Thesis (Ph.D.)--University of Western Sydney, 2007. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Natural Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (M.S.)--Florida State University, 2006. / Advisor: Timothy S. Moerland, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Sept. 15, 2006). Document formatted into pages; contains x, 33 pages. Includes bibliographical references.

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The microbial growth, colour stability and pH changes for black wildebeest (Connochaetes gnou) meat under chilled (4.2±0.8°C) vacuum storage were investigated. The investigation centred on the role of ultimate muscle pH on shelf life of the meat. Although bacterial growth was observed over time for both DFD (pH >6) and Normal (pH <6) meat, DFD meat exhibited higher growth rates for lactic acid bacteria (LAB), total viable counts (TVC) and total coliforms. This was attributed to the combination of high pH and possibly the depletion of glucose in the DFD muscles. On the other hand, the growth rate of total coliforms was less than what was observed for the other microorganisms tested. It was assumed that chilled vacuum storage in combination with the high levels of LAB inhibited the growth of total coliforms. Salmonella was not detected in any of the samples analysed. There were no changes in pH during the 12 days storage period for DFD meat whereas pH for Normal meat decreased towards the end of storage possibly due to lactic acid production by LAB. The colour changes were more noticeable in Normal meat (more browning) than in DFD meat after blooming for 30 min. The conclusion for this study was that DFD meat spoiled faster than Normal meat. The meat was further subjected to preservation by oregano essential oil (1% v·v-1). In this case, there was an initial inhibition of TVC, LAB and total coliforms. Furthermore, the growth rates for TVC and LAB were lower (p<0.05) in the oregano oil treatment group than in the control. For total coliforms however, there was only an initial inhibition observed and no effect on the growth rate. Addition of oregano essential oil also resulted in a significant lowering of meat pH. This may have added to the microbial inhibition observed. Based on TVC values, addition of oregano essential oil extended the shelf-life of black wildebeest meat by 3 days. At the beginning of the study, the lipid oxidation (TBARS) values were above the threshold for detection. Also, the percentage of metmyoglobin had exceeded the levels at which browning becomes visible. Therefore, conclusions on the effects of oregano essential oil on the colour and lipid oxidations were not made in this study. However, oregano essential oil inhibited microbial growth and stabilised TBARS throughout the 9 day storage period. Therefore there is potential to use oregano essential oil as a preservative for black wildebeest meat, although more research is needed. / AFRIKAANSE OPSOMMING: In hierdie studie word die mikrobiese groei, stabiliteit en pH kleur verandering ondersoek vir swartwildebeestevleis onder verkoelde (4.2 ± 0.8 ° C) vakuum berging. Die ondersoek is spesifiek gefokus op die rol van die eind-spier pH op die raklewe van die vleis. Alhoewel mikrobiese groei vir beide DFD (pH >6) en Normal (pH <6) vleis waarneembaar was met verloop van tyd, het die DFD vleis hoër groeitempo vir melksuurbakterieë (MSB) en totale lewensvatbare tellings (LVT) getoon. Dit was as gevolg van die kombinasie van hoë pH en die moontlikheid van die vermindering van die glukose in die DFD spiere. Aan die ander kant was dit waargeneem dat die groeikoers van die totale kolivormig bakterieë minder was, teenoor die ander mikro-organismes wat getoets was. Dit was aangeneem dat die verkoelde vakuum stoor die groei van die totale kolivormig bakterieë geïnhibeer het. Salmonella was nie opgespoor in enige van die geanaliseerde monsters nie. Daar was geen verandering in pH tydens die stoor tydperk vir DFD vleis nie, maar die pH vir normale vleis het tydens die einde van die stoor tydperk afgeneem. Die kleur verandering onder vakuum stoor was meer waarneembaar in die normale vleis as wat dit was in die DFD vleis. Die gevolgtrekking van hierdie studie was dat DFD vleis baie vinniger bederf teenoor normale vleis. Maar daar was variasie op die gewig van die oorspronklike mikrobiese lading en dit kon die bakteriese groeitempo van die normale vleis beïnvloed. Die vleis is verder behandel met oregano essensiële olie ( 1 % v·v-1) vir preservering . In hierdie geval, was daar 'n aanvanklike inhibisie van LVT, MSB en totale kolivormig bakterieë. Verder was die groeitempo vir LVT en MSB aansienlik laer (p<0.05 ) in die behandelings groep teenoor die in die kontrole . Vir die totale kolivormig bakterieë was daar egter net 'n aanvanklike inhibisie waargeneem en geen effek op die groeikoers nie. Die byvoeging van oregano essensiële olie het ook gelei tot 'n beduidende verlaging van die pH. Dit kon gelei het tot die mikrobiese inhibisie wat waar geneem was. Gebaseerd op die LVT, het die byvoeging van oregano essensiële olie gelei tot die verlenging van die swartwildebeeste vleis se raklewe met 3 dae. Aan die begin van hierdie studie was die lipied oksidasie (TBARS) waardes bo die drumpel van opsporing. Ook, die persentasie van metmyoglobin het die vlakke waarop verbruining sigbaar word, oorskry. Daar is potensiaal vir die gebruik van oregano essensiële olie as n middel vir die verlenging van swartwildebees vleis, maar nog navorsing is nodig.

Made available in DSpace on 2016-07-01T13:10:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-14. Added 1 bitstream(s) on 2016-07-01T13:14:20Z : No. of bitstreams: 1 000866925.pdf: 1632439 bytes, checksum: 5b0c9806c2ca74639544b85aa3d9fe2b (MD5) / Cada vez mais vemos o crescimento da exigência do consumidor para alimentos cárneos, principalmente as que se referem a características como maciez, a suculência e o odor, que estão diretamente ligadas a constituição proteica do produto, juntamente com os lipídeos e os carboidratos. No atual panorama do consumo de produtos cárneos no Brasil, os produtos de origem avícola apresentam significativo aumento, e a elaboração de produtos derivados tem acompanhado este avanço. Neste sentido, identificar se existe misturas cárneas indevidas nos subprodutos é de vital importância. Para a avaliar a qualidade destes, a avaliação da origem e da qualidade das proteínas musculares é um fator de grande importância, assim como a avaliação das perdas proteicas ocorridas no processo de rigor mortis. As principais técnicas para quantificação das proteínas são as eletroforéticas e densitométricas. Para avaliação de suas perdas, pode ser utilizado ainda, a técnica de histologia dos tecidos musculares. Para realização deste trabalho, partiu-se de que as proteínas musculares de frangos e perus apresentam diferentes características estruturais devido à expressão gênica e suas estruturas primárias podem ser identificadas mesmo em misturas, por técnicas eletroforéticas e, através da densitometria, quantificadas. Foram colhidos fragmentos de músculo peitoral de aves de cada espécie abatidas em laboratórios, para além da avaliação da mistura de extratos proteicos, realizar a avaliação das perdas proteicas ocorridas durante a resolução do rigor mortis. Os fragmentos obtidos foram avaliados em técnicas histológicas e eletroforéticas, tendo sido coletados em tempos correntes de 0, 30, 60, 90, 120 e 150 minutos após o abate. Ainda foi realizada a extração com outra parte dos mesmos fragmentos por maceração e centrifugação, e posterior mistura de extratos na proporção 80, 60, 40 e 20%, para posterior submissão a... / We see the growing consumer demand for meat foods, particularly those relating to features such as tenderness, juiciness and odor, which are directly related to the constitution of the protein product, together with the lipids and carbohydrates. In the current panorama of consumption of meat products in Brazil, the poultry origin show a significant increase, and the development of derivatives has followed this advance. In this sense, identify whether there is undue meats mixtures of by-products is vitally important. To assess the quality of the assessment of the origin and quality of muscle proteins is a major factor as well as the evaluation of protein losses during rigor mortis process. The main techniques for protein quantification are electrophoretic and densitometric. To evaluate the protein losses can still be used the histology of muscle tissues technique. For this work, he left that the muscle proteins of chickens and turkeys have different structural characteristics due to gene expression and their primary structures can be identified even in mixtures, by electrophoretic techniques, and through densitometry quantified. Pectoral muscle fragments poultry slaughtered were collected in the laboratory, in addition to the evaluation of protein extracts mixing, accomplish the evaluation of the loss occurred during the resolution of rigor mortis. The fragments obtained were evaluated for histological and electrophoretic technique, currents being collected at times 0, 30, 60, 90, 120 and 150 minutes after the slaughter. Protein extraction with another of the same fragments by maceration and centrifugation, and subsequent mixture of extracts in the proportion 80, 60, 40 and 20% yet been performed for later submission to IEF technique on Phast-Gel. The results showed that the protein loss in the resolution of rigor mortis were not significant, even with the total transformation of the tissue at post mortem. Furthermore, the protein mixtures...

Orientador: Roberto de Oliveira Roça / Coorientador: Paulo Roberto Rodrigues Ramos / Banca: Noeme Souza Rocha / Banca: Renée Laufer Amorim / Banca: Ana Maria Lopes / Banca: Marice Thereza Correa Domingues / Resumo: Cada vez mais vemos o crescimento da exigência do consumidor para alimentos cárneos, principalmente as que se referem a características como maciez, a suculência e o odor, que estão diretamente ligadas a constituição proteica do produto, juntamente com os lipídeos e os carboidratos. No atual panorama do consumo de produtos cárneos no Brasil, os produtos de origem avícola apresentam significativo aumento, e a elaboração de produtos derivados tem acompanhado este avanço. Neste sentido, identificar se existe misturas cárneas indevidas nos subprodutos é de vital importância. Para a avaliar a qualidade destes, a avaliação da origem e da qualidade das proteínas musculares é um fator de grande importância, assim como a avaliação das perdas proteicas ocorridas no processo de rigor mortis. As principais técnicas para quantificação das proteínas são as eletroforéticas e densitométricas. Para avaliação de suas perdas, pode ser utilizado ainda, a técnica de histologia dos tecidos musculares. Para realização deste trabalho, partiu-se de que as proteínas musculares de frangos e perus apresentam diferentes características estruturais devido à expressão gênica e suas estruturas primárias podem ser identificadas mesmo em misturas, por técnicas eletroforéticas e, através da densitometria, quantificadas. Foram colhidos fragmentos de músculo peitoral de aves de cada espécie abatidas em laboratórios, para além da avaliação da mistura de extratos proteicos, realizar a avaliação das perdas proteicas ocorridas durante a resolução do rigor mortis. Os fragmentos obtidos foram avaliados em técnicas histológicas e eletroforéticas, tendo sido coletados em tempos correntes de 0, 30, 60, 90, 120 e 150 minutos após o abate. Ainda foi realizada a extração com outra parte dos mesmos fragmentos por maceração e centrifugação, e posterior mistura de extratos na proporção 80, 60, 40 e 20%, para posterior submissão a... / Abstract: We see the growing consumer demand for meat foods, particularly those relating to features such as tenderness, juiciness and odor, which are directly related to the constitution of the protein product, together with the lipids and carbohydrates. In the current panorama of consumption of meat products in Brazil, the poultry origin show a significant increase, and the development of derivatives has followed this advance. In this sense, identify whether there is undue meats mixtures of by-products is vitally important. To assess the quality of the assessment of the origin and quality of muscle proteins is a major factor as well as the evaluation of protein losses during rigor mortis process. The main techniques for protein quantification are electrophoretic and densitometric. To evaluate the protein losses can still be used the histology of muscle tissues technique. For this work, he left that the muscle proteins of chickens and turkeys have different structural characteristics due to gene expression and their primary structures can be identified even in mixtures, by electrophoretic techniques, and through densitometry quantified. Pectoral muscle fragments poultry slaughtered were collected in the laboratory, in addition to the evaluation of protein extracts mixing, accomplish the evaluation of the loss occurred during the resolution of rigor mortis. The fragments obtained were evaluated for histological and electrophoretic technique, currents being collected at times 0, 30, 60, 90, 120 and 150 minutes after the slaughter. Protein extraction with another of the same fragments by maceration and centrifugation, and subsequent mixture of extracts in the proportion 80, 60, 40 and 20% yet been performed for later submission to IEF technique on Phast-Gel. The results showed that the protein loss in the resolution of rigor mortis were not significant, even with the total transformation of the tissue at post mortem. Furthermore, the protein mixtures... / Doutor

A Tropomiosina (Tm) está diretamente envolvida no processo de regulação da contração muscular, que é controlado por um mecanismo alostérico que envolve Ca2+, troponina (Tn), actina (Ac) e miosina. A Tm é uma proteína flexível, de estrutura \"coiled-coil\", constituída de duas &#945;-hélices com 284 aminoácidos cada uma. A molécula de Tm faz interações do tipo \"cabeça-cauda\" com outra molécula de Tm através da sobreposição de aproximadamente 8 a 15 resíduos da extremidade N- terminal de uma molécula com 8 a 15 resíduos da extremidade e-terminal da outra molécula de Tm. Desta maneira, em baixas forças iônicas, formam-se filamentos lineares através de um processo de polimerização. A estabilidade de regiões específicas da Tm pode ser importante para a sua função no controle da regulação da contração muscular. Além disso, a Tm pode ser usada como um modelo relativamente simples e de ocorrência natural para entendermos as interações intra- e intermoleculares que governam a estabilidade das \"coiled-coils\". Assim sendo, nós produzimos oito fragmentos recombinantes de Tm (Tm143-284(50HW), Tm189-284(50Hw), Tm189-284, Tm220-284(50HW), Tm220-284, Tm143-235, Tm167-260 e Tm143-260) e um peptídeo sintético (Ac-Tm215-235) com a finalidade de investigar as estabilidades conformacionais relativas das diferentes regiões derivadas da metade C-terminal da proteína, a qual é conhecida por sua interação com o complexo troponina. Experimentos de ultracentrifugação analítica mostram que os fragmentos que incluem os últimos 24 resíduos da molécula (Tm143-284(50HW), Tm189-284(50HW), Tm220-284(50HW), Tm220-284) estão completamente dimerizados a 10 &#181;M (concentração do dímero em 50 mM de tampão fosfato, pH 7,0; 100 mM de NaCI; 0,5 mM de DTT e 0,5 mM de EDTA, 10°C), enquanto que fragmentos que não possuem o e-terminal nativo (Tm143-235, Tm167-260 e Tm143-260) se encontram em equilíbrio monômero-dímero nestas condições. A presença de trifluoroetanol promove uma diminuição na razão [&#952;]222/[&#952;]208, observada por dicroísmo circular, em todos os fragmentos e induz a formação de trímeros estáveis apenas para aqueles contendo os resíduos 261-284. Estudos de desnaturação por uréia, acompanhados por dicroísmo circular e fluorescência, mostram que os resíduos 261-284 da tropomiosina são muito importantes para estabilidade da metade C-terminal da molécula. Além do mais, a ausência desta região promove um aumento na cooperatividade do desenovelamento induzido por uréia. Os experimentos de desnaturação por temperatura e por uréia mostram que o fragmento Tm143-235 é relativamente instável quando comparado com outros fragmentos de mesmo tamanho. Nós identificamos alguns fatores que podem estar contribuindo para a particular instabilidade desta região, incluindo repulsões inter-hélices entre resíduos em posições g e e\' da repetição heptapeptídica, um resíduo carregado na interface hidrofóbica da \"coiled-coil\" e por fim, uma grande fração de resíduos &#946;-ramificados localizados em posições d. Sabe-se que a não acetilação do N-terminal da molécula de Tm, bem como a ausência de alguns resíduos na sua extremidade C-terminal, fazem com que a Tm deixe de sofrer polimerização. Entretanto, trabalhos anteriores realizados em nosso laboratório haviam mostrado que um fragmento de Tm recombinante (ASTm1-260), contendo a fusão dipeptídica Ala-Ser (que é conhecida por restaurar a capacidade de polimerização de Tm não acetiladas no N-terminal), polimerizava-se mais do que a proteína recombinante de tamanho integral (ASTm), apesar da deleção dos últimos 24 aminoácidos C-terminais. Para investigar com mais detalhes a natureza da interação cabeça-cauda, nós construímos dois fragmentos que compreendem a metade N-terminal da molécula de Tm, ASTm1-142 e nfTm1-142, o primeiro deles contendo uma fusão dipeptídica AS no N-terminal e o segundo com o N-terminal não acetilado, sem a fusão dipeptídica. Estes dois fragmentos foram empregados em ensaios de interação cabeça-cauda, realizados através de ensaios de desnaturação térmica acompanhados por dicroísmo circular, juntamente com três fragmentos da região C-terminal da Tm. Dois dos fragmentos e-terminais acabam na posição 260 (Tm167-260 e Tm143-260) e um deles termina na posição 284 (Tm220-284), que corresponde ao C-terminal nativo da proteína. Os resultados mostram que ocorre uma interação cabeça-cauda entre o fragmento N-terminal ASTm1-142 e todos os fragmentos C-terminais utilizados neste estudo. As moléculas recombinantes de Tm que terminam na posição 260 são, de fato, capazes de fazer interações cabeça-cauda independentemente do fato de elas se apresentarem instáveis nas condições estudadas. Inclusive, após a interação, ocorre um aumento considerável na estrutura a-hélice, o que se dá preferencialmente nos fragmentos C-terminais. / Tropomyosin (Tm) participates in the process of muscle contraction, which is controlled by an allosteric mechanism involving Ca2+, troponin (Tn), actin (Ac) and myosin. Tm is a coiled-coil flexible molecule composed by two &#945;-helices with 284 amino acids each. Tm molecule performs head-to-tail interactions with another Tm molecule through the overlap of approximately 8 to 15 N-terminal residues of one molecule with 8 to 15 C-terminal residues of the other molecule. Thus Tm forms linear filaments in low ionic strengths, which is characteristic for a polymerization process. The stability of specific regions of Tm may be important to its function in controlling the regulation of muscle contraction. Besides, Tm can be used as a relatively simple model and of natural occurrence to understand the intra- and intermolecular interactions that govern the stability of \"coiled-coils\". We therefore produced eight recombinant fragments of Tm (Tm143-284(50HW), Tm189-284(50HW), Tm189-284, Tm220-284(50HW), Tm220-284, Tm143-235, Tm167-260 and Tm143-260) and one synthetic peptide (Ac-Tm215-235) to investigate the relative conformational stabilities of different regions derived from the C-terminal end of the molecule, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that fragments comprising the last 24 residues of the molecule (Tm143-284(50Hw), Tm189-284(50HW), Tm220-284(50Hw), Tm220-284) are completely dimerized at 10 &#181;M (dimer concentration in buffer containing 50 mM phosphate, pH 7.0, 100 mM NaCI, 0.5 mM DTT and 0.5 mM EDTA, 10ºC), whereas the fragments that do not posses the native C-terminal portion (Tm143-235, Tm167-260 and Tm143-260) present a monomer-dimer equilibrium at the same conditions. Trifluoroethanol promoted a decrease in the ratio [&#952;]222/[&#952;]208 for all fragments (observed by circular dichroism), and induced the formation of stable trimers for the fragments comprising the residues 261-284. Urea denaturation studies, followed by circular dichroism and fluorescence, show that residues 261-284 of Tm are very important for the stability of the C-terminal half of the molecule. Still, the absence of this region promotes an increase in the cooperativity of unfolding induced by urea. Urea and temperature denaturation experiments showed that the fragment Tm143-235 is relatively unstable when compared to other fragments of the same size. We identified some factors that may be contributing to the particular instability of this region, including interhelix repulsions between residues in positions g and e\' of the heptad repeat, a charged residue in the hydrophobic interface of the coiled-coil and a great fraction of (&#946;-branched residues located at d positions. It is known that the non-acetylation of the N-terminus of the Tm molecule, as well as, the absence of some residues in the C-terminus of the molecule cause Tm to loose its ability to undergo polymerization. Former works performed in our laboratory showed that a fragment of recombinant Tm (ASTm1-260) with the dipeptide fusion Ala-Ser (which is known by its ability in restores the polymerization of non-acetylated Tms), despite the absence of the C-terminal 24 amino acids, polymerizes to a much greater extent than the corresponding full length recombinant protein. To better investigate the nature of the head-to-tail interaction we constructed two fragments that comprise the N-terminal half of the Tm: ASTm1-142 and nfTm1-142, the former containing the dipeptide AS (Ala-Ser) fusion to its N-terminus, and the latter presenting a bare non-acetylated N-terminus without the dipeptide fusion . These two fragments were employed in thermal denaturation assays followed by circular dichroism to test their head-to-tail interaction along with three fragments comprising Tm\'s C-terminal region. Two of the C-terminal fragments ends at position 260 (Tm167-260 and Tm143-260), whereas one ends at position 284 (Tm220-284). Results show that there is a head-to-tail interaction between the N-terminal fragment ASTm1-142 and all other e-terminal fragments employed in this study. Recombinant Tm molecules ending at position 260, are capable of performing head-to-tail interactions, regardless of their stability under the studied condition. Upon interaction, an increase in the a-helix content occurs preferentially in the e-terminal fragments.

Uma série de mutantes recombinantes da tropomiosina com a sonda fluorescente 5-hidroxitriptofano inserida em várias posições ao longo da seqüência primária vêm sendo utilizados em nosso laboratório para o estudo em nível molecular do controle do processo de contração muscular. Estes mutantes têm sido produzidos em cepas de bactéria auxotróficas para triptofano em meio mínimo ao qual adicionamos 5-hidroxitriptofano, um análogo do aminoácido triptofano. Esta sonda fluorescente apresenta a vantagem de absorver energia numa faixa de comprimentos de onda não absorvida pelo triptofano (temos usado com sucesso o comprimento de onda de 312 nm para excitar seletivamente o 5-hidroxitriptofano). Desta forma, o ambiente da sonda pode ser estudado em situações nas quais outras proteínas que possuam triptofanos, como, por exemplo a actina, estejam presentes. Procedemos a uma caracterização dos mutantes utilizados quanto à sua ligação à actina na ausência e presença de troponina (+/- Ca2+) e na presença de acrilamida por meio de ensaios de co-sedimentação com actina. O comportamento funcional dos diversos mutantes foi investigado pela análise da regulação da atividade Mg2+ -ATPásica da acto-S1 miosina destes mutantes na ausência e na presença de troponina (+/- Ca2+). Estudos da estabilidade destes mutantes da tropomiosina por meio de desnaturação por temperatura seguida por dicroísmo circular foram realizados para analisarmos o efeito das mutações na estabilidade global da molécula. Ensaios de desnaturação por uréia seguidas por fluorescência também foram realizados para analisarmos a estabilidade da molécula próxima à região da sonda fluorescente. O presente estudo compreendeu ainda a análise da supressão de fluorescência pelos supressores extrínsecos acrilamida e iodeto para diversos mutantes da tropomiosina na presença e ausência de outras proteínas do filamento fino, o que permitiu a obtenção de informações sobre o grau de exposição da sonda fluorescente nas diversas situações estudadas bem como sobre o ambiente eletrostático ao redor das sondas nestas diferentes situações. Estes resultados permitiram a obtenção de um modelo para a ligação da tropomiosina ao filamento de actina na presença de troponina (+/- Ca2+). Este modelo propõe que as bandas &#945; do padrão de repetição &#945;/&#946; inicialmente proposto por Mclachlan e Stewart (1975 e 1976) se liguem ao filamento na ausência de íons cálcio. A introdução de cálcio no sistema é capaz de induzir uma rotação e um deslocamento na molécula de tropomiosina com relação ao filamento de actina. Nesta situação, seriam as bandas &#946; da tropomiosina as responsáveis pela ligação desta molécula ao filamento de actina. / Several recombinant mutants of tropomyosin with the fluorescent probe 5-hydroxytryptophan inserted at several positions along the primary sequence are being used in our laboratory for studying the control of the muscular contraction process at a molecular level. These mutants are produced in bacterial strains auxotrophic to tryptophan in minimal medium to which 5- hydroxytryptophan, a tryptophan analogue, is added. This fluorescent probe has the advantage of absorbing light in a range of wavelengths not absorbed by the amino acid tryptophan (we have been successfully using the wavelength of 312 nm to selectively excite the 5-hydroxytryptophan). The environment of the probe can thus be studied even when other tryptophan containing proteins, such as actin, are present. We have characterized the mutants as to their ability to bind to actin in the absence and in the presence of troponin (+/- Ca2+) and in the presence of acrylamide through actin co-sedimentation assays. The functional behavior of the several mutants constructed was assessed by analyzing their ability to regulate the acto-S1 myosin Mg2+-ATPase activity in the absence and in the presence of troponin (+/- Ca2+). Studies on the stability of these tropomyosin mutants by thermal denaturation followed by circular dichroism were performed to analyze the effect of the mutations on the molecule\'s global stability. Urea denaturation assays followed by fluorescence were also performed to allow us to investigate the stability of the molecule in the vicinity of the fluorescent probe. The present study also involved the analysis of the fluorescence quenching by the extrinsic quenchers acrylamide and iodide for divers tropomyosin mutants in the presence and in the absence of other thin filament proteins. This allowed us to obtain information on the degree of exposure of the fluorescent probe in the several conditions studied as well as on the electrostatic microenvironment surrounding the probes in these different situations. These results enabled us to propose a model for the binding of tropomyosin to the actin filament in the presence of troponin (+/Ca2+). In this model the &#945;-bands from the &#945;/&#946; repetition pattern initially proposed by Mclachlan and Stewart (1975 and 1976) would be binding the filament in the absence of calcium ions. The introduction of calcium to the system would induce a rolling motion in and a displacement of the tropomyosin molecule with relation to the actin filament. In this situation tropomyosin\'s &#946;-bands would be responsible for the binding of this molecule to the actin filament.

Muscular dystrophy is a genetic disease which affects the morphology, physiology and biochemical nature of the muscle fiber. This study was designed to examine the progressive effects of muscular dystrophy on the differentiation process of skeletal muscle. Chapter 1 examines the neonatal development of muscle spindles and their intrafusal fibers in the soleus and extensor digitorum longus (EDL) of genetically dystrophic mice according to histochemical, quantitative, and ultrastructural parameters. Despite alterations in the surrounding extrafusal fibers, muscle spindles and their intrafusal fibers appeared enzymatically and histologically unaffected in incipient stages of murine dystrophy. In the second chapter the distribution and concentration of parvalbumin (PV), a calcium-binding protein, in 32 and 2-week-old dystrophic mice was mapped by immunohistochemical and biochemical procedures. The number of parvalbumin-immunoreactive fibers was significantly reduced in the adult dystrophic EDL but slightly increased in the adult dystrophic soleus. No differences between strains were observed in the 2-week samples. These findings were supported by routine myosin ATPase histochemistry. Parvalbumin was isolated on SDS-PAGE gels and the concentration of PV was estimated by a RIA. These results confirmed the immunohistochemical data in that PV content was dramatically reduced in the adult dystrophic EDL and significantly increased in the dystrophic soleus. No changes were detected in the samples of the 2-week-old muscles. The similarity in the distribution and content of PV between the fast and slow dystrophic muscles at 32 weeks of age suggests an alteration in the distribution and phenotypic expression of fiber types in muscular dystrophy and supports the hypothesis that dystrophy alters the normal differentiation process of skeletal muscle. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

An affinity column for cathepsin D was prepared making use of the strong affinity of pepstatin for cathepsin D. Pepstatin is an N-acylated pentapeptide from Actinomycetes with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy- 6-methyl heptanoic acid. A relatively rapid and efficient method for cathepsin D purification has been developed; Steps include homogenization, ammonium sulfate fractionation, and chromatography on pepstatin-Sepharose column. The final preparation has a specific activity of 38 units/mg. and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate corresponding to a subunit molecular weight of 42,000. Polyacrylamide gel electrophoresis studies did not reveal any impurities. The proteolytic activity of isolated cathepsin D on bovine myofibrils and myosin was examined at pH 3.80, 37 °C. The heavy chains of myosin, as well as other smaller regulatory proteins of the myofibrils were degraded. Actin was degraded less rapidly than myosin heavy chain. Degradation became more extensive when the substrate-enzyme incubation time was increased.