Orientador : Valdemiro Carlos Sgarbieri / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-27T19:15:11Z (GMT). No. of bitstreams: 1 Chaud_SaulaGoulart_D.pdf: 1321140 bytes, checksum: ab67e4743a9713d2e1ac51a83eb769d1 (MD5) Previous issue date: 2004 / Resumo: O presente trabalho teve por objetivo o fracionamento e a caracterização química da parede celular (PC) de levedura obtida como subproduto do processo industrial da produção de etanol, visando avaliar as propriedades funcionais e fisiológicas do material fracionado. A PC foi fracionada em glicoproteína (GP), glicana mais manana (G+M), glicana insolúvel (GI), glicana solúvel (GS) e manana (M) que foram caracterizadas quanto à composição centesimal, perfil de aminoácidos, minerais, ácidos graxos e quantificação de monossacarídeos. A proporção entre os componentes da PC foi, fibra (77,8%), com predominância (74%) de fibra solúvel, permanecendo no material 18 a 20% de proteína. O teor de açúcares totais da PC foi 80,8 g/100g e o teor de lipídios foi de 2,00%. Dentre os ácidos graxos saturados predominaram palmítico e esteárico, monoinsaturados palmitoléico e oléico, poliinsaturados linoléico e a¿ linolénico. Os principais elementos minerais foram cálcio, fósforo, potássio e ferro, sendo que a PC representa uma excelente fonte de ferro e cobre. As frações obtidas da PC foram M, 25,13% e GI, 42,92% p/p. As frações com maior conteúdo de fibra foram M, 70,34% de fibra solúvel, GI, 75,20% de fibra insolúvel, G+M, 60,23% fibra solúvel e GS, 70,73% fibra solúvel. Teores de açúcares redutores, M, 70%, com 48,4% de manose, GI, 28,95% e GS, 25,07% glicose. A fração G+M continha 28,08 g/100g de carboidratos totais e 20,0% de redutores totais. A GS apresentou a maior solubilidade, a G+M a menor solubilidade do N e de sólidos totais e a GI apresentou o menor índice de solubilidade. A GS apresentou a maior capacidade de retenção de água (14,36 g/g de amostra) e o menor índice (0,06%) de solubilidade em água. A fração GP reteve (5,33 g/g de amostra) e 83,83% de solubilidade. A viscosidade (95oC) e a viscosidade final (50oC) obedeceram à seguinte ordem: GS > GI > G+M > M > GP e PC. A GI (12% e 14% p/v) apresentou o maior índice de dureza de gel. As frações GP e M apresentaram as melhores propriedades emuslificantes que melhoraram significativamente com a adição de 0,2 , 0,4 e 0,6% (p/v) de ovoalbumina nas suspensões a 1% do material em estudo. Avaliou-se os efeitos da substituição da fibra de uma dieta padrão (AIN-P) por 10% de fibra de cada uma das frações da PC, formulando-se as dietas G+M, M, GI, GS. Ao final de 28 dias, os animais em dieta G+M ganharam menos peso que nas demais. As dietas AIN-M (padrão modificada com 10% celulose) e M foram as que proporcionaram um maior ganho de peso, seguidas das dietas padrão (AIN-P) e GI. O QEA da dieta G+M foi o menor ao longo dos 28 dias. Os maiores índices de digestibilidade da proteína foram observados nas dietas AIN-M, AIN-P e M. Com exceção da dieta G+M observou-se efeitos hipocolesterolêmico e hipolipidêmico em todas as dietas, porém, efeito hiperglicêmico em relação ao valor inicial (T0). Não houve modificações significativas na microflora intestinal dos animais em nenhuma das dietas. As quantidades de lipídios totais e colesterol excretados (fezes) variaram bastante, sendo que a dieta M apresentou maior especificidade para excreção do colesterol. Dentre os AGV, o ácido acético foi o predominante, seguido do propiônico e do butírico, em todas as dietas estudadas. As concentrações de ácido butírico nas dietas contendo M, G+M e G foram significativamente superiores aos padrões (AIN-P e AIN-M) / Abstract: The objectives of this work were the fractionation and chemical characterization of yeast cellular wall (YCW) from yeast biomass obtained as byproduct in the industrial production of ethanol, as well as the evaluation of functional and physiological properties of the various fractions. YCW was fractionated to obtain glycoprotein (GP), a faction containing glycan plus mannan (G+M), insoluble glycan (IG), soluble glycan (SG), and mannan (M), which were characterized for percent composition, amino acid profile, minerals, fatty acids, and monosaccharides. Proportions between YCW components included fiber (77.8%), with predominance (74%) of soluble fiber, remaining in the material 18 to 20% protein. Total sugar content of YCW was 80.8%/100 g and lipid content was 2.00%. Among the saturated fatty acids, the palmitic and stearic were predominant, among the monounsaturated acids, the palmitoleic and the oleic, and among the polyunsaturated acids, the linoleic acid and a¿linolenic acid. The most abundant mineral elements were calcium, phosphorous, potassium, and iron and the YCW showed to be an excellent source of iron and copper. Fractions obtained from YCW were: M 25.13% and IG 42.92% w/v. Fiber content in the isolated fractions were: M 70.34% soluble fiber, IG 75.2% insoluble fiber, G+M 60.23% soluble fiber, and SG 70.73% soluble fiber. Percentages of reducing sugars were M 70% with 48.4% mannose, IG 28.95% and SG 25.07% glucose. The fraction G+M contained 28.0 g/100g total carbohydrate and 20% reducing sugars. The SG presented the highest solubility and G+M the lowest N solubility and the IG presented the lowest total solids solubility. GS showed the highest water retention capacity (14.36 g/g sample) and the lowest (0.06%) solubility in water. The fraction GP showed retention of 5.33 g/g sample and solubility in water of 83.83%. The SG showed the highest viscosity at 50oC (heating phase). The viscosity at 95oC and the final viscosity (50oC) obeyed the following order: SG > IG > G+M > M > GP and YCW. The IG (12% and 14% w/v) presented the highest gel hardness. The fractions GP and M exhibited the best emulsifying properties, which improved significantly by adding 0.2, 0.4, and 0.6 ovoalbumin to the emulsifying media at 1%. Effects of fiber substitution were evaluated through alteration of control diet (AINP), substituting each YCW fraction with 10% of fiber, which resulted in the diets G+M, M, IG, SG. After 28 days, animals on a G+M diet had gained less body weight than the others. AIN-M (standard modified with 10% cellulose) and M diets promoted the highest growth, followed by AIN-P and IG. The DER of the G+M diet was the lowest along 28 days of experiment. Higher protein digestibility indexes were found for AIN-P, AIN-M, and M. With exception of the G+M diet, hypocolesterolemic and hypolipidemic effects were found for all the diets, compared to the starting point (T0). No significant modification of the intestinal microbiota was verified in any dietary treatment. Great variation was verified among the diets for fecal lipids and cholesterol excretion. The M diet showed greater capacity to excrete cholesterol in the rat feces. Among the SCFA, acetic acid predominated in all treatments, followed by propionic acid and butiric acid. Butiric concentrations were significantly higher in the M, G+M, and G diets compared with AIN-P and AIN-M / Doutorado / Doutor em Alimentos e Nutrição

Gene delivery via lentiviruses can yield long term expression of transgenes. Specificity of host cell targeting by viral vectors occurs primarily through viral glycoprotein (GP)/cellular receptor interactions. Ebola virus (EBOV) GP has broad tropism for a variety of cell types making this viral GP a potentially useful reagent for delivery of gene therapy. However, titers of EBOV GP pseudotyped lentiviruses are insufficient for practical use in clinical applications. Enhancement of EBOV-GP pseudotyped titers by as little as half a log might yield clinically applicable titers. In an alanine scanning study, we identified 19 residues in EBOV-GP1 that increased transduction efficiency two to three fold. When mapped onto the crystal structure of EBOV GP, these residues were primarily located at the interface of GP1/GP2 suggesting these residue substitutions may confer conformational changes in the protein structure thereby enhancing transduction efficiency. To determine if combinations of these alanine substitutions might further enhance transduction, we have introduced the changes into EBOV GP in a stepwise manner. To date, introduction of some combinations of alanine substitutions resulted in as much as an eight-fold increase in transduction over WT GP, this being our super mutant combination, whereas other combinations eliminated transduction. Identification of 5 additional mutations via 3D modeling of the glycoprotein uncovered an additional mutation in GP2, located at the GP1/GP2 interface, which also enhances EBOV GP transduction. Transduction of cell lines important for gene therapy including hepatocytes and porcine airway cells confirmed an enhancement in transduction as well. Other cell populations, specifically fibroblasts and renal cells, were also transduced but enhanced transduction was not observed indicating this phenomenon may be cell type specific. The in vivo studies were inconclusive because no expression was detected from any of the EBOV GP pseudovirions. Even expression of the positive control, GP64 particles, waned after 3 weeks post inoculation indicating insufficient quantities or poor quality pseudovirions were used. These EBOV GPs should prove useful for future gene therapy studies by providing an alternate glycoprotein that is as effective as GP64 at producing high titer lentiviral vectors.

A major problem associated with cellular cryopreservation is the recovery of cellular viability upon thawing. Current cryopreservation techniques use additives such as DMSO, sucrose and fetal bovine serum. However, each have their own respective cytotoxic issues. A significant factor in cryotoxicity is the formation of large ice crystals which can damage cellular components and cause dehydration. This has significant impacts for applications such as food preservation, scientific research, and tissue preservation. To this end, our laboratory is interested in synthesizing biologically-relevant compounds that can act as cryoprotectants by preventing the formation of large ice crystals in sub-zero temperatures. Our lab has previously synthesized structural analogues of native antifreeze glycoproteins (AFGPs, found in the blood of Antarctic cod), that possess the unique ability to inhibit ice-recrystallization. However, the mechanism by which they inhibit ice recrystallization is unclear. This thesis focuses on efforts made to understand this mechanism, and synthesize molecules that are more potent in ice recrystallization inhibition (IRI) activity compared to previously synthesized analogues. By assessing the IRI activity of various mono- and disaccharides, we have shown that the density of water molecules that surround a carbohydrate (Hydration Index) is directly proportional to the ability of sugars to inhibit ice crystal growth. In an effort to design functional AFGP analogues, various C-linked analogues were synthesized that contained different spacer lengths between the carbohydrate and the peptide backbone. Analyses of the solution conformations of these analogues showed that IRI-active AFGP analogues contain a distinct conformation in which the carbohydrate is oriented to form a hydrophobic pocket with the side chain. We hypothesize that this change in glycoconjugate hydration is responsible for disturbing its surrounding waters, thereby preventing water from adding to the ice lattice required for ice growth. Finally, SAR studies showed that threonine-containing AFGP and antifreeze proteins are more potent in antifreeze activity than serine-containing analogues. As the most potent AFGP analogue previously synthesized by our lab contains a C-linked-α-galactosyl -serine residue, we hypothesized that the analogous glycopeptide containing a C-α-galactosyl-threonine residue will be more potent in antifreeze activity. The final section describes efforts to synthesize a C-linked α-galactosyl-threonine glycoconjugate.

No description available.

A major problem associated with cellular cryopreservation is the recovery of cellular viability upon thawing. Current cryopreservation techniques use additives such as DMSO, sucrose and fetal bovine serum. However, each have their own respective cytotoxic issues. A significant factor in cryotoxicity is the formation of large ice crystals which can damage cellular components and cause dehydration. This has significant impacts for applications such as food preservation, scientific research, and tissue preservation. To this end, our laboratory is interested in synthesizing biologically-relevant compounds that can act as cryoprotectants by preventing the formation of large ice crystals in sub-zero temperatures. Our lab has previously synthesized structural analogues of native antifreeze glycoproteins (AFGPs, found in the blood of Antarctic cod), that possess the unique ability to inhibit ice-recrystallization. However, the mechanism by which they inhibit ice recrystallization is unclear. This thesis focuses on efforts made to understand this mechanism, and synthesize molecules that are more potent in ice recrystallization inhibition (IRI) activity compared to previously synthesized analogues. By assessing the IRI activity of various mono- and disaccharides, we have shown that the density of water molecules that surround a carbohydrate (Hydration Index) is directly proportional to the ability of sugars to inhibit ice crystal growth. In an effort to design functional AFGP analogues, various C-linked analogues were synthesized that contained different spacer lengths between the carbohydrate and the peptide backbone. Analyses of the solution conformations of these analogues showed that IRI-active AFGP analogues contain a distinct conformation in which the carbohydrate is oriented to form a hydrophobic pocket with the side chain. We hypothesize that this change in glycoconjugate hydration is responsible for disturbing its surrounding waters, thereby preventing water from adding to the ice lattice required for ice growth. Finally, SAR studies showed that threonine-containing AFGP and antifreeze proteins are more potent in antifreeze activity than serine-containing analogues. As the most potent AFGP analogue previously synthesized by our lab contains a C-linked-α-galactosyl -serine residue, we hypothesized that the analogous glycopeptide containing a C-α-galactosyl-threonine residue will be more potent in antifreeze activity. The final section describes efforts to synthesize a C-linked α-galactosyl-threonine glycoconjugate.

Thesis (Ph. D.)--Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 2000. / "February, 2001." "Funding was provided by National Institute of Health grant ES-07381 and a Rinehart Coastal Research Center grant." Includes bibliographical references (p. 174-177).

Thesis--University of Kentucky (Ph. D.), 2003. / Title from document title page. Document formatted into pages; contains vii, 65 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 60-65).

P-glycoprotein (Pgp; ABCB1) is a well known transporter involved in energy-dependent-drug efflux activity. At the brain capillary endothelium, its luminal membrane location is ideal for its ascribed role in the physiological efflux of a wide array of structurally and functionally diverse compounds from the brain. This is a critical issue in regards to the delivery of central nervous system (CNS)-acting therapeutics. Moreover, a dysregulation of Pgp has been implicated in specific CNS disease states, including Alzheimer's disease, epilepsy, and brain cancer where an upregulation of Pgp has been well established as a mediator of multi-drug resistance. Inflammation is a common component in all of these conditions. Previously our laboratory has reported changes in BBB molecular and functional properties during inflammatory pain (Huber et al. 2001). This has led us to investigate the effects of peripheral inflammatory pain on Pgp efflux transport properties at the BBB, in vivo. In the present study we examined the effects of lambda-carrageenan-induced inflammatory pain (i.e. hyperalgesia; CIP) on the molecular and functional properties of Pgp at the BBB. Western blots using enriched fractions of isolated rat brain microvessels revealed that Pgp expression at the BBB was increased by CIP and that this increase occurred predominantly within the membrane region of the cell. Additionally, both in situ brain perfusions and whole body antinociceptive profiling of the Pgp substrate and opioid analgesic, [3H] morphine, indicate that changes in Pgp at the BBB, mediated by peripheral inflammation, can impact brain uptake of morphine. To further elucidate the mechanism(s) behind the rapid upregulation (3 h) of Pgp at this region, we explored regulation of Pgp at the plasma membrane. Our findings show that CIP induces a movement of Pgp within these domains and that Pgp co-localizes with caveolin-1 and clathrin, key structural proteins associated with caveolae and clathrin-pit lipid rafts, respectively. Our data indicate for the first time that peripheral inflammatory pain induces functional and molecular changes in Pgp, a critical efflux transporter, at the BBB in vivo and that these alterations may be mediated in part via a proteolipidic re-organization mechanism.

The development of multidrug resistance (MDR) in tumour cells to a wide range of anticancer drugs has become a major obstacle in the chemotherapeutic treatment of cancer. Molecular characterization of MDR tumour cells has led to the identification of several cell-based genetic alterations including the overexpression of a membrane protein, P-glycoprotein (P-gp). P-gp is a ATP dependent drug efflux pump and P-gp ATPase activity has been demonstrated to be essential in drug transport. In an effort to understand how P-gp ATPase activity is coupled to drug binding and transport, we examined the effects of N-ethylmaleimide (NEM), a potent inhibitor of P-gp ATPase, on P-gp drug binding and transport. Our results show that short term treatment of MDR cells with NEM led to a concentration-dependent increase in P-gp drug binding and phosphorylation. In addition, NEM increases [3H]-vinblastine accumulation in drug resistant cells but not in sensitive cells. Our study suggests that inhibition of P-gp ATPase activity, and not increased phosphorylation of P-gp by NEM, is responsible for the observed increase in P-gp-drug binding. / Selection of tumour cell lines in vitro has led to multiple cellular changes that may mediate drug resistance to anticancer drugs. The role of other mechanisms, in addition to P-gp and multidrug resistance protein (MRP) in drug resistance, is supported by evidence from studies with tumour cell lines and clinical tumours. In an effort to identify other cellular changes that may be important in tumour drug resistance to anticancer drugs, we have used a differential immunodot blot method to isolate monoclonal antibodies that bind to proteins in drug resistant but not in drug sensitive cells. By using the immunodot blot method, we have isolated a monoclonal antibody (IPM96) which recognized a 40 kDa protein (P-40) in several MDR cell lines. The expression of P-40 is concurrent with the level of drug resistance. Biochemical characterization showed P-40 to be associated with the cell membrane and in the soluble fraction. Molecular cloning of P40 cDNA revealed that P-40 is identical to annexin I, a substrate for the epidermal growth factor receptor tyrosine kinase. The observed increase in P-40 (or annexin I) protein levels in drug resistant cells is due to the elevation of P-40 transcripts. The pharmacological characterization of P-40 cDNA transfectants (P-40-MCF-7) has demonstrated that overexpression of P-40 in drug sensitive cells is capable of conferring drug resistance to adriamycin, actinomycin D, Taxol and cisplatin. Taken together, our study provides convincing evidence that annexin I is important in the development of drug resistance in cancer cells. In addition, it suggests a novel mechanism of drug resistance that is different from the ATP-dependent drug efflux pumps that mediate P-gp- and MRP-associated MDR

Resistance to ivermectin (IVM) in Haemonchus contortus has developed in many countries and its mechanism is still under investigation. P-glycoproteins (P-gp) are transmembrane proteins that can transport drugs out of cells. Researchers have found that there is polymorphism in a P-gp gene from H. contortus between IVM-selected and unselected worms. Three main P-gp polymorphs were identified, polymorph A was found to be related to IVM selection, while polymorphs B and X were associated with susceptibility. The purpose of this research is to investigate the genetic variations in P-glycoprotein that are associated with IVM selection or susceptibility in H. contortus. Total RNA and genomic DNA were extracted from individual male adult worms of IVM-selected and unselected strains of H. contortus. A fragment of the P-gp gene was amplified from the genomic DNA of individual worms and RFLP analysis was performed on the PCR product to genotype the corresponding worms. The homozygous worms that possessed polymorph A, B or X were identified.