Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of three mitochondrial and one cytosolic forms of biotin-dependent carboxylases in humans. Patients suffering from this autosomal recessive disease have Multiple Carboxylase Deficiency (MCD) with symptoms of life-threatening metabolic acidosis which, in almost all cases, can be successfully treated with pharmacologic doses of oral biotin. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS maybe targeted to the cytoplasm and mitochondria or that carboxylases are biotinylated in the cytoplasm prior to import into the mitochondria. In order to resolve the compartmentalization of HCS, 5' HCS cDNA sequences have been examined for a targeting signal and a candidate sequence was tested for its capacity to target mitochondria. Analysis of 5' cDNA reveals complex alternative splicing, none of which appear to contain mitochondrial targeting sequences. In addition, antibodies have been developed in order to perform immunochemical analysis of the subcellular distribution of HCS. Polyclonal antisera were raised against full length HCS as well as two peptides corresponding to a 20 amino acid region in the N-terminus and to the 20 amino acids preceding the stop codon. Immunohistochemical staining of human fibroblasts with the antibody to full length HCS gives cytosolic, mitochondrial and nuclear localization. Interestingly, analysis with the N-terminal antiserum reveals a large punctate staining pattern exclusively localized to the nucleus. The corresponding C-terminal antiserum reveals solid nuclear staining with some mitochondrial co-localization. Taken together, these results indicate the ubiquitous nature of HCS in human cells and also allude to a potential role for HCS in the nucleus of human cells.

Bibliography: leaves 130-159. / 159, [61] leaves, [7] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the physical and catalytic properties of a biotin protein ligase and aims to understand further the interaction of this enzyme with its protein substrates. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2000?

Thesis (Ph. D.)--University of Wisconsin--Madison, 1951. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

University, Diss., 2005--Bielefeld.

Orientador: Maria del Pilar Taboada Sotomayor / Resumo: A biotina (vitamina B7) pode ser acoplada a diferentes tipos de moléculas e, ainda assim, ser reconhecida seletivamente pelas proteínas avidina e estreptavidina devido à alta constante de afinidade que existe entre elas (Ka = 1,0×1015 L mol-1). Assim, essa interação proporciona excelente aumento na sensibilidade de várias análises; o que leva ao desenvolvimento métodos analíticos para determinação de diferentes compostos que são biotinilados. O uso de materiais biológicos, como anticorpos ou proteínas, em análises de rotina se torna muitas vezes dispendioso e, devido à baixa estabilidade das biomoléculas em condições adversas às mesmas, iniciou-se a síntese de materiais que possam mimetizar as interações biológicas de reconhecimento; o que vem sendo feito com sucesso pelos polímeros molecularmente impressos (MIP). Esses polímeros apresentam vantagens em relação às biomoléculas por possuirem baixo custo, fácil preparação e alta resistência mecânica e térmica. Assim, esta dissertação aborda a síntese, caracterização, otimização e aplicação de diferentes MIP que possuem biotina como molécula alvo. Os estudos iniciais focaram na síntese do MIP magnético (MMIP) com acesso restrito (RAMIP) para realizar exclusão proteica pela modificação do polímero com albumina do soro bovino (BSA) e polietileno glicol (PEG). Enquanto o RAMIP recoberto com BSA não apresentou bons resultados nos experimentos, o RAMIP recoberto com PEG, após otimização, apresentou resultados altamente satisfatórios ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Biotin (vitamin B7) can be attached with different types of molecules and still be selectively recognized by avidin/streptavidin proteins due to the high affinity constant between them (Ka = 1,0×1015 L mol-1). Thus, this interaction provides an excellent increase in the sensitivity of analyzes; which leads to the development of analytical methods for the determination of different biotinylated compounds. The use of biological materials, such as antibodies or proteins, in analyzes is often expensive and has low stability of biomolecules under adverse conditions, which has led to the synthesis of materials that can mimic the biological interactions of recognition; which has been successfully made by molecularly printed polymers (MIP). These polymers have advantages over biomolecules because they have low cost, easy preparation and high mechanical and thermal resistance. Thus, this dissertation explores the synthesis, characterization, optimization and application of different MIP that has biotin as the target molecule. Initial studies focused on the synthesis of magnetic MIP (MMIP) with restricted-access (RAMIP) to evaluate protein exclusion by modifying the polymer with bovine serum albumin (BSA) and polyethylene glycol (PEG). While RAMIP coated with BSA didn't present good results in the experiments, the RAMIP coated with PEG, after optimization, presented highly satisfactory results in protein exclusion (with values ranging from 98 to 99.4% of protein exclusion) and good res... (Complete abstract click electronic access below) / Mestre

In this study, water soluble CdTe QDs of different emission wavelengths were prepared. Microwave synthesis was used to prepare CdTe QDs coated with mercaptopropionic acid (MPA). MPA ensures biocompatibility and good water solubility of QDs. Therefore QDs can be used directly in bioanalytics. Further, QDs modified with biotinylated glutathione (GSH) were prepared. In this case mercapto group (--SH) ensures a bonding of GSH to QDs surface and biotin serves as high specific linker for streptavidin, avidin or neutravidin modified analyte. Finally, peptide mediated immuno-quantum dots were prepared. CdTe QDs were coated with heptamer HWRGWVC which is able to bind Fc region of human immunoglobulin G.

La première partie de cette thèse est dédiée à la modification de polyélectrolytes pour former des films de multicouche de polyélectrolytes (PEM) ayant des propriétés d’adhésion de protéine et de cellules bien contrôlées et modifiables par étirement. L’acide polyacrylique a été modifié avec des groupes latéraux phosphorylcholine (PC) à des taux de 25 % (PAA-PC) ou avec des chaînes oligo(éthylène oxyde) terminées par la biotine : (EO)nBiotine (n = 0, 3, 9 et 18) avec de taux de modification de 1, 5, 10 ou 25 %. Des PEM incorporant ces polymères lient spécifiquement la streptavidine et repoussent tout autre protéine. Les propriétés d’adsorption et la sélectivité de ces PEM ont été mesurées par microbalance à quartz. Sur un substrat de PDMS étirables, on a construit des PEM terminés par un PAA portant des RGD recouvert par deux couches contenant PAA-PC. Au repos, seuls les PC sont exposés et inhibent l’adhésion cellulaire ; sous étirement, les groupes RGD sous-jacents sont exposés et déclenchent l’adhésion de fibroblastes.La deuxième partie est consacrée à l‘étude d’acide polyméthacrylique modifié hydrophobiquement avec des chaînes alkyle liées par des esters à la chaîne principale. 3 chaînes différentes ont été greffées : -C12H25 ; -C18H35 et C4H8-OOC- C11H23 avec des taux de 1, 5 and 10 %. Ces polymères sont associatifs et forment des hydrogels dans des tampons physiologiques pour des taux de modifications de 5% et des concentrations supérieures à 4% en poids. Ces gels ont été caractérisés par des mesures rhéologiques. Leur incubation avec des lipases provoque une baisse de leur viscosité, interprétable par une coupure des esters. Quand les gels faits à partir du PAA-C12 sont incubés avec une culture de Pseudomonas aeruginosa, la viscosité baisse également, ce qui montre que les chaînes sont également coupées in vivo. / The first part of this thesis is dedicated to the modification of polyelectrolytes to form polyelectrolyte films with controlled and stretch responsive cell and protein adsorption properties. Poly(acrylic acid) (PAA) was modified with side phosphorylcholine groups (PC) at rates of 25 % or with oligo(ethylene oxide) chains ended by biotin ((EO)nBiotin, (n =0, 3, 9 and 18) at 1, 5, 10 and 25 % modification rates. Polyelectrolytes multilayer films (PEM) containing these polyelectrolytes bind selectively streptavidin but repel all other proteins. The adsorption properties and selectivity were measured by quartz crystal microbalance. On a stretchable PDMS substrate, we have built PEM ended by PAA bearing RGD, covered by two PAA-PC layers on the top. Under rest, only the PC groups are exposed and prevent cell adhesion; when the film is stretched, the underlying RGD groups are exposed, and trigger adhesion of fibroblasts.The second part was consecrated to the study of poly(methacrylic acid) hydrophobically modified with alkyl chains connected through an ester moiety to the main chain. Three different chains were grafted -C12H25; -C18H35 and -C4H8- OOC-C11H23 with a rate of 1, 5 and 10 %. These polymers associate in water and form hydrogels in physiological buffer, for modification rates higher than 5 % and polymer concentrations higher than 4 wt. %. The gels were characterized by rheology. Their incubation with lipases resulted in a decrease of their viscosity, which could be interpreted by the cleavage of the hydrophobic side chains, by rheological tests. When the gels with PAA-C12 were incubated with a culture of Pseudomonas aeruginosa, their viscosity decreased, which shows that alkyle chains are also cleaved in vivo.

Orientador: Gabriela Alves Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T18:55:36Z (GMT). No. of bitstreams: 1 Ohara_Andre_M.pdf: 6935792 bytes, checksum: 553cf8492d7af096c35b5c595ec6e3a1 (MD5) Previous issue date: 2014 / Resumo: A bioprospecção de micro-organismos representa um grande potencial tecnológico para produção de biocompostos de interesse comercial. Dentre estes compostos estão as enzimas lipolíticas, como as lipases (triacilglicerol acil-hidrolases EC 3.1.1.3) que apresentam um enorme potencial para aplicações biotecnológicas. Outro tipo de biocomposto de relevante interesse comercial são as vitaminas do complexo B, as quais possuem função essencial na atividade de enzimas que regulam reações do metabolismo. Desta forma, o objetivo desse trabalho foi isolar e selecionar linhagens de leveduras do solo de diferentes biomas do Estado de São Paulo com potencial para produção de lipase e de vitaminas do complexo B (biotina e riboflavina). Para tanto, foram isoladas 132 leveduras de amostras de solos provenientes da Mata Atlântica e da região de transição (Mata Atlântica e Cerrado). Essas linhagens foram depositadas na coleção de micro-organismos do laboratório de Bioquímica de Alimentos da FEA-UNICAMP, que já contava com 300 leveduras provenientes da região de Cerrado. As 432 linhagens presentes na coleção foram então avaliadas quanto ao potencial para a produção de lipase extracelular através de seleção em meio sólido diferencial e cultivo em meio líquido, sendo a atividade de lipase determinada no sobrenadante por titulometria de neutralização. O potencial para a produção de biotina e riboflavina extracelular foi avaliada por meio de seleção em meio de cultivo líquido, sendo as concentrações das vitaminas determinadas no sobrenadante por espectrofotometria e fluorimetria. Desta forma 33 leveduras apresentaram potencial para produção de lipase alcançando valores de atividade enzimática que variaram de 6,51 U/mL a 21,44 U/mL. Em relação à produção das vitaminas do complexo B, 38 linhagens apresentaram concentrações de biotina no sobrenadante do cultivo que variaram de 0,28 µg/mL a 18,61 µg/mL e 64 linhagens apresentaram concentrações de riboflavina que variaram de 0,03 µg/mL a 0,77 µg/mL. A linhagem RP.C153 apresentou potencial para produção de lipase e riboflavina, enquanto a linhagem RP.J1308 para produção de lipase e biotina. A linhagem RP.C153 foi classificada como Pichia caribbica e a linhagem RP.J1308 como Candida oleophila / Abstract: The bioprospection of microorganisms represents a great technological potential for the production of biocompounds of commercial interest. Among these compounds are the lipolytic enzymes, such as lipases (triacilglicerol acilhidrolases EC 3.1.1.3) which have currently a huge potential for biotechnological applications. B vitamins are another type of biocompound with relevant commercial interest, which have essential role in the activity of enzymes that regulate metabolic reactions in living organisms. Therefore, the aim of this work was to isolate and select strains of yeasts from the soil of different biomes present in the State of São Paulo for the biotechnological production of lipase and B vitamins (biotin and riboflavin). Thus, 132 yeasts were isolated from soil samples of the Atlantic Forest (Ilha Bela - SP) and the transition region between the Atlantic Forest and Savanna (Campinas - SP). These strains were deposited in the collection of microorganisms of the Food Biochemistry Laboratory FEA - UNICAMP, which already had 300 yeasts from the Savanna region (Ribeirão Preto - SP). Therefore 432 strains present in the collection were then evaluated for their potential to produce extracellular lipase by selection on solid selective differential and liquid medium culture, and the enzyme activity in the supernatant was determined by neutralization titration. The potential production of extracellular biotin and riboflavin was assessed by selection in medium liquid culture, and the concentrations of those vitamins were measured in the supernatant by spectrophotometry and fluorimetry. Therefore 33 yeasts demonstrated potential for the production of lipase, reaching values of enzymatic activity ranging from 6.51 U/mL to 21.44 U/mL. In relation to the production of B vitamins, 38 strains presented concentrations of biotin in the supernatant culture ranging from 0.28 µg/mL to 18.61 µg/mL and 64 strains demonstrated concentrations of riboflavin ranging from 0.03 µg/mL to 0.77 µg/mL. The strain RP.C153 presented potential for production of lipase and riboflavin, while strain RP.J1308 for production of lipase and biotin. The RP.C153 strain was classified as Pichia caribbica and RP.J1308 strain as Candida oleophila / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Indiana University-Purdue University Indianapolis (IUPUI) / The nuclear protein SETMAR has been reported to be involved in many processes such as non-homologous end joining (NHEJ), di-methylation (arguably) of K36 of histone H3, restart of stalled replication forks, chromosome decatenation, enhancing of TOPII inhibitors which results in resistance to chemotherapeutics in cancer patients, etc. All these purported functions are impossible to execute without interaction with other proteins. It is established that SETMAR binds specifically to DNA at terminal inverted repeat sequences and can loop DNA. This DNA sequence specific pull-down exploits this attribute to identify possible protein interactors of SETMAR. As a result of this experiment several proteins have been identified for further research: BAG2, c12orf45, PPIA, XRCC5/6, and ZBTB43, all of which are found in higher statistical abundances in full length SETMAR samples.