1c möglich. Als Transduktor diente eine Pt-Elektrode, an der das von der GOD generierte H2O2 umgesetzt und ein mit der HbA1c-Konzentration korrelierendes Stromsignal erzeugt wurde. Die Immunosensoren zeigten Ansprechzeiten von 3 s. Mit dem Immunosensor auf Basis des indirekt-kompetitiven Testprinzips wurde eine Kalibrationskurve für HbA1c im Bereich von 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %) mit Assayzeiten von 60 min und mit dem Immunosensor im Sandwich-Format eine Kalibrationskurve im Bereich von 0,5-5 &#181;g/ml (7,8-78 nM; 5-50 % HbA1c vom Gesamt-Hb, CV 6-10 %, 3 h) aufgenommen. / Hemoglobin-A1c (HbA1c) is a hemoglobin subtype formed by non-enzymatic reaction of glucose with the N-terminus of the beta-polypeptide chains. As it reflects the glycemic status of diabetics over the preceding 8-12 weeks, the determination of HbA1c has become an established procedure in the management of diabetes mellitus. It is measured as the percentage of total hemoglobin. Up to 5 % HbA1c are considered as normal whereas in diabetic subjects it could be elevated from 5-20 %. In addition to amperometric biosensors for glucose self monitoring which have been successfully applied in diabetes management, biosensors for HbA1c would be an useful supplement for a comprehensive diabetes control. <br /> <br /> Objective of this work was to develop and compare amperometric biosensors for determination of HbA1c based on enzymatic and immunochemical methods. <br /> <br /> For the enzyme based HbA1c assay a novel fructosamine oxidase (FAO) derived from marine yeast Pichia pastoris, strain N1-1 was utilized. It recognizes and oxidatively degrades fructosyl-valine (FV) which corresponds to the glycated N-terminus of the beta-chain of HbA1c and therefore is regarded as a model compound for HbA1c. Hydrogen peroxide which is liberated by the FAO during FV conversion was indicated optically in a horseradish peroxidase (POD) coupled reaction and electrochemically. For the biosensor the FAO was embedded in polyvinyl alcohol-stylbazole (PVA-SbQ) and fixed it in front of a Pt-electrode. So far, the measuring range of FV did not cover the clinically relevant range of HbA1c. Low specificity was assumed since enzyme activity also was obtained with glycated peptides, e.g. fructosyl-valine-glycine, not corresponding to the glycated N-terminus of the hemoglobin-beta-chain.<br /> <br /> For the immunosensor two immunoassays formats - heterogeneous sandwich and heterogeneous competitive - were tested. The assays were designed as follows: The competitive immunoassay was based on the immobilized synthetic glycated pentapeptide fructosyl-valine-histidine-leucine-threonine-proline (glkPP) utilized as HbA1c analogue. The peptide has an amino acid sequence corresponding to the N-terminus of the hemoglobin beta-chains and is capable for competition together with the HbA1c of the sample for the amount of a glucose oxidase (GOD)-labelled anti-HbA1c antibody. In the sandwich-type assay haptoglobin (Hp), a natural hemoglobin binding molecule with antibody characteristic properties, was used as bioreceptor for enrichment of total hemoglobin onto the surface. In a subsequent step the HbA1c fraction was quantified by a GOD-labelled HbA1c specific antibody. <br /> <br /> Cellulose dialysis membrane was used as the solid support for immobilization of Hp and glkPP near the sensor surface. For activation of the membrane two reagents, 1,1&prime;-carbonyldiimidazole (CDI) and 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), were compared with respect to the degree of activation and coupling efficiency. Site-directed immobilization of Hp and glkPP was achieved by coupling Hp via its carbohydrate residue and glkPP via its C-terminus to the activated membrane using a bis-amine or bis-hydrazide spacer. <br /> <br /> The affinity membranes were placed in front of a modified Clark-type hydrogen peroxide electrode in an electrochemical measuring cell and HbA1c analysis was carried out within the stirred cell. Detection of the bound GOD-label was achieved by measurement of the electrocatalytic oxidation of hydrogen peroxide at +600 mV vs. Ag/AgCl. The indication was done in only 3 s. For the competitive principle a typical inhibition curve with a linear range between 0,25-30 &#181;g/ml (3,9-465 nM, CV 3-9 %, 60 min per sample) HbA1c was obtained. Due to the high functional stability of the peptide multiple regeneration of the affinity surface was possible without loss of binding capacity. With the sandwich assay configuration the clinically relevant range could easily be covered (calibration curve: 5-50 % HbA1c corresponding to 7,8-78 nM, CV 6-10 %, 3 h per sample).

No description available.

Genetic as well as environmental factors are believed to be of importance in the etiology of rheumatoid arthritis (RA). There are a number of previous studies of genetic markers in RA, but so far no genetic linkage and only a few associations have been found. Of the associations only one (with the HLA antigen DR4) appears to be well documented. In most previous association studies the patients have not been divided according to sex and family history of RA. In this investigation the HLA antigens A, B and DR and five serum protein systems (Bf, C3, Pi, Hp and Tf) were studied in patients with erosive rheumatoid arthritis (RA), from northern Sweden. Special attention was paid to variations in the strength of associations accord­ing to sex and family history of polyarthritis. The following results were found:  The frequency of the HLA antigen B27 was significantly increased in the North-Swedish population (16.6%) and among patients with a family history of polyarthritis (42.6%). In agree­ment with previous investigations a significantly increased frequency of the DR4 antigen was found in the RA patients.  In the properdin factor B (Bf) system the S phenotype was found to be significantly in­creased in male patients and in patients with a family history of polyarthritis, more severe form of RA and high titres of rheumatoid factor.  No significant differences with respect to phenotype or gene frequencies were found in the C3 complement system. Thus, the association between RA and C3 found in previous investiga­tions was not confirmed.  A significant increase of rare alpha-1-antitrypsin (Pi) types (MS, MZ, MF and SZ) was found among RA patients. However, the increase concerned mainly Z heterozygotes and was more strongly pronounced among male patients.  In the haptoglobin system a significant increase of the Hp^ gene and the Hp2-2 type was found among patients with a family history of polyarthritis, more pronounced among males.  A significant increase of the transferrin gene and of the 2 type was found among male RA patients, more pronounced among patients with a family history of polyarthritis. In 6 out of 8 gene loci studied significant associations were found, which is in agreement with a multifactorial etiology of RA. The results were largely in agreement with the hypothesis that associations would be expected to be stronger in males and in patients with a family history of polyarthritis. A notable finding was the high frequency of first degree relatives (around 40%) with symmetric peripheral polyarthritis of which more than 70% had a diagnosis of RA verified by hospital records. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 6 uppsatser.</p> / digitalisering@umu

Orientador: Maria de Fátima Sonati / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T22:02:31Z (GMT). No. of bitstreams: 1 Fernandes_ThalesAllyrioAraujodeMedeiros_D.pdf: 2937928 bytes, checksum: 301442af6cf4af176a07dec075589b81 (MD5) Previous issue date: 2014 / Resumo: As doenças falciformes apresentam uma heterogeneidade fenotípica substancial e vários fatores, tanto genéticos quanto ambientais, contribuem para esta variabilidade. A co-herança com a talassemia ? e os haplótipos ?S são considerados importantes moduladores genéticos da doença, mas outros fatores hereditários podem também influenciar os perfis clínicos e laboratoriais dos pacientes. Estudos têm sugerido que os genótipos da haptoglobina (Hp) poderiam estar entre esses fatores. Assim, o presente estudo objetivou avaliar o efeito da presença da talassemia ?, dos diferentes haplótipos ?S e dos genótipos da haptoglobina na evolução clínica e nas características laboratoriais de pacientes com doença falciforme provenientes do estado do Rio Grande do Norte (RN). Foram analisados 155 indivíduos não aparentados (82 homens e 73 mulheres), com idades variando de 7 meses a 48 anos (mediana de 12 anos), provenientes de diversos municípios do RN e atendidos nos centros de referência de tratamento de doenças hematológicas para acompanhamento ambulatorial. Todos os pacientes, ou seus responsáveis, foram informados a respeito dos objetivos e procedimentos da pesquisa, e responderam a um questionário padronizado. Ao final, coletaram-se alíquotas de sangue periférico para a realização das análises hematológicas, bioquímicas e moleculares. Posteriormente, foram examinados os prontuários médicos arquivados nestes centros, de onde foram obtidas as informações relativas à evolução clínica (número de internações e transfusões nos últimos 12 meses, necessidade de estabelecimento de terapia transfusional crônica, desenvolvimento de infecções bacterianas graves, síndrome torácica aguda, sequestro esplênico, alterações cerebrovasculares, priapismo, úlcera de perna, necrose óssea, problemas cardiovasculares, renais e oftalmológicos, colelitíase, déficit ponderal e retardo no crescimento). Predominaram os indivíduos com idades inferiores a 12 anos, que se autodeclararam mulatos, que moravam em pequenas cidades relativamente distantes dos centros de referência e que possuíam baixo nível educacional e socioeconômico. Os pacientes com idades inferiores a 10 anos foram diagnosticados com a doença mais precocemente. Quase 50% dos indivíduos analisados faziam uso de hidroxiuréia, 91,4% relatou ter recebido vacinação pneumocócica/meningocócica e 76,1% já tinha feito uso profilático de penicilina alguma vez na vida. A co-herança com a talassemia alfa foi encontrada em 11,6% dos pacientes e não mostrou associação significativa com nenhuma das complicações clínicas avaliadas. No entanto, os pacientes ?-talassêmicos apresentaram maiores níveis de hemácias e hematócrito, e menores valores de hemoglobina corpuscular média (HCM). Os haplótipos ?S mais frequentes foram o CAR (77,5%), Benin (11,9%) e Camarões (5,5%). A homozigoze do haplótipo CAR esteve associada à maior incidência de retardo no crescimento, enquanto as demais combinações de haplótipos apresentaram valores significativamente maiores de hemoglobina e hemácias. A distribuição dos genótipos da haptoglobina estava em equilíbrio de Hardy-Weinberg em todos os grupos considerados e o tipo predominante foi o Hp2-1 (47,7%). As frequências dos alelos HP*1 (0,503) e HP*2 (0,497) foram muito semelhantes. A herança do alelo HP*2 se correlacionou significativamente com a necessidade de implementação da terapia transfusional crônica ao longo da vida e a níveis mais elevados de ferritina. Já os homozigotos do alelo HP*1 (18 pacientes) apresentaram níveis mais elevados de LDH e AST, que deixaram de ser significativos quando se incluiu na análise os indivíduos que faziam uso da hidroxiuréia (10 pacientes). Nossos resultados sugerem que os fatores genéticos aqui avaliados influenciaram em algum grau a evolução clínica e/ou os perfis laboratoriais dos pacientes desta amostra populacional. A ausência de associação significativa com as demais complicações aqui investigadas reflete a natureza multifatorial da doença e a necessidade de ampliação do tamanho amostral em estudo / Abstract: Sickle cell disease presents a significant phenotypic heterogeneity, and both genetic and acquired factors contribute to this variability. Co-inheritance of alpha-thalassemia and ?S haplotypes are the major genetic modifiers of the disease, but others inherited features can influence the clinical and laboratorial profile of the patients. Reports have suggested that haptoglobin genotypes could be one. Therefore, this study aimed to evaluate the effect of alpha thalassemia, ?S haplotypes and genotypes of haptoglobin in the clinical outcome and laboratorial characteristics of patients with sickle cell disease of Rio Grande do Norte State (RN). We analyzed 155 non-related individuals (82 men and 73 women) with sickle cell disease from various municipalities of RN, ages ranging from 7 months to 48 years (median age 12 years), who went to referral centers for outpatient visits. All the patients, or their caregivers, were informed about the research procedures and objectives, and answered a standardized questionnaire. After this, we collected blood samples for hematological, biochemical, and molecular analyses. Additionally, clinical data (number of blood transfusions and hospital admission in the last 12 months, need for chronic transfusion therapy, development of severe bacterial infections, acute chest syndrome, splenic sequestration, cerebrovascular disease, priapism, leg ulcers, osteonecrosis, cardiovascular, renal, and ophthalmologic problems, gallstones, weight deficit and stunted growth) were obtained from the patients¿ medical records archived in these referral centers. The patients were predominantly younger than 12 years old, self-declared as mulatto, lived in small cities relatively distant from the referral center, and had a low education and socio-economic level. Individuals who were 10 or younger were diagnosed at an earlier age. Almost 50% of the patients were taking hydroxyurea, 91.4% reported having received pneumococcal/meningococcal vaccination and 76.1% have ever done prophylactic use of penicillin. The co-inheritance of alpha thalassemia was found in 11.6% of patients and presented no significant association with any clinical complication of sickle cell disease. However, patients with this genetic feature had higher red blood cell (RBC) counts and packet cell volume (PCV), and lower values of mean corpuscular hemoglobin (MCH). The ?S haplotypes more frequent were CAR (77.5%), Benin (11.9%) and Cameroon (5.5%). The homozygous CAR/CAR haplotype was associated with higher incidence of stunted growth, while the other haplotype presented higher hemoglobin and RBC. The distribution of haptoglobin genotype were in Hardy-Weinberg equilibrium in all the groups, and the predominant type was Hp2-1 (47.7%). The frequencies of HP*1 (0.503) and HP*2 (0.497) alleles were similar. The inheritance of HP*2 allele was significantly correlated to the requirement of chronic transfusion therapy and higher levels of ferritin. On the other hand, the Hp1-1 patients (18 individuals) had higher levels of LDH and AST, that were not significant when we included in the analysis the individuals who were using hydroxyurea (10 individuals). Our results suggest that the genetic characteristics evaluated in this study influenced in a certain extent the clinical outcome and/or laboratorial profile of patients with sickle cell disease from RN. The absence of significant association with the others clinical complications reflect the multifactorial nature of these events and the need to increase the analyzed sample size / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas