The purposes for which the fractionation of proteins are carried out are quite varied and manyfold. However, one of the more important reasons is that of determining the nature and the extent of autolysis or proteolysis on the bovine muscle proteins during post mortem aging. Hence, the development of a procedure for the adequate fractionation of muscle proteins would greatly stimulate the interest and research progress in this difficult field of study. The research reported herein pertains to a study of the fractionation of fresh bovine muscle proteins by ion exchange chromatography. A KCl-phosphate buffer, pH 7.5 and an ionic strength of 0.55, was used to extract the proteins from the muscle. This extract was then diluted to specific ionic strengths in order to separate the gross fractions (actomyosin, myosin, sarcoplasmic + actin) from the total KCl-phosphate soluble proteins. The major protein fractions were then separated by diethylaminoethyl-cellulose (DEAE-cellulose) ion exchange chromatographic procedure. A non-linear gradient elution schedule was used throughout the chromatographic procedure. The disc electrophoresis technique was used to determine the homogeneity of the various protein fractions and sub-fractions. The total KCl-phosphate soluble proteins were fractionated into 9-12 fractions by DEAE-cellulose ion exchange chromatography. The number of fractions differed from one sample to another. The disc electrophoresis results paralleled those of the chromatographic procedure. Some of the fractions appeared to be homogeneous while others were not. The KCl-phosphate soluble proteins minus actomyosin were fractionated by column chromatography into 9-10 different protein components. The disc electrophoresis results also indicated that this fraction contained 9-10 components. The sarcoplasmic + actin proteins were fractionated into six fractions by the chromatographic procedure. These fractions appeared to be electrophoretically homogeneous. Although some success was attained in the separation of the actomyosin proteins, the myosin fraction was quite resistant to chromatographic separation. In spite of the ineffectiveness of the DEAE-cellulose column chromatographic technique to fractionate the myosin and actomyosin proteins, the procedure appears to have some value for studying the other protein fractions during autolysis. Moreover, further work on the adoption of this procedure might provide the necessary information to perfect the technique for muscle protein fractionation. / Graduation date: 1963

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1966. / Bibliography: leaves 88-91. / xii, 91 l illus., tables

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1969. / Bibliography: leaves [174]-183. / xiii, 183 l illus

This thesis investigated the helical structure of core histone octamers to discover interacting surfaces and their relevance to the compaction of nucleosome arrays into the chromating fiber.

A fi d'analitzar la contribució de la regió C-terminal proposada com a iniciadora del plegament (CFIS 106-118) a l'estabilitat de l'RNasa A, els residus alifàtics d'aquesta regió es van substituir, mitjançant mutagènesi dirigida, per altres residus en els quals la cadena lateral alifàtica era rogressivament escurçada. La major part de les substitucions projectades suposaven delecions no disruptives de grups metil(è). A més, es va reemplaçar la Tyr115 per un Trp, de manera que, potencialment, s'introduïa una única sonda fluorescent, no desestabilitzant, per tal de seguir els canvis conformacionals que es poguessin generar en la regió durant el procés de legament/desplegament de la proteïna. Tant els paràmetres cinètics, com els espectres d'FTIR i CD, determinats per cadascuna de les ribonucleases variants, indiquen que els reemplaçaments aminoacídics efectuats presenten, en general, poc o cap efecte en l'estructura nativa i en l'activitat de l'enzim. Es va emprar l'espectroscòpia d'absorció a l'ultraviolat de quarta derivada, la fluorescència (per la variant amb Trp) i l'espectroscòpia d'infraroig per transformada de Fourier, per tal de seguir i caracteritzar, en condicions d'equilibri, les transicions conformacionals de cada variant en funció de la pressió i de la temperatura. Els resultats es van comparar amb els que es van obtenir per la proteïna salvatge. Per determinar més a fons les característiques del procés de desplegament de la variant Y115W, les transicions de desnaturalització induïdes per urea d'aquesta variant i de la proteïna salvatge, van ésser examinades per mitjà d'electroforesi en gradient d'urea i espectroscòpia de fluorescència. Curiosament, els canvis conformacionals que resulten de la desnaturalització per pressió són molt semblants als que s'obtenen per temperatura. Enfront d'un augment gradual tant de pressió com de temperatura, l'estructura terciària i els elements d'estructura secundària de les proteïnes estudiades es perden de manera conjunta i reversible. Aquestes variacions estructurals que es promouen descriuen un procés de desplegament molt cooperatiu i en dos estats. Atès que ambdues tècniques (UV i FTIR) utilitzen cadascuna un règim de concentració proteica molt diferent, els resultats indiquen que el procés de desplegament per pressió i per temperatura és intramolecular. Els resultats obtinguts suggereixen que la hidrofobicitat i el volum de les cadenes laterals del CFIS, juntament amb les interaccions de van der Waals entre elements d'estructura secundària intervenen de manera molt notable en l'estabilització de la proteïna. Entre els diferents aminoàcids alifàtics que pertanyen al CFIS C-terminal, la Val108 és el residu més important per tal de preservar la integritat estructural de l'estat natiu. Els reemplaçaments en aquesta posició causen petites alteracions conformacionals i una gran desestabilització de la proteïna (per exemple, el punt mig de la transició de desnaturalització per pressió i per temperatura de la variant V108G disminueix uns 592 MPa i 25ºC, respectivament, respecte a la proteïna salvatge). D'acord amb els resultats obtinguts, la variant Y115W ofereix una sonda útil per tal de seguir la cinètica de plegament/desplegament de l'RNasa A. / Para analizar la contribución de la región C-terminal propuesta como iniciadora del plegamiento (CFIS 106-118) en la estabilidad de la RNasa A, los residuos alifáticos de esta región fueron sustituidos, mediante mutagénesis dirigida, por otros residuos en los cuales la cadena lateral alifática era progresivamente recortada. La mayor parte de las sustituciones proyectadas suponían deleciones no disruptivas de grupos metilo(eno). Además, se reemplazó la Tyr115 por un Trp, de forma que, potencialmente, se introducía una única sonda fluorescente, no desestabilizadora, para seguir los cambios conformacionales que se pudieran generar en la región durante el proceso de plegamiento/desplegamiento de la proteína. Tanto los parámetros cinéticos, como los espectros de FTIR y CD, determinados para cada una de las ribonucleasas variantes, indican que las sustituciones aminoacídicas efectuadas presentan, en general, poco o ningún efecto en la estructura nativa y en la actividad de la enzima. Se utilizó la espectroscopia de absorción ultravioleta de cuarta derivada, la fluorescencia (para la variante con Trp) y la espectroscopia de infrarrojo por transformada de Fourier, para seguir y caracterizar, en condiciones de equilibrio, las transiciones conformacionales de cada variante en función de la presión y de la temperatura. Los resultados se compararon con los que se obtuvieron por la proteína salvaje. Para determinar más a fondo las características del proceso de desplegamiento de la variante Y115W, las transiciones de desnaturalización inducidas por urea de esta variante y de la proteína salvaje, fueron examinadas mediante electroforesis en gradiente de urea y espectroscopia de fluorescencia. Curiosamente, los cambios conformacionales que resultan de la desnaturalización por presión son muy parecidas a los que se obtuvieron por temperatura. Enfrente de un aumento gradual tanto de presión como de temperatura, la estructura terciaria i los elementos de estructura secundaria de las proteínas estudiadas se pierden de forma conjunta y reversible. Estas variaciones estructurales que se promueven describen un proceso de desplegamiento muy cooperativo y en dos estados. Dado que las dos técnicas (UV y FTIR) utilizan cada una un régimen de concentración proteica muy diferente, los resultados indican que el proceso de desplegamiento por presión y por temperatura es intramolecular. Los resultados obtenidos sugieren que la hidrofobicidad y el volumen de las cadenas laterales del CFIS, junto con las interacciones de van der Waals entre elementos de estructura secundaria intervienen de forma muy notable en la estabilización de la proteína. Entre los diferentes aminoácidos alifáticos que pertenecen al CFIS C-terminal, la Val108 es el residuo más importante para preservar la integridad estructural del estado nativo. Las sustituciones en esta posición causan pequeñas alteraciones conformacionales y una gran desestabilización de la proteína (por ejemplo, el punto medio de la transición de desnaturalización por presión y por temperatura de la variante V108G disminuye unos 592 MPa y 25ºC, respectivamente, respecto a la proteína salvaje). De acuerdo con los resultados obtenidos, la variante Y115W ofrece una sonda útil para seguir la cinética de plegamiento/desplegamiento de la RNasa A. / To analyze the contribution of the postulated carboxy terminal chain-folding initiation site (CFIS 106-118) on ribonuclease A stability, the aliphatic side chains in this region were progressively truncated using site-directed mutagenesis. Most amino acid substitutions were designed to be non-disruptive deletions of methyl and methylene groups. In addition, replacement of Tyr 115 with Trp was designed for its potential as a unique and non-destabilizing fluorescent label of conformational changes local to the region. Steady-state kinetic parameters for the enzyme reaction, FTIR and CD spectra of each RNase A variant indicate that amino acid replacements performed in this region have, in general, little or no effect on the native structure and function of the enzyme. Fourth derivative UV absorbance, fluorescence (for the Trp variant) and Fourier transform infrared spectroscopy were used to detect and characterize the conformational transitions of each variant, as a function of both pressure and temperature. The results were compared with those presented for the wild-type protein. To further determine the unfolding properties of the Y115W variant, the unfolding transitions induced by urea of RNase A wild-type and Y115W variant, were examined by urea gradient gel electrophoresis and fluorescence spectroscopy. Interestingly, conformational changes resulting from pressure denaturation do not differ considerably from those obtained during temperature treatment. Simple two- state, reversible unfolding transitions were observed, suggesting that the disruption of tertiary and secondary structure of each protein at high pressure or temperature is strongly cooperative. Spectral changes occur at about the same values using both low- and high-protein concentration regime techniques, indicating that the observed unfolding events are intramolecular, and that breakdown of the tertiary and secondary structures occurs concomitantly. The results obtained reveal that hydrophobicity and volume of the CFIS side chains, together with van der Waals interactions between secondary structural elements play an important role in stabilizing the protein. Among the aliphatic amino acids belonging to the C-terminal CFIS, V108 is the most critical residue. Replacements performed in this site cause small conformational differences in the native state, and a dramatic destabilization of the protein (i.e. the pressure and temperature midpoint denaturation values of the V108G variant decrease by 592 MPa and by 25ºC, respectively, relative to the wild-type RNase A). According to the results obtained in the current study, the Y115W labeled variant offers a useful probe for the folding/unfolding kinetics.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

Appendix on leaves 193-197. / Additional notes pasted on back fly-leaf. / Copies of authors previously published articles in pocket inside back cover. / Bibliography: leaves 147-192. / 197, [114] 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. / Enforced-expression of various forms of Myb and Kit in primary murine haemopoietic cells were investigated to determine the functions of these two oncoproteins separately and in synergy in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?

Typescript (photocopy) / 172 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Zoology, 1982

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006