Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Title from title-page of electronic thesis. Prepared for: Dept. of Microbiology & Immunology. Bibliography: leaves 289-332.

Thesis (M.S.)--Youngstown State University, 1995. / Includes bibliographical references (leaves 44-48).

Thesis (Ph.D.)--Kent State University, 2009. / Title from PDF t.p. (viewed April 9, 2010). Advisor: John Chiang. Includes bibliographical references (p. 225-256).

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 289-332. Also available online via the Internet.

Thesis--Uppsala.

In order to gain an understanding of the interaction of probe molecules with NaCh aggregates, steady-state and time-resolved photophysical methods were used. By employing several probe molecules, an understanding of how the structure of the probe affected binding to the two distinct sites in the aggregates was investigated. The first half of the thesis examined the reactivity of benzophenone (Bp) and 4,4-dimethylbenzophenone (DMBp) with NaCh aggregates by studying the kinetics of both the ketone triplet excited states and the ketone ketyl radicals. There were three species of triplet excited states observed in the presence of primary aggregates. One of the ketone triplet excited state species was located within the primary aggregate, and it reacted to form ketyl radicals. Other triplet states included in the primary aggregate were found to be long-lived, while a third species of triplet states was present in the aqueous phase. At higher bile salt concentrations, and in the presence of secondary aggregates, a layer of complexity was added. The binding dynamics for the triplet excited state with the secondary binding sites were much faster than those observed for the primary binding site. Hydrogen abstraction did not compete with other deactivation pathways in the presence of secondary aggregates, and thus only self-quenching and exit of the excited state probe from the secondary site were observed. Ketyl radical recombination took place in water and in the secondary sites. The second half of the research focused on the study a series of naphthalene (Np) derivatives in order to look at the effects of shape and hydrophobicity of probe molecules on the interactions between these probes and the host NaCh aggregates. 1-Ethylnaphthalene, 2-ethylnaphthalene, 1-acetonaphthone, 2-acetonaphthone, 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol were studied. 1-Ethylnaphthalene and 2-ethylnaphthalene were Ethylnaphthalene, 2-ethylnaphthalene, 1-acetonaphihone, 2-acetonaphthone, 1-naphthyl- 1-ethanol and 2-naphthyl-1-ethanol were studied. I-Ethylnaphthalene and 2- ethylnaphthalene were contained within the primary binding site, while 1-naphthyl-1-ethanol, 2-naphthyl-1-ethanol, 1-acetonaphthone and 2-acetonaphthone were contained within the secondary binding site. The effect of the position of the substituent was only noticed when the probe molecules formed weak interactions with the outside of the primary aggregate, and not when the probe was complexed to one of the binding sites present in the NaCh system. The naphthalene probe molecules were also used to study the effect of ionic strength on NaCh aggregate formation. It was found that primary aggregation occurred at lower NaCh concentration as the ionic strength was increased. No effect of ionic strength was observed on the formation of secondary aggregates. All the findings in this study are consistent with an aggregation model in which two distinct binding sites are present. The shape of the probe as well as its hydrophobicity are critical to its interaction with the NaCh aggregates. From these dynamic studies it was found that only a small number of NaCh monomers (6-13) are needed to define both the primary and secondary binding sites. / Graduate

Orientadores: Luiz Sergio Leonardi, Michel Cremer, Luis Alberto Magna / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T11:06:50Z (GMT). No. of bitstreams: 1 Trentini_ElianeAnrain_M.pdf: 2064169 bytes, checksum: 0b3a3597ddde6867786067aded166323 (MD5) Previous issue date: 2006 / Resumo: Não há, ainda, um modelo ideal de reconstrução das lesões extensas da via biliar. A reconstrução em Y de Roux é clássica, com vantagens como baixa incidência de refluxo de conteúdo intestinal para as vias biliares. Porém, como não é anatômica, ela dificulta enormemente ou impede o acesso endoscópico à via biliar. Uma técnica fisiológica para a substituição do colédoco é a interposição de segmento pediculado de jejuno entre a via biliar e o duodeno, descrita desde 1950 e realizada com êxito em número expressivo de pacientes. Contudo, esta técnica não se tornou amplamente utilizada. Com o intuito de reconstruir a via biliar de maneira fisiológica foi proposta a aplicação do princípio de Monti às vias biliares, que já está estabelecido em humanos para vias urinárias. Nesta técnica, faz-se a detubularização e retubularização transversa de um segmento de jejuno, promovendo uma modificação no sentido das pregas mucosas, tornando-as longitudinais o que facilita o fluxo de líquidos no seu interior. Ao aplicá-la na reconstrução das vias biliares possibilita-se, sobretudo, o acesso endoscópico, diagnóstico e terapêutico, às vias biliares. No presente estudo, foram operados 13 cães: inicialmente foi realizada ligadura laparoscópica do colédoco dos cães para provocar dilatação da via biliar e icterícia obstrutiva. Após uma semana, foi realizada derivação biliodigestiva por laparotomia com a interposição do tubo jejunal acima descrito entre a via biliar dilatada e o duodeno. Os cães foram submetidos a dosagens bioquímicas de transaminases glutâmico-pirúvica e glutâmico-oxalacética, bilirrubinas totais, fosfatase alcalina e gamaglutamiltransferase no pré-operatório das cirurgias e semanalmente, até a eutanásia, realizada seis semanas após a derivação biliodigestiva, quando foi realizada nova laparotomia e ressecção da peça via biliar-tubo jejunal-duodeno em monobloco para análise macroscópica. Foi coletada bile dos cães por ocasião da derivação biliodigestiva e no sacrifício. Dos 13 submetidos a ligadura laparoscópica de colédoco, um foi excluído porque não alcançou significativa dilatação da via biliar. Após a derivação biliodigestiva três cães morreram; destes três, apenas um apresentou peritonite à necropsia. Portanto, nove cães tiveram seus dados submetidos à análise estatística. Eles apresentaram icterícia obstrutiva após sete dias de ligadura do colédoco, comprovada por exames bioquímicos. Todos os nove animais apresentaram redução gradativa, estatisticamente significativa, de sua colestase após derivação biliodigestiva com a interposição do tubo jejunal pediculado e mantiveram-se saudáveis até o término do experimento. Os valores médios de bilirrubina total, fosfatase alcalina e gamaglutamiltransferase uma semana após ligadura da via biliar foram: 4,39; 3251,7 ; 66,1. Os valores dessas variáveis seis semanas após a derivação biliodigestiva foram 0,11 ; 323,1 ; 10,7, respectivamente. Concluiu-se que o tubo de segmento jejunal interposto entre o colédoco previamente ligado e o duodeno foi eficaz na descompressão da via biliar. A análise macroscópica das peças coletadas mostrou boa integração via biliar-tubo e tubo-duodeno. Com a abertura longitudinal das peças observou-se ótima cicatrização das estruturas anastomosadas e perviedade do tubo jejunal / Abstract: An ideal model for reconstruction of extensive lesions of the biliary tract has not been found so far. The Roux-en-Y reconstruction is a classic reconstruction and presents advantages as the low incidence of intestine contents reflux to the biliary tract. However, since this is not anatomical, it impedes or impairs the endoscopic access to the biliary tract. A physiological technique to replace the common bile duct is the interposition of a pediculated segment of jejunum between the biliary tract and the duodenum, described since 1950 and successfully performed in several patients. However, this technique has not been widely adopted. The present experimental study was proposed for reconstruction of the biliary tract in a physiological manner, by application of the Monti principle to the biliary tract, which is well established in humans for the urinary tract. This technique comprises detubulization and transverse retubulization of a segment of jejunum, changing the mucosal folds in longitudinal direction, thus enhancing the flow of liquids inside it. Its application for reconstruction of the biliary tract would allow endoscopic access to the biliary tract, for both diagnostic and therapeutic purposes. Thirteen dogs were operated in the present study; initially, laparoscopic ligation of the common bile duct of dogs was performed to induce extrahepatic cholestasis. After one week, biliodigestive derivation was performed by laparotomy with interposition of the aforementioned jejunal tube between the dilated biliary tract and the duodenum. The dogs were submitted to biochemical dosage of alanine and aspartate transaminases, total bilirrubin, alkaline phosphatase and gamma-glutamyltransferase preoperatively and weekly for six weeks postoperatively. Another laparotomy was then performed with resection of a monoblock specimen from the biliary tract-jejunal tube-duodenum for macroscopic analysis and the animals were killed. Bile was collected from the dogs upon biliodigestive derivation and upon killing. From the 13 animals submitted to laparoscopic ligation of the common bile duct, one was excluded because significant dilation of the biliary tract was not achieved. Three dogs died after biliodigestive derivation; among these, only one exhibited peritonitis upon autopsy. Thus, data on nine dogs were submitted to statistical analysis. These dogs exhibited obstructive jaundice at seven days after ligation of the common bile duct, as demonstrated by biochemical examinations. All nine animals presented statistically significant gradual reduction of cholestasis after biliodigestive derivation by interposition of a pediculated jejunal tube and were healthy until study completion. The mean values of total bilirubin, alkaline phosphatase and gamma glutamyltransferase at one week after ligation of the biliary tract were: 4.39; 3251.7; and 66.1. The values of these variables at six weeks after biliodigestive derivation were 0.11 ; 323.1 and 10.7, respectively. It was concluded that interposition of a jejunal segment tube between the previously ligated common bile duct and the duodenum was effective for decompression of the biliary tract. Macroscopic analysis of the collected specimens revealed good integration between the biliary tract and the tube and between the tube and the duodenum. Longitudinal sectioning of the specimens revealed optimal healing of the anastomosed structures and patency of the jejunal tube / Mestrado / Cirurgia / Mestre em Cirurgia

Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.

Orthotopic liver transplantation is the treatment of choice for many patients with end-stage liver disease. Despite advances in immunosuppression, acute rejection remains common (up to 70%) and results in significant patient morbidity. It is frequently difficult to distinguish abnormal liver function due to rejection from that due to infection, biliary obstruction or ischaemic injury without performing invasive procedures such as a liver biopsy or angiography which may be Clinically, the diagnosis of rejection is usually once the immunological process is already hazardous. made late, established. In this study, we evaluated standard biochemical parameters and cytokine concentrations (IL-1, IL-6 and TNF-alpha) in serial samples of bile obtained post-operatively via the Ttubes of patients following orthotopic liver transplantation in order to determine whether there are any biochemical or immunological pointers to the early diagnosis of rejection which would enable earlier administration of appropriate antirejection therapy. Biliary cytokines did not prove to be useful and reliable markers of early rejection. Serial measurement of biliary bilirubin levels showed an early and significant decrease a few days prior to rejection, and were a more sensitive marker of graft function than serum bilirubin levels.