Frequência dos mutantes C282Y e H63D do gene HFE e sua influência no metabolismo do ferro e na expressão da beta talassemia heterozigota

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biodisponibilidade relativa do ferro de fonte orgânica para leitões desmamados

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência dos níveis de ferritina sérica, das mutações no gene da beta globina e dos valores das enzimas de detoxificação Catalase e Glutationa S -Transferase na expressão fenotípica de Beta Talassemia Heterozigota

Pós-graduação em Genética - IBILCE

Clonagem e expressão de fragmentos de anticorpos (scFV) contra o vírus da leucemia felina (FeLV) por phage display

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Investigation of Human Albumin-Induced Circular Dichroism in Dansylglycine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Relative bioavailability of iron from organic sources for weanling piglets

Some authors consider minerals from organic sources more bioavailable for pig nutrition in comparison with inorganic sources. To evaluate the relative iron bioavailability from the organic source iron carbo-amino-phospho-chelate (ICAPC) to weanling piglets, it was conducted an experiment with 126 commercial piglets, using iron sulfate monohydrate (S) as standard. The experiment had a randomized block design with seven treatments (diet without adding specific source of iron, diet with 50, 100 and 150 ppm iron from S and diet with 50, 100 and 150 ppm iron from ICAPC), six replications and three animals per experimental unit. Performance parameters (average daily gain - ADG, feed: gain ratio - F:G) and blood variables (hemoglobin - Hb, hematocrit - Ht, transferrin - TR, latent iron-binding capacity - LIBC, total iron-binding capacity - TIBC, serum iron - Fe and transferrin saturation index - TSI) were evaluated. At the end of the experiment a piglet from each experimental unit was slaughtered and its liver and spleen removed for assessment of iron concentration by flame atomic absorption spectrometry (FAAS). The evaluated sources of iron yielded similar results for the variables of interest, but the increase in iron intake was followed by a linear increase in ADG, Hb, Ht, Fe and TSI as well as a linear decrease in the values of F:G, TR, LIBC and TIBC. Iron bioavailabilities from both ICAPC and S sources are similar for weanling piglets.

Oxidation of Bovine Albumin by Hypochlorous and Hypobromous Acids: Structural and Functional Alterations

Aims: Hypochlorous (HOCl) and hypobromous (HOBr) acids are among the most powerful oxidants produced by the innate immune cells. Albumin is the predominant protein in most body fluids and is considered the most important antioxidant of blood plasma. Study Design: Oxidation of bovine albumin (BSA) and study of its structural and functional alterations. Place and Duration of Study: Faculty of Science and Faculty of Pharmaceutical Science, University of the State of Sao Paulo UNESP, between June and December 2012. Methodology: BSA was oxidized with excess of HOCl or HOBr and its structural and functional alterations were analyzed by spectroscopic techniques as UV-Vis absorption, intrinsic and synchronous fluorescence, fluorescence quenching, Rayleigh scattering and circular dichroism. Results: Both oxidants were able to deplete the intrinsic fluorescence of BSA, but HOBr was more effective than HOCl. The alterations in the synchronous fluorescence, UV-Vis absorption, and the appearance of a fluorescence band centered at 450 nm confirmed the difference between the oxidants. The oxidation did not induce aggregation of BSA as measured by Rayleigh scattering. The far-UV circular dichroism spectra showed a loss in the helical content and the near-UV-circular dichroism showed an alteration in the tertiary structure; HOBr was the more effective of the oxidants in this case. However, the oxidations did not induce significant alterations in the binding capacity of BSA, which was evaluated using hydrophobic (norfloxacin) and hydrophilic (ascorbic acid) drugs. Conclusion: These results suggest that, although highly susceptible to oxidation, the alterations did not inhibit BSA’s physiological function as a transport protein.

Effects of Protein A in Detection of Canine Distemper Virus through immunosensor construction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

Regulation of corticosterone function during early weaning and effects on gastric cell proliferation

Objectives: The development of the gastrointestinal tract depends on many elements, including glucocorticoids. In the current study, we evaluated the effects of early weaning on corticosterone function and the growth of rat gastric mucosa. Methods: By using Wistar rats submitted to early weaning at 15 d, we analyzed plasma corticosterone, corticosteroid-binding globulin (CBG), and glucocorticoid receptor (GR) distribution in the gastric epithelium. Results: With the use of radioimmunoassay, we found that early weaning increased corticosterone concentration at day 16 and 17 in test subjects as compared with controls, whereas it was equivalent between groups at day 18. CBG binding capacity decreased during treatment, and it was significantly lower at day 18. At this age, GR levels and distribution in the gastric mucosa were also reduced as compared with suckling counterparts. To reduce corticosterone activity during early weaning and to explore cell proliferation responses, we administered RU486 to 15-d-old pups. We found that cytoplasmic GR reached a peak after 48 h, whereas nuclear levels remained constant, thereby confirming the inhibition of receptor function. Next, by checking gastric proliferative responses, we observed that RU486 induced higher DNA synthesis and mitotic indices in test subjects as compared with control groups. Conclusions: We demonstrated that early weaning changed corticosterone activity by increasing hormone levels, reducing CBG binding capacity, and decreasing GR distribution in the gastric epithelium. These modifications seem to be important to the reorganization of gastric growth after the abrupt interruption of suckling.

Studio Sperimentale e Modellazione della Separazione di Proteine con Membrane di Affinità

Chromatography represents one of the most important and widely used unit operation in the biotechnology industry. However this technique suffers from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. In this work, affinity membranes with improved capacity have been considered as an alternative technology for the capturing step in antibody manufacturing. Several affinity membranes have been prepared starting from various membrane supports. Different affinity ligands have been utilized like Protein A, the natural ligand of choice for antibodies, as well as synthetic ligands that exhibit affinity for the Fc portion of antibodies. The membranes have been characterized in detail: binding and elution performance was evaluated in adsorption experiments using pure IgG solutions, while membrane selectivity was evaluated using complex solutions like a cell culture supernatant. The most promising affinity membranes were extensively tested in dynamic experiments. The effects of operating parameters like feed concentration and flow rate on separation performances like binding capacity, selectivity and process yield have been studied in detail in order to find the optimal conditions for binding and elution steps. The membranes have been used over several complete chromatographic cycles to evaluate the effects of ageing and of membrane regeneration on dynamic binding capacity. A novel mathematical model is proposed that can describe all the chromatographic steps involved in the membrane affinity chromatography process for protein purification. The mathematical description is based on the species continuity equation coupled with a proper binding kinetic equation, and suitable to describe adequately the dispersion phenomena occurring both in the micro-porous membranes as well as in the extra-column devices used in the system. The model considers specifically all the different chromatographic steps, namely adsorption, washing and elution. The few relevant fitting parameters of the model were derived from a calibration with the experimental affinity cycles performed with pure IgG solutions, then the model is used to describe experimental data obtained in chromatographic cycles carried out with complex feeds as the cell culture supernatant. Simulations reveal a good agreement with experimental data in all the chromatography steps, both in the case of pure IgG solutions and for the cell culture supernatant considered.

Proteinuria e sindrome nefrosica nel cane: studio reptrospettivo di 338 casi

La sindrome nefrosica (SN) è definita come la presenza concomitante di una proteinuria maggiore di 3.5g/24 h, ipoalbuminemia, ipercolesterolemia e presenza di edemi. I pazienti con SN sono più a rischio di quelli che presentano una nefropatia glomerulare non nefrosica (NNGD) per lo sviluppo di ipertensione, ipernatremia, complicazioni tromboemboliche e comparsa di insufficienza renale. In Medicina Veterinaria, la Letteratura riguardante l’argomento è molto limitata e non è ben nota la correlazione tra SN e gravità della proteinuria, ipoalbuminemia e sviluppo di tromboembolismo. L’obiettivo del presente studio retrospettivo è stato quello di descrivere e caratterizzare le alterazioni cliniche e clinicopatologiche che si verificano nei pazienti con rapporto proteine urinarie:creatinina urinaria (UPC) >2 con lo scopo di inquadrare con maggiore precisione lo stato clinico di questi pazienti e individuare le maggiori complicazioni a cui possono andare incontro. In un periodo di nove anni sono stati selezionati 338 cani e suddivisi in base ad un valore cut-off di UPC≥3.5. Valori mediani di creatinina, urea, fosforo, albumina urinaria, proteina C reattiva (CRP) e fibrinogeno sono risultati al di sopra del limite superiore dell’intervallo di riferimento, valori mediani di albumina sierica, ematocrito, antitrombina al disotto del limite inferiore di riferimento. Pazienti con UPC≥3.5 hanno mostrato concentrazioni di albumine, ematocrito, calcio, Total Iron Binding Capacity (TIBC), significativamente minori rispetto a quelli con UPC<3.5, concentrazioni di CRP, di urea e di fosforo significativamente maggiori. Nessuna differenza tra i gruppi nelle concentrazioni di creatinina colesterolo, trigliceridi, sodio, potassio, cloro, ferro totale e pressione sistolica. I pazienti con UPC≥3.5 si trovano verosimilmente in uno “stato infiammatorio” maggiore rispetto a quelli con UPC<3.5, questa ipotesi avvalorata dalle concentrazioni minori di albumina, di transferrina e da una concentrazione di CRP maggiore. I pazienti con UPC≥3.5 non presentano concentrazioni di creatinina più elevate ma sono maggiormente a rischio di anemia.

Charakterisierung zellulärer Interaktionspartner des großen Hüllproteins des Hepatitis-B-Virus

Im Mittelpunkt dieser Arbeit stand das große L-Hüllprotein (L) des Hepatitis B - Virus. L bildet eine ungewöhnliche duale Topologie in der ER-Membran aus, welche auch im reifen Viruspartikel erhalten bleibt. In einem partiellen, posttranslationalen Reifungsprozess wird die sogenannte PräS-Region von der zytosolischen Seite der Membran aus in das ER-Lumen transloziert. Aufgrund seiner dualen Topologie und der damit verbundenen Multifunktionalität übernimmt L eine Schlüsselfunktion im viralen Lebenszyklus. Ein Schwerpunkt dieser Arbeit lag deshalb darin, neue zelluläre Interaktionspartner des L-Hüllproteins zu identifizieren. Ihre Analyse sollte helfen, das Zusammenspiel des Virus mit der Wirtszelle besser zu verstehen. Hierfür wurde das Split - Ubiquitin Hefe - Zwei - Hybrid System eingesetzt, das die Interaktionsanalyse von Membranproteinen und Membran-assoziierten Proteinen ermöglicht. Zwei der neu identifizierten Interaktionspartner, der v-SNARE Bet1 und Sec24A, die Cargo-bindende Untereinheit des CoPII-vermittelten vesikulären Transports, wurden weitergehend im humanen Zellkultursystem untersucht. Sowohl für Bet1 als auch für Sec24A konnte die Interaktion mit dem L-Hüllprotein bestätigt und der Bindungsbereich eingegrenzt werden. Die Depletion des endogenen Bet1 reduzierte die Freisetzung L-haltiger, nicht aber S-haltiger subviraler Partikel (SVP) deutlich. Im Gegensatz zu Bet1 interagierte Sec24A auch mit dem mittleren M- und kleinen S-Hüllprotein von HBV. Die Inhibition des CoPII-vermittelten vesikulären Transportweges durch kombinierte Depletion der vier Sec24 Isoformen blockierte die Freisetzung sowohl L- als auch S-haltiger SVP. Dies bedeutet, dass die HBV - Hüllproteine das ER CoPII-vermittelt verlassen, wobei sie aktiv Kontakt zur Cargo-bindenden Untereinheit Sec24A aufnehmen. Der effiziente Export der Hüllproteine aus dem ER ist für die Virusmorphogenese und somit für den HBV - Lebenszyklus essentiell. rnEin weiterer Schwerpunkt dieser Arbeit basierte auf der Interaktion des L-Hüllproteins mit dem ER-luminalen Chaperon BiP. In der vorliegenden Arbeit wurde überprüft, ob BiP, ähnlich wie das zytosolische Chaperon Hsc70, an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt ist. Hierfür wurde BiP durch die ektopische Expression seiner Ko-Chaperone BAP und ERdj4 in seiner Substrat-bindenen Kapazität manipuliert. ERdj4, ein Mitglied der Hsp40 - Proteinfamilie, stimuliert die ATPase-Aktivität von BiP, was die Substratbindung stabilisiert. Der Nukleotid - Austauschfaktor BAP hingegen vermittelt die Auflösung des BiP - Substrat - Komplexes. Die Auswirkung der veränderten in vivo-Aktivität von BiP auf die posttranslationale PräS-Translokation wurde mit Proteaseschutz - Versuchen untersucht. Die ektopische Expression des positiven als auch des negativen Regulators von BiP resultierte in einer drastischen Reduktion der posttranslationalen PräS-Translokation. Ein vergleichbarer Effekt wurde nach Manipulation des BiP ATPase - Zyklus durch Depletion der zellulären ATP - Konzentration beobachtet. Dies spricht dafür, dass das ER-luminale Chaperon BiP, zusammen mit Hsc70, eine zentrale Rolle in der Ausbildung der dualen Topologie des L-Hüllproteins spielt. rnZwei weitere Proteine, Sec62 und Sec63, die sich für die posttranslationale Translokation in der Hefe als essentiell erwiesen haben, wurden in die Analyse der dualen Topologie des L-Hüllproteins einbezogen. Interessanterweise konnte eine rein luminale Ausrichtung der PräS-Region nach kombinierter Depletion des endogenen Sec62 und Sec63 beobachtet werden. Dies deutet an, dass sowohl Sec62 als auch Sec63 an der Ausbildung der dualen Topologie des L-Hüllproteins beteiligt sind. In Analogie zur Posttranslokation der Hefe könnte Sec62 als Translokon-assoziierter Rezeptor für Substrate der Posttranslokation, und damit der PräS-Region, dienen. Sec63 könnte mit seiner J-Domäne BiP zum Translokon rekrutieren und daraufhin dessen Substrat-bindende Aktivität stimulieren. BiP würde dann, einer molekularen Ratsche gleich, die PräS-Region durch wiederholtes Binden und Freisetzen aktiv in das ER-Lumen hereinziehen, bis eine stabile duale Topologie des L-Hüllproteins ausgebildet ist. Die Bedeutung von Sec62 und Sec63 für den HBV - Lebenszyklus wird dadurch untermauert, dass sowohl die ektopische Expression als auch die Depletion des endogenen Sec63 die Freisetzung L-haltiger SVP deutlich reduziert. rn

Electrostatic supramolecular assembly of charged dendritic polymers and their biological application

The aim of this study was the development of functional multilayer films through electrostatic layer by layer (LbL) assembly of dendritic macromolecules, the investigation of the fundamental properties of these multilalyered films and the study of their biological applications. rnThe synthesis of the anionic hyperbranched polyglycerols (hbPG) and the preparation of multilayers made of hbPG/phosphorus dendrimer as well as the influences of deposition conditions on multilayers were reported. The thicknesses of multilayer films increase with a decrease of molecular weight of anionic hbPGs. The multilayer films fabricated by low molecular weight hbPGs grow less regularly due to the less charged carboxylic acid groups providing the relative weaker electrostatic forces for the deposition. The thicknesses of multilayer films are reduced with increasing pH values and decreasing the concentration of NaCl. The observed changes of multilayer thickness and surface morphology could be interpreted with the aid of theories regarding the charge density and conformation of the anionic hbPG chains in solution. rnBesides the study of fundamental properties of hbPG/phosphorus multilayer films, antifouling thin films derived from hbPG layers were developed. The antifouling properties of hbPG layers were found to correlate with factors of the molecular weight of anionic hbPG and the film thickness. It was demonstrated that anionic hbPG single layer with highest molecular weight can reduce non specific protein adsorption more efficiently than single layer with lower molecular weight and all the hbPG bilayers possessed excellent property of antifouling. rnPhosphorus dendrimer multilayers were successfully prepared as the platforms to detect DNA immobilization and hybridization. The effect of NaCl concentration on the multilayer film thickness was evaluated to obtain the optimized film thickness. Making use of the multilayer deposited at the optimized condition as a substrate, a high loading of DNA probes was achieved through covalent coupling of probe DNA with the as-formed multilayer films. The hybridization of target DNA with immobilized probe DNA was then carried out and studied by SPFS. The limit of detection upon hybridization was estimated on various dendrimer multilayer platforms. The minimum detection concentration for DNA hybridization is in the same order of magnitude compared with other neutral phosphorus dendrimer systems. Furthermore, the LbL deposition of phosphorus dendrimer multilayers provided a mild and simple way to prepare platforms as DNA microarrays. rnBased on the phosphorus dendrimer multilayer systems, dendritic star polymers were employed which have more reactive groups than that phosphorus dendrimers. The as-assembled dendritic star polymer multilayer films exhibited such distinct morphology characteristics that they underwent extensive structural reorganization upon post-treatment under different pH conditions. Kinetic binding of probe DNA molecules on the outermost negatively charged dendritic surface was studied by SPR as well. The binding capacities of probe DNA on the multilayer surfaces fabricated from the first-generation and the second-generation of dendritic star polymers were compared. The improved binding capacity was achieved from the second-generation of dendritic star polymer multilayer films due to their more reactive groups. DNA hybridization reaction on dendritic multilayer films was investigated by SPFS. The similar hybridization behaviors were found on both multilayer surfaces. Meanwhile, the hybridization kinetic affinities were compared with that of phosphorus dendrimer multilayer surfaces and showed improved detection sensitivity than phosphorus dendrimer multilayer films.rn