Dissertations / Theses on the topic 'CALPAIN5'

Les cellules souche cancéreuses contribuent au développement des sarcomes, mais le manque de marqueurs spécifiques empêche leur caractérisation et la possibilité de cibler ce type de cellules. Nous avons utilisé la séquence régulatrice de la calpaïne-6 dans des systèmes rapporteurs pour identifier les cellules exprimant la calpaïne-6. Ces cellules étaient des cellules initiatrices de tumeurs et se comportaient comme des cellules souche, au sommet de la hiérarchie cellulaire. L'expression de la calpaïne-6 dépend d’un programme génique de cellules souche qui implique Oct4, Nanog et Sox2 et est activée par l'hypoxie. L’inhibition de la calpaïne-6 a bloqué le développement tumoral et a induit la diminution du nombre de cellules souche cancéreuses dans les sarcomes osseux. L’expression de la calpaïne-6 était inversement corrélée à l'expression de marqueurs de sénescence mais était associé à un flux autophagique dynamique. L’inhibition de la calpaïne-6 a induit l'entrée des cellules en sénescence et a supprimé le flux autophagique. Nos résultats révèlent que le calpaïne-6 identifie les cellules souche des sarcomes et joue un rôle important dans le maintien des cellules souche cancéreuses en contrôlant les processus d’autophagie et de sénescence. La calpaïne-6 semble être une cible thérapeutique prometteuse pour éradiquer les cellules souche dans les sarcomes<br>Cancer stem cells contribute to sarcoma development, but lack of specific markers prevents their characterization and the possibility of targeting. We used the regulatory sequence of calpain-6 in reporter constructions to identify calpain-6–expressing cells. These cells were tumor-initiating cells and behaved like stem cells at the apex of the cellular hierarchy. Calpain-6 expression depended on the stem-cell transcription network that involves Oct4, Nanog, and Sox2 and was activated by hypoxia. Calpain-6 knockdown blocked tumor development and induced depletion of sarcoma stem cells. Calpain-6 was inversely associated with expression of senescence markers but was associated with a dynamic autophagy flux. Calpain-6 knockdown induced cell entry into senescence and suppressed autophagy flux. Our results reveal that calpain-6 identifies sarcoma stem-cell and plays an important role as a regulator of cancer cell fate driving a switch between autophagy and senescence. Calpain-6 may be a promising therapeutic target to eradicate sarcoma stem cells

O objetivo deste trabalho foi avaliar o potencial de utilização de marcadores moleculares em genes candidatos da calpaína (CAPN) e calpastatina (CAST) como ferramenta auxiliar para programas de melhoramento de características relacionadas ao crescimento e maciez da carne. Foram avaliados 605 bovinos da raça Nelore, pertencentes à Agropecuária CFM Ltda, com idade média ao abate de 24 meses. Após a extração do DNA de amostras de sangue, por desproteinização em presença de NaCl, a identificação e determinação do polimorfismo para os marcadores moleculares CAPN316, CAPN530, CAPN4751, CAPN4753 e UOGACAST1, foi realizada pelo sistema de detecção TaqManTM utilizando-se PCR em Tempo Real. A análise de maciez da carne, aos 7, 14 e 21 dias de maturação, foi realizada com amostras de carne do Longissimus dorsi, retiradas entre a 12ª e 13ª costela e cisalhadas utilizando-se um Warner Bratzler Shear Force. Nenhum efeito significativo dos marcadores avaliados foi observado para as características de crescimento. Foi verificado efeito significativo, em relação à maciez da carne, para os seguintes polimorfismos: aos 7, 14 e 21 dias de maturação para o marcador CAPN4751; aos 21 dias para o marcador CAPN4753 e aos 14 e 21 dias para o marcador UOGCAST1. Em relação aos efeitos das combinações genotípicas para os marcadores dois a dois, os resultados foram significativos para a combinação CAPN4751/UOGCAST1 nos três tempos de maturação. Para a combinação de marcadores CAPN4753/UOGCAST1 também foram verificados resultados significativos para carnes maturadas aos 14 e 21 dias. Os resultados observados neste trabalho sugerem a possibilidade da utilização de seleção assistida por marcadores (MAS), visando o aumento da qualidade da carne em bovinos da raça Nelore.<br>The objective of this study was to evaluate the potential utilization of molecular markers on candidate calpain and calpastati n genes as an auxiliary tool for breeding programs on traits related to growth and meat tenderness. A total of 605 Nellore animals, raised by CFM Agro-pecuária Ltda, were used in this study and slaughtered with 24 months in average. After DNA blood samples extraction, by desproteinization in presence of NaCl, the polymorphism (CAPN316, CAPN530, CAPN4751, CAPN4753 and UOGACAST1) identification and determination was realized by TaqManTM detection system using real time PCR. The meat tenderness analysis, at the 7, 14 and 21 days of maturation was realized with Longissimus dorsi meat samples, taken at the 12th and 13th rib interval and Warner Bratzler Peak Shear Force measurements were used. There were no significant effects of molecular markers in growth traits. There were significant effects, regarding to meat tenderness, for following polymorphisms: at 7, 14 and 21 days of maturation, for CAPN4751 marker; at 21 days of maturation, for CAPN4753, and finally, at 14 and 21 days of maturation, for UOGCAST1 marker. In respect to genotypic combination effects analysis for pairwise marker, the results were significant for CAPN4751/UOGCAST1 in three days of maturation. In combination effects for CAPN4753/UOGCAST1 markers, significant effects were also observed for meat tenderness at 14 and 21 days. Theses results suggest that marked selection assisted (MAS) can be used to improve meat quality in Nellore beef cattle.

L'Atròfia Muscular Espinal (AME) és una malaltia neurodegenerativa greu i la primera causa genètica de mort infantil. S'origina per la pèrdua o mutació del gen Survival Motor Neuron 1 (SMN1) que causa una deficiència de la proteïna de Survival Motor Neuron (SMN). La reducció d'aquesta proteïna condueix principalment a la degeneració de les motoneurones (MNs) de la medul·la espinal i, en conseqüència, produeix atròfia i feblesa del múscul esquelètic. Actualment, només es coneix parcialment quins mecanismes cel·lulars i moleculars exactes són els responsables de la pèrdua de funció de les MNs. La reducció de SMN causa degeneració de les neurites i mort cel·lular sense característiques apoptótiques clàssiques. L'autofàgia és un procés important i altament regulat, essencial per a l'eliminació d'orgànuls danyats i substàncies o proteïnes tòxiques a través de la degradació amb els lisosomes. L'autofàgia és especialment important en cèl·lules post-mitòtiques, com les MNs, on l'acumulació d’autofagosomes provoca la interrupció del transport axonal, la interferència del trànsit intracel·lular i la degeneració de les neurites. El que és ben sabut en l'AME és que el nivell intracel·lular de proteïna SMN defineix l'inici i la gravetat de la malaltia i això està parcialment determinat pel nombre de còpies del gen SMN2, la duplicació centromérica de SMN i el principal modificador de l'AME. Per aquesta raó, comprendre els processos que regulen la degradació de SMN amb la finalitat d'identificar compostos que augmentin els nivells de proteïnes és el principal objectiu en el desenvolupament terapèutic per a l’AME. Les calpaínes són una família de proteases dependents de calci que s'han relacionat amb trastorns musculars i malalties neurodegeneratives. Específicament, s'ha descrit en el múscul que SMN pot ser proteolizada per calpaína. L'activitat de la calpaína també està involucrada en la regulació de l'autofàgia mitjançant la modulació de múltiples de les proteïnes involucrades en el procés. L'objectiu en el present treball ha estat analitzar la desregulació de l'autofàgia i determinar la participació de la calpaína en la regulació de la proteïna SMN en les MNs per a aprofundir en l'origen de la neurodegeneración i desenvolupar un nou enfocament terapèutic per a l'AME. Per aquesta finalitat, hem analitzat marcadors autofágics en diferents models in vitro d’AME, tant de ratolí com d'humà. Els resultats van mostrar que, tant els autofagosomes com els nivells de LC3 es troben augmentats en les mostres d’AME en comparació amb els controls, la qual cosa suggereix una desregulació del procés d'autofàgia al llarg de la progressió de la malaltia. A més, la reducció dels nivells endògens de calpaína utilitzant un shRNA van mostrar un augment dels nivells de Smn i LC3, alhora que prevenia la degeneració neurítica que es produeix en les MNs de ratolí afectats per AME. Es van obtenir resultats similars en experiments in vitro utilitzant un inhibidor farmacològic de calpaína, la calpeptina. Tanmateix, l'activació de la calpaína produïda per condicions despolarizants induïa la proteólisis de l’α-fodrina i de SMN, la qual cosa confirma que calpain regula directament els nivells de proteïna SMN en les MNs. A més, el tractament amb calpeptina in vivo va millorar significativament l'esperança de vida i la funció motora de dos models de ratolins amb AME, la qual cosa demostra la utilitat potencial dels inhibidors de la calpaína en la teràpia per a la malaltia. Finalment, l'anàlisi de la via de la calpaína en ratolins i models cel·lulars humans d’AME va indicar un augment de l'activitat de la calpaína en les MNs amb nivells reduïts de SMN. Per tant, els nostres resultats demostren que l'activitat de la calpaína es troba sobreactivada en les MNs d’AME i que la seva inhibició pot tenir un efecte beneficiós sobre el fenotip de la malaltia a través de l'augment de SMN i la regulació del procés d'autofàgia en les MNs de la medul·la espinal.<br>La atrofia muscular espinal (AME) es una enfermedad neurodegenerativa grave y la primera causa genética de muerte infantil. Se origina por la pérdida o mutación del gen Survival Motor Neuron 1 (SMN1) que causa una deficiencia de la proteína de Survival Motor Neuron (SMN). La reducción de esta proteína conduce predominantemente a la degeneración de las motoneuronas (MNs) de la médula espinal y, en consecuencia, produce atrofia y debilidad del músculo esquelético. Actualmente, solo se conoce parcialmente que mecanismos celulares y moleculares exactos son los responsables de la pérdida de función de las MNs. La reducción de SMN causa degeneración de neuritas y muerte celular sin características apoptóticas clásicas. La autofagia es un proceso importante y altamente regulado, esencial para la eliminación de orgánulos dañados y sustancias o proteínas tóxicas a través de la degradación con los lisosomas. La autofagia es especialmente importante en células post-mitóticas, como las MNs, donde la acumulación de autofagosomas provoca la interrupción del transporte axonal, la interferencia del tráfico intracelular y la degeneración de las neuritas. Lo que es bien sabido en la AME es que el nivel intracelular de proteína SMN define el inicio y la gravedad de la enfermedad y esto está parcialmente determinado por el número de copias del gen SMN2, la duplicación centromérica de SMN y el principal modificador de la AME. Por esa razón, comprender los procesos que regulan la degradación de SMN con la finalidad de identificar compuestos que aumentan los niveles de proteínas es el principal objetivo en el desarrollo terapéutico de AME. Las calpaínas son una familia de proteasas dependientes de calcio que se han relacionado con trastornos musculares y enfermedades neurodegenerativas. Específicamente, se ha descrito en el músculo que SMN puede ser proteolizada por calpaína. La actividad de la calpaína también está involucrada en la regulación de la autofagia mediante la modulación de múltiples de las proteínas involucradas en el proceso. El objetivo en el presente trabajo ha sido analizar la desregulación de la autofagia y determinar la participación de la calpaína en la regulación de la proteína SMN en las MNs para profundizar en el origen de la neurodegeneración y desarrollar un nuevo enfoque terapéutico para la AME. Con este fin, hemos analizado marcadores autofágicos en diferentes modelos in vitro de AME, tanto de ratón como de humano. Los resultados mostraron que los autofagosomas y los niveles de LC3 se encuentran aumentados en las muestras de AME en comparación con los controles, lo que sugiere una desregulación del proceso de autofagia a lo largo de la progresión de la enfermedad. Además, la reducción de los niveles endógenos de calpaína utilizando un shRNA muestraron un aumento de los niveles de Smn y LC3, a la vez que previene la degeneración neuritica que se produce en las MNs de ratón afectados por AME. Se obtuvieron resultados similares en experimentos in vitro utilizando un inhibidor farmacológico de calpaína, la calpeptina. Asimismo, la activación de calpaína producida por condiciones despolarizantes inducia la proteólisis de α-fodrina y de SMN, lo que confirma que calpain regula directamente los niveles de proteína SMN en las MNs. Además, el tratamiento con calpeptina in vivo mejoró significativamente la esperanza de vida y la función motora de dos modelos de ratones con AME, lo que demuestra la utilidad potencial de los inhibidores de la calpaína en la terapia para la enfermedad. Finalmente, el análisis de la vía de la calpaína en ratones y modelos celulares humanos de AME indicó un aumento de la actividad de la calpaína en las MNs con niveles reducidos de SMN. Por lo tanto, nuestros resultados demuestran que la actividad de la calpaína se encuentra sobreactivada en las MNs de AME y su inhibición puede tener un efecto beneficioso sobre el fenotipo de la enfermedad a través del aumento de SMN y la regulación del proceso de autofagia en las MNs de la médula espinal.<br>Spinal Muscular Atrophy (SMA) is a severe neurodegenerative disease and the first genetic cause of infant death. It is originated by the deletion or mutation of Survival Motor Neuron 1 (SMN1) gene causing a Survival Motor Neuron (SMN) protein deficiency. The reduction of this protein predominantly leads to the degeneration of spinal cord motoneurons (MNs) and consequently produces skeletal muscle atrophy and weakness. The exact cellular and molecular mechanisms responsible for MN loss of function are only partially known. SMN reduction causes neurite degeneration and cell death without classical apoptotic features. Autophagy is an important and highly regulated process, essential for the removal of damaged organelles and toxic substances or proteins through lysosome degradation. This mechanism is specifically important in post-mitotic cells like MNs where autophagosome accumulation causes axonal transport disruption, interference of intracellular space trafficking, and neurite degeneration. What is well known in SMA is that intracellular SMN protein levels are critical to define the disease onset and severity, and this is partially determined by the number of copies of SMN2, the centromeric duplication of the SMN gene and the main modifier of SMA. For that reason, understanding the processes of SMN stability and degradation to identify compounds that increase protein levels is a major goal in SMA therapeutics development. Calpains are a family of calcium-dependent proteases that have been related to muscle disorders and neurodegenerative diseases. Specifically, it has been described in muscle that SMN can be a proteolytic target of calpain. Calpain activity is also involved in autophagy regulation by modulation of multiple proteins involved in the process. The objectives in the present work have been to analyze the autophagy deregulation and determine the involvement of calpain in SMN protein regulation on MNs, in order to deepen in the origin of neurodegeneration and to develop a new therapeutic approach for SMA disease. To this end, we have analyzed autophagic markers in different mouse and human SMA in vitro models. The results showed that autophagosomes and LC3 levels were increased in SMA samples compared to controls, suggesting a deregulation of the autophagy process throughout the disease progression. Moreover, calpain knockdown using an shRNA approach showed an increase of both, Smn and LC3 levels and prevented neurite degeneration occurred in SMA affected mouse MNs. Similar results were obtained in in vitro experiments using a pharmacological calpain inhibitor, calpeptin. Likewise, calpain activation produced by depolarized conditions induced α-fodrin and SMN proteolysis, confirming that calpain directly regulates the SMN protein level in MNs. Additionally, calpeptin in vivo treatment significantly improved the lifespan and motor function of two severe SMA mouse models, demonstrating the potential utility of calpain inhibitors in SMA therapeutics. Finally, the analysis of calpain pathway members in mice and human cellular SMA models indicated an increase of calpain activity in SMN-reduced MNs. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on the SMA phenotype through the increase of SMN and the regulation of the autophagy process in spinal cord MNs.

Thesis (M.S.)--West Virginia University, 1999.<br>Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 72-80).

Calpaïne 3 est une cystéine protéase retrouvée principalement au niveau du tissu musculaire. Cette enzyme joue un rôle clef dans le maintient de l’intégrité des fibres musculaires. En effet, des mutations au niveau du gène de calpaïne 3 ont été identifiées comme étant responsables d’une dystrophie musculaire autosomale récessive, la LGMD2A (Limb-girdle muscular dystrophy type 2A), caractérisée par une atrophie progressive des muscles des ceintures scapulaires et pelviennes. Nos travaux montrent que calpaïne 3 inhibe l’activité transcriptionnelle de MyoD. Ce facteur de transcription myogénique (MRF) joue un rôle central dans le contrôle de la myogenèse aussi bien au cours du développement embryonnaire que chez un individu adulte au cours du processus de régénération musculaire. Cette diminution d’activité transcriptionnelle a lieu aussi bien dans des cellules myoblastiques (C2C12) que fibroblastiques (C3H10T1/2). Par contre calpaïne 3 ne modifie pas l’activité transcriptionnelle des autres MRFs (Myf5, myogénine ou MRF4). Nous avons pu montrer que calpaïne 3 affecte spécifiquement l’activité transcriptionnelle de MyoD en entraînant une diminution de son niveau protéique (Western-blot, microscopie confocale), sans affecter son niveau d’ARNm (RT-QPCR). De plus, des expériences de détermination de la demi-vie protéique ont pu montrer que calpaïne 3 intervenait sur la dégradation protéique de MyoD. Des expériences sont en cours afin de déterminer si calpaïne 3 hydrolyse directement ou non le facteur MyoD. Nos travaux montrent que l’hydrolyse de MyoD induite par calpaïne 3 représente une voie parallèle à celle du système protéolytique protéasome ubiquitine-dépendant connu pour être impliqué dans sa dégradation. Nous avons également montré qu’une modification de l’expression de calpaïne 3, soit par surexpression soit par inhibition avec des siRNA spécifiques, entraîne une perturbation du processus de différenciation myogénique. Cet effet a été plus particulièrement étudié au sein d’une sous-population de cellules qui reste indifférenciée dans les cellules C2C12 induites en différenciation. Ces cellules, appelées cellules de réserve, s’apparentent aux cellules satellites intervenant dans la régénération musculaire. Nous avons montré que calpaïne 3 participe à la régulation du nombre des cellules de réserve au cours de la différenciation des cellules C2C12. Ce rôle de calpaïne 3 pourrait être lié à son intervention dans la dégradation du facteur MyoD. L’ensemble de ces résultats suggère ainsi que calpaïne 3 pourrait jouer un rôle in vivo dans le maintien d’un stock de cellules satellites au cours de la régénération musculaire<br>Calpain 3 (CAPN3) is a calcium-dependent cysteine protease mainly expressed in skeletal muscle. This protease plays a key role in maintaining the integrity of muscular fibers. Indeed, mutations in CAPN3 encoding gene cause limb-girdle muscular dystrophy type 2A, an autosomal recessive muscular dystrophy characterized by progressive atrophy and weakness of the proximal limb muscles. Our work reveals an inhibitory effect of CAPN3 directed against the myogenic regulatory factor (MRF), MyoD. We have shown that CAPN3 inhibits the transcriptional activity of MyoD either in myoblastic cells (C2C12 cells) or in fibroblastic ones (C3H10T1/2 cells). On the contrary, no variation in the transcriptional activity of the other members of the MRFs family (Myf5, myogenin, or MRF4) was observed. CAPN3 affects the transcriptional activity of MyoD by decreasing the quantity of the endogenous protein MyoD (Western-blots, confocal microscopy experiments), without affecting its mRNA level (RT-QPCR). Moreover, half-life determination experiments showed that CAPN3 induce MyoD degradation acts on MyoD by a proteic degradation. Experiments are in progress to determine whether CAPN3 acts directly or not on MyoD. Furthermore, the inhibitory effect of CAPN3 on MyoD is independent of the ubiquitin-proteasome proteolytic pathway that is known to play a role during MyoD degradation. Indeed, MyoD mutants resistant to proteolytic degradation by the proteasome are sensitive to CAPN3 action. Interestingly, we have shown that modifications in CAPN3 expression, induced by overexpression or downregulation (siRNA), cause perturbations in myogenic differentiation. CAPN3 appears as a regulator of myogenic differentiation by modulating the quantity of MyoD available for progressing in differentiation. In addition, we have highlighted a potential role of CAPN3 in maintaining a pool of reserve cells along C2C12 cells differentiation. These cells share numbers of similarities with satellite cells present in the adult muscles. In conclusion, we have shown that CAPN3 acts as a regulatory molecule on myogenic differentiation, and probably have implications in the area of regeneration

Thesis (Ph. D.)--Medical College of Ohio, 2003.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Ronald Mellgren. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 92-124).

Introdução: A hipertrofia cardíaca induzida por sobrecarga hemodinâmica crônica (HC) é caracterizada por espessamento das paredes do ventrículo esquerdo e do tecido intersticial. As atividades aumentadas de calpaína-1 e metaloproteinase de matriz(MMP)-2 são observadas em diferentes modelos de hipertensão arterial e estão relacionadas com as mudanças fisiopatológicas na HC. Por outro lado, a atividade de MMP-2 parece ser modulada positivamente por ativação de calpaína-1 em diferentes modelos. O objetivo deste trabalho é analizar se a calpaína-1 contribui para o aumento da atividade de MMP-2 no coração e se esse mecanismo resulta nas mudanças crônicas cardíacas na hipertensão renovascular. Métodos: Ratos Wistar submetidos ao modelo de 2-rins-1 clipe (2R-1C)(180-200g) e seus respectivos controles (Sham) foram tratados com verapamil (VRP), um bloqueador de canais para cálcio tipo L (BCCL, 8mg/kg/bid) ou veículo durante 8 semanas. O BCCL reduz as concentrações intracelulares de cálcio, o que leva à diminuição da ativação de calpaína-1, e então à possível modulação da atividade e expressão proteica de MMP-2. Pressão arterial sistólica (PAS) dos ratos foi monitorada durante 10 semanas de hipertensão por pletismografia de cauda. O ventrículo esquerdo (VE) foi analisado por histologia e ecocardiografia para avaliação das dimensões ventriculares. A atividade de calpaína-1 e MMP-2 foi avaliada por zimografia em gel. A expessão proteica de calpaína-1 e MMP-2 foi avaliada por western blot e imunofluorescência. Os corações foram submetidos à avaliação funcional por Langendorff. Todos os protocolos foram aprovados pelo Comitê de Ética em Pesquisa Animal da Faculdade de Medicina de Ribeirão Preto (43/2017). Resultados: Após 10 semanas, a PAS teve um aumento sustentado nos animais 2R-1C e o tratamento com VRP não foi capaz de reduzí-la em nenhum tempo de hipertensão. O peso corporal não apresentou diferença significativa entre os grupos. O grupo hipertenso teve um aumento da massa cardíaca quando comparado ao sham e o tratamento com verapamil reduziu esse parâmetro. A análise da espessura do ventrículo esquerdo demonstra que o VRP é capaz de reverter a HC induzida por sobrecarga pressórica nos animais hipertensos. Os animais 2R-1C apresentaram um aumento singificativo na expressão proteica e atividade de calpaína-1 e o VRP foi capaz de diminuir esses níveis. Foi observado aumento da atividade das isoformas de MMP-2 nos ratos 2R-1C quando comparados aos controles e o VRP foi capaz de reduzir a atividade da isoforma de 64kDa. A contratilidade cardíaca intrínseca dos animais 2R-1C sugere uma disfunção cardíaca quando comparados aos controles sham, embora a fração de ejeção desses animais esteja preservada. O VRP não foi capaz de alterar esses parâmetros. Conclusão: O VRP pode contribuir para a redução da hipertrofia cardíaca por diminuir a expressão proteica de calpaína-1 e MMP-2 na hipertensão renovascular. Apoio financeiro: Capes, CNPq, FAPESP<br>Introduction: The chronic hemodynamic overload-induced cardiac hypertrophy (CH) is characterized by thickening of the left ventricle walls and hypertrophy of the cardiomyocytes and interstitial tissue. Increased activity of calpain-1 and matrix metalloproteinase(MMP)-2 was observed in different models of arterial hypertension models and contributes to the pathophysiologic changes shown in CH. On the other hand, MMP-2 activity is also positively modulated by activation of calpain-1 in different animal models of cardiovascular diseases. The objectives here are to analyze whether calpain-1 contributes to increase the activity of MMP-2 in the heart and whether this mechanism results in chronic cardiac changes in the renovascular hypertension. Methods: Two kidney-one clip (2K1C) hypertensive male Wistar rats (180-200g) and their respective controls (Sham) were orally treated with verapamil (VRP), a L-type calcium channels blocker (LCCB, 8mg/kg/bid), or vehicle during 8 weeks. The LCCB reduces the intracellular concentration of calcium, thus decreasing the activation of calpain-1, and then may modulate the activity of MMP-2. Systolic blood pressure (SBP) was monitored in the rats during 10 weeks of hypertension. Left ventricle (LV) was analyzed by histology and echocardiography to evaluate ventricle thickening. Calpain- 1 and MMP-2 activities were analyzed by zymography and their expression by immunofluorescence and western blot. Hearts were submitted to functional evaluation by Langendorff. All the protocols were approved by the Ethical Committee in Animal Research of Ribeirao Preto Medical School (43/2017). Results: After 10 weeks, the systolic blood pressure had sustained increase and treatment with VRP was not able to decrease it in any time of hypertension. The body weight did not present significant changes between the groups. Hypertensive group had significant increase in the ventricle/body weight ratio (VW/BW) when compared to sham and treatment with VRP decreased it. Analysis of ventricle thickening showed that VRP is able to revert CHinduced pressure overload. The 2K-1C rats showed a significant increase in the activity and expression of calpain-1 in the heart and VRP reverted it. It was also observed increased activity of MMP-2 forms in the hypertensive rats and VRP decreased the 64kDa MMP-2 activity. The 2K-1C group had cardiac dysfunction when compared to controls groups, and VRP did not alter it. The ejection fraction was not changed in 2K- 1C rats. Conclusion: VRP decreased expression and activity of calpain-1 and MMP-2 in the hearts of 2K-1C rats and then contributed to ameliorate hypertension-induced cardiac hypertrophy

O tipo biológico influenciado pela genética Bos taurus indicus garante maior adaptação aos animais em regiões com altas temperaturas, no entanto exibe alguns atributos de carcaça e associados à palatabilidade da carne inferiores quando comparados ao tipo biológico predominantemente Bos taurus taurus. Existe a hipótese de que tais diferenças estejam associadas ao conteúdo mitocondrial na musculatura destes animais. Objetivou-se avaliar as diferenças no metabolismo oxidativo, pela medida de respiração mitocondrial em fibras permeabilizadas submetidas a duas temperaturas (38,5 e 40 °C) utilizando respirômetro de alta resolução (Oroboros Instruments), do Longissimus lumborum imediatamente após abate, entre os tipos biológicos Brahman e Angus com taxa e extensão da proteólise distintas ao longo de 14 dias de maturação. Foram investigadas as atividades enzimáticas de lactato desidrogenase e citrato sintase, pH, bem como autólise de &mu;-calpaína e degradação de proteínas miofibrilares de 18 animais das raças Brahman (6), Brangus (6) e Angus (6). A taxa de acidificação das carcaças foi diferente entre os tipos biológicos (P = 0,049) e entre os tempos postmortem (P < 0,001), sendo que as carcaças dos animais Brahman às 6h postmortem apresentaram pH tão alto quanto as carcaças de Brangus e Angus às 3h posmortem. A atividade de citrato sintase 1h postmortem também foi diferente (P = 0,086) entre os tipos biológicos, sendo que maior atividade (P = 0,075) foi identificada em Brahman comparado a Brangus. O fluxo de consumo de oxigênio mitocondrial no estado 4 (ST4_O), apresentou interação (P = 0,050) entre os tipos biológicos e temperatura, assim como a respiração mitocondrial do sistema de transporte de elétrons desacoplado da produção de ATP (P = 0,047), ambos superiores para Brahman a 38,5 °C na comparação entre tipos biológicos. Todavia, ocorreu uma queda da ST4_O, apenas para os animais Brahman, quando a temperatura foi aumentada. A maior resistência a acidificação e maior respiração mitocondrial imediatamente após o abate para o tipo biológico Brahman, foram acompanhadas pela redução (P = 0,058) da autólise completa de &mu;-calpaína às 24h postmortem. A degradação da titina após 7d de maturação foi reduzida (P = 0,056) e se manteve menor (P = 0,003) aos 14d para Brahman comparado aos outros tipos biológicos. O aparecimento da banda 150 kDa da alfa-II-espectrina às 24h postmortem também é menor (P = 0,011) para Brahman, assim como a degradação da desmina (P = 0,019) e troponina-T (P = 0,001). As duas últimas proteínas tiveram sua degradação reduzida ao longo dos 14d postmortem para este tipo biológico. Portanto, o tipo biológico Brahman tem um mecanismo muscular adaptativo que envolve maior conteúdo mitocondrial, com maior vazamento de prótons pelo sistema de transporte de elétrons e que pode estar relacionado a redução da taxa de ativação da &mu;-calpaína, redução da degradação proteica e, consequentemente, a redução da taxa e da extensão do amaciamento da carne destes animais.<br>Biological type influenced by Bos taurus indicus ensures higher animals adaptation to warm environment, however exhibit less desirable carcass and palatability traits when compared to predominant Bos taurus taurus biological types. There is a hypothesis that muscle mitochondrial content is associated with those differences. It was aimed to evaluate the oxidative metabolism, measuring mitochondrial respiration in permeabilized fibers sumitted to two temperatures (38.5 and 40 °C) using high resolution respirometry (Oroboros Instruments), in Longissimus lumborum imediately after slaughter between biological types Brahman and Angus with different proteolysis rate and extension during 14d aging. Were investigated lactate dehydrogenase and citrate sinthase activities, pH, as well as &mu;-calpain autolysis and protein degradation between 18 animals from breeds Brahman (6), Brangus (6) and Angus (6). The carcass pH decline is different between biological types (P = 0.049) and between time postmortem (P < 0.001), with Brahman carcasses showing pH at 6h postmortem as high as Brangus and Angus pH at 3h postmortem. Citrate sinthase activity 1h postmortem is different (P = 0,086) between biological types, with Brahman showing higher (P = 0.075) values than Brangus animals. The mitochondrial oxygen flux consumption at state 4 (ST4_O) showed interaction (P = 0.050) between biological types and temperature, as well as respiration for electron transport system uncopled from ATP production (P = 0.047), both higher for Brahman at 38.5 °C in biological types comparison. However, decreased ST4_O was observed only for Brahman animals when temperature was increased. Higher resistance to pH decline and higher mitochondrial respiration after slaughter to Brahman cattle were followed by reduced (P = 0.058) complete &mu;-calpain autolysis at 24h postmortem. Titin degradation after 7d aging was reduced (P = 0.056) and kept lower (P = 0.031) after 14d to Brahman when compared to other biological types. The 150 kDa band from alpha-IIspectrin degradation at 24h postmortem is lower (P = 0.011) to Brahman, as well as desmin degradation (P = 0.019) and troponin-T (P = 0.001). Desmin and troponin-T had its degradation reduced extended until 14d aging for this biological type. Therefore, Brahman biological type has an adaptive muscular mechanism that involves higher mitochondrial content, with higher proton leak in electron transport system and that can be related with reduced rate for &mu;-calpain activation, reduced protein degradation and, consequentely, reduced rate and extension to beef tenderization for these animals.

This thesis reports the development of potent and selective inhibitors of m-calpain for the treatment of cataract. SJA6017 has been proven to prevent lens opacity in rat and has been our lead compound. A series of Val-Leu peptidyl aldehyde inhibitors (33a-e, 33g, 33i and 35) have been designed, synthesized, and tested for therapeutic potential as cataract inhibitors. Chapter 1 is an introduction to calpain and the diseases associated with it's over activation. A review of the literature on calpain inhibition is given. Structure activity relationship (SAR) theory is presented. The techniques that have been applied in our research group to drug design include molecular modeling, synthesis, assay and animal studies which are all briefly discussed. The importance of a -strand conformation for an inhibitor to bind to calpain is discussed. Chapter 2 describes the synthesis of m-calpain inhibitors. This comprises the preparation of the Val-Leu dipeptide core 29, Val-Leu dipeptidyl alcohols 31a-g and 31i, and the synthesis of dipeptidyl aldehydes 33a-e, 33g, 33i and 35. The choice of coupling regents and conditions in the coupling reactions is investigated. Sulfur trioxide pyridine oxidation for the conversion of Val-Leu dipeptidyl alcohols to aldehydes is discussed. The molecular modeling and biological assay results are presented.

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF-hand motif characteristic of the classical calpains, calpain 1 and 2, but retains catalytic and Ca2+ binding non EF domains. Tra-3, an ortholog of CAPN5, is involved in necrotic cell death in C.elegans; although specific role of CAPN5 has not been investigated in the mammalian CNS. I compared relative mRNA levels of calpains in rat CNS, which revealed that CAPN5 is the second most highly expressed calpain. We examined relative levels of CAPN5 from late embryonic day 18 to postnatal day 90 and found lower mRNA but higher protein levels during CNS development. Using X –gal staining in Capn5 +/- mice, immunostaining of rat brain sections and SH-SY5Y cells, and subcellular fractionation of rat brain cortex, we found that CAPN5 is a non-cytoplasmic calpain localized in the nucleus and enriched in synaptic mitochondria. Proteinase K treatment of mitochondria and mitoplasts from B35 rat neuroblastoma cells and rat synaptic mitochondria revealed CAPN5 was localized on the inner mitochondrial membrane and released from mitochondria on membrane permeabilization with alamethicin. We used immunolabelling, confocal imaging, nuclear subfractionation and transient transfections to evaluate the subnuclear localization of CAPN5. CAPN5 was detected in punctate domains and associated with promyelocytic leukemia (PML) protein, a tumor suppressor protein. We further demonstrated that CAPN5 carries a nonconventional bipartite nuclear localization signal. Together, these findings demonstrate that CAPN5 is a non-cytosolic calpain, abundant in the CNS and localized to the mitochondria inner membrane and nuclear PML bodies.

Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and the adoption of such information by the meat industry. It is essential that we understand the mechanism of meat tenderisation of which, the calpain protease system is believed to play a central role. This thesis focuses on three aspects; characterisation of calpain activity, the effect of porcine μ-calpain on myofibril degradation and the effect of μ-calpain on specific proteins desmin and troponin-T. To study the effect of calpain activity, fluorogenic assays were used to determine: μ-calpain concentration for optimal peptide cleavage; calcium requirements and the effect of chelating substances on the activity of μ-calpain. In addition, the affinity of μ-calpain for substrates CalS-I and CalS-III were assessed. The effect of μ-calpain on myofibril degradation was evaluated through the use of myofibrillar fragmentation index and density marker beads. Myofibrils were digested at three different temperatures for varying time periods. Conflicting results were displayed and it was concluded that these methods are not accurate, thus further research should be conducted to ensure inconsistencies are eliminated. Specific proteins desmin and troponin-T have previously been shown to exhibit degradation in the presence of calcium and μ-calpain. SDS-polyacrylamide electrophoresis, western blotting and densitometry measurements were utilized to investigate this effect. It was concluded that μ-calpain plays a significant role in the post mortem proteolysis of myofibrillar protein. This thesis provides information and strives to give a better understanding of the proteolytic changes that occur within muscle. Understanding how these mechanisms affect meat on a cellular level, can help to control the influence they inflict on meat quality.

Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.

Trypanosomatid parasites are unicellular eukaryotes characterised by the presence of a subpellicular array of microtubules, a single flagellum, and a kinetoplast (containing the condensed mitochondrial DNA). The majority of trypanosomatid species undergo complex life-cycles, alternating between a mammalian host and an insect vector. Progression through this life-cycle requires the differentiation of trypanosomatids into distinct, niche adapted developmental forms. Differentiation into each life-cycle stage involves important biochemical and morphological changes, including the remodelling of the subpellicular cytoskeleton that defines cell shape. In higher eukaryotes, proteins from the calpain superfamily are involved in developmentally- and environmentally-regulated remodelling of the cytoskeleton and the dynamic organisation of signal transduction cascades. Interestingly, trypanosomatids contain unusually large families of calpain-related proteins, but there is little knowledge about the functional roles of these molecules during the life-cycle of trypanosomatid parasites. In this thesis, I present the results of the bioinformatic analysis of calpain-like proteins in three trypanosomatid parasites, Trypanosoma brucei, Leishmania major and Leishmania infantum. From this analysis, I selected several calpain-related proteins tor RNAi functional analysis, on the bases of their domain composition and conservation across different species. The detailed analysis of the resulting RNAi phenotypes revealed the essential function of some calpain-like proteins for the correct morphogenesis of specific developmental forms of T. brucei, shedding some light on the mechanisms that regulate this parasite differentiation and cytoskeletal remodelling, and providing new putative therapeutic targets for African sleeping sickness.

The calpain system is associated with cancer chemotherapeutic response in both in vivo and in vitro studies. Previous immunohistochemistry (IHC) data conducted in our group indicated that high calpain-2 expression was associated with both the resistance to platinum-based adjuvant chemotherapy and worse patient outcome; moreover calpain-2 appeared as an independent prognostic factor in multivariate analysis. To test the hypothesis that conventional calpain subunits, especially calpain-2, are associated with the chemo-resistance of ovarian cancer cells to platinum-based chemotherapy (cisplatin and/or carboplatin), five ovarian cancer cell lines, with varying platinum-based chemotherapy sensitivities, were chosen as in vitro models: the platinum-sensitive A2780 cells and its resistant counterpart A2780-cis cells; the platinum-resistant SKOV3 cells; and the platinum-sensitive PEO1 cells and its platinum-resistant counterpart PEO4 cells. Western blotting was used to assess the expression of the conventional calpain subunits (i.e. calpain-1, -2 and -4) and calpastatin in this panel of cell lines. Calpain activity was regulated by inhibitor calpeptin and calpain-2 short hairpin RNA (shRNA) was used in attempt to specifically downregulate calpain-2 expression. Calpain activity was assessed by an activity assay using fluorogenic peptidase substrate t-BOC. The role of calpain in proliferation and resistance to platinum-based chemotherapy were examined in vitro using growth curves and colony formation. Moreover the study of calpain-4 expression was added into the current project, in addition to verifying the association between the expression of calpain-1, -2 and calpastatin and clinicopathologic variables (e.g. chemo-resistance and patient outcome) by standard immunohistochemistry (IHC) with a larger patient cohort (n=575). To test the hypothesis that conventional calpain subunits and calpastatin are associated with ovarian tumour metastasis, the effect of calpain inhibition (by calpain inhibitors) and activation (by calcium ionophore) on ovarian cancer cell migration was examined using haptotaxis (scratch wound) migration assay. Based on information from 2016 FASEB calpain conference, microtubule-associated protein 4 (MAP4) and spleen tyrosine kinase (Syk) appeared as potential calpain-related proteins associated with angiogenesis and epithelial-mesenchymal transition. Using IHC, their protein expression (i.e. MAP4 and Syk) was assessed and their associations with clinicopathological variables in ovarian cancer patient samples were studied; besides, their correlations with conventional calpain subunits and calpastatin, together with EMT (epithelial-to-mesenchymal transition)-associated proteins and angiogenesis-associated proteins (n=87, data provided by Dr S. Deen) were analysed. Significant variations of calpain system protein expression levels were observed between the different cell lines. Among the 5 cell lines, A2780 and A2780-cis cells (likely to be endometrioid carcinoma cell lines) expressed very low levels of the conventional calpain subunits and calpastatin; whilst PEO1 and PEO4 cells (high-grade serous carcinoma cell lines) expressed comparatively higher level of these proteins. Thus, different expression of the calpain system seemed to be associated with ovarian cancer histological subtypes, which was supported by the IHC study. No significant difference of the calpain system expression was detected and calpeptin caused a similar inhibition of cell proliferation between chemo-sensitive ovarian cancer cells and their resistant counterparts. Because SKOV3 and PEO4 cells expressed the highest levels of calpain-2, shRNA was used for specific knockdown of calpain 2 in these two cell lines, unfortunately numerous attempts proved unsuccessful. Although with the optimised concentration and treatment duration, calpeptin could inhibit approximately 30% of calpain activity, down-regulation of calpain activity via calpeptin could not sensitise ovarian cancer SKOV3, PEO1 and PEO4 cells to cisplatin and carboplatin. Hence, to revisit the question as to whether conventional calpains and calpastatin are associated with chemoresponse and patient survival, a larger cohort was used to validate the previous study. In the current study, the expression of the conventional calpain subunits and calpastatin were positively associated with each other. Calpain-2, -4 and calpastatin expression were associated with overall survival (OS) but none of them was an independent marker of OS in multivariate analysis. Neither the conventional calpain subunits nor calpastatin expression was found associated with resistance to platinum-based adjuvant chemotherapy. Since low calpain-1 expression was associated with low tumour stage, cellular processes that involved in cancer spread were studied. Again calpeptin was used for the inhibition of calpain activity, but no significant inhibition was observed on ovarian cancer cell migration. In contrast, upregulating calpain activity by A23187 using optimised concentration was found able to significantly inhibit the migration of SKOV3 cells. The recently found calpain-related proteins MAP4 and Syk were then included into the current study. Like calpain-1, neither MAP4 nor Syk expression was associated with patient outcome. Next, cases were grouped by clinicopathological variables for the examination of the association between the protein expression and survival; in such a case only high nuclear Syk expression was significantly associated with better patient outcome in certain subgroups. Low calpain-1 expression was associated with low tumour stage, so were MAP4 and Syk expression. MAP4, Syk and calpain-1 expression were significantly associated with tumour histological subtypes and their expression were significantly correlated with each other. Integrin α2β3 was moderately correlated with calpain-1, MAP4 and cytoplasmic DARC expression. In conclusion, although in both the previous and the current study, calpain-2 expression was adversely associated with OS of ovarian cancer patients, the current results did not support the initial hypothesis that calpain can sensitise ovarian cancer cells to cisplatin/carboplatin. The roles that calpain system played in cancer cell haptotactic migration appeared to vary with cell context. Calpain-1, MAP4 and Syk expression were significantly correlated with each other and were closely related to ovarian cancer spread.

This thesis summarises the progress made in the design and synthesis of conformationally constrained β-strand peptidomimetic compounds using ring closing metathesis methodology under microwave irridation conditions. The best macrocycle were elaborated into an inhibitor for a specific protease target. Calpain was used as an example of protease targeting cataract disease. Chapter One introduces proteases in general centring on the general context of protease inhibitor design. The significant of the β-strand 'bioactive' conformation is discussed in details in particular the exploitation of conformationally constrained to potential lock the 'bioactive' conformation. Chapter Two illustrates in silico methods used to design a series of β-strand macrocycle 2.1-2.7. The analysis of these is performed using molecular modelling software Schrodinger suite (2005). A brief discussion of ring closing metathesis methodology is also included. Chapter Three describes the synthesis of the precursor required for RCM reactions (tripeptides dienes). Various types of allylated amino acid side chains were synthesised. The tripeptides were obtained using standard peptide coupling methodology utilising reagents such as HATU, EDC and HOAT. Chapter Four describes the application of ring closing metathesis for the synthesis of β-strand macrocycles. The development of a new reaction conditions to optimise the ring closing metathesis reaction is discussed. In particular the effect of the use of a Lewis acid (chlorodicyclohexylborane) additive in RCM reactions is investigated. Chapter Five discusses the mechanism of cataract formation, cataract treatment and the potential development of calpain inhibitors. One of the macrocycles synthesised in chapter 4 is elaborated into a calpain inhibitor. The in-vitro assay result of this is presented and this compound is currently undergoing in vivo evaluation.

The calcium activated cysteine protease calpain-I has a pivotal role in a variety of physiological processes within the human body. In particular calpain-I enables the cell spreading and subsequent chemotaxis behaviour of neutrophils in response to tissue damage. Neutrophils are linked to the pathological condition rheumatoid arthritis and so calpain-I is considered be a valuable therapeutic target. Many inhibitors of calpain-I are highly non-selective with the exception of two small molecule synthetic inhibitors. A phenyl and an indole-based α-mercaptoacrylic acid have shown a slight selectivity towards calpain-I over other cysteine proteases. In this work 24 novel monohalogenated α-mercaptoacrylic acid inhibitors were prepared based on these lead structures using Vilsmeier-Haack chemistry followed by Knoevenagel condensation of the resulting aromatic aldehydes as key steps. The thiols within the α-mercaptoacrylic acid moiety demonstrated a tendency to form disulfide bridges in solution. Analysis of this disulfide formation through 1H NMR spectroscopy, UV-Vis spectrophotometry and HPLC showed that the monomeric form was active under the reducing conditions used in subsequent assays. The analogues were tested as inhibitors of calpain-I revealing that bromoindole based inhibitors were the most potent. Selected compounds showed ~10 fold selectivity towards calpain-I versus calpain-II. In live neutrophils they were capable of slowing the cell spreading process by up to 70%. When live neutrophils containing the inhibitor were irradiated with 410 nm light, the cells completely lost the ability to spread. To show that these compounds were allosteric inhibitors the calcium binding domain PEF(S) was expressed in E. coli and purified using anion exchange chromatography and size exclusion chromatography. Solution of X-ray co-crystal structures of the calpain PEF(S) domain with two different inhibitors revealed that they bind to the protein in a similar fashion as an α-helical domain of calpastatin, the endogenous inhibitor of calpain.

The work in this thesis reports studies directed to developing a calpain cysteine protease inhibitor that could be of value in slowing cataract development in humans. The work focuses on the development of macrocyclic compounds which can have advantages over acyclic compounds due to their resistance to proteolytic hydrolysis, improved selectivity, bioavailability and membrane permeability. A review of X-ray crystal structures of natural and synthetic calpain inhibitors complexed with the cysteine protease calpain show the inhibitors generally bind in the enzyme active site in an extended β-strand conformation. The calpain inhibitor SJA-6017 has been identified as a suitable lead compound. The importance of the para-fluoro group in SJA-6017 has been investigated. Modifications have been made to constrain this basic structure within a macrocycle and restrict the peptide chain as a β-strand conformation. Macrocycle CAT811 is a potent calpain 1 and 2 inhibitor and shows promise in slowing the progression of cortical cataract in trials with sheep having a hereditary propensity towards the development of cataract. In this thesis I report studies directed to improve the yield of the key RCM macrocyclisation step in the synthesis of aldehyde CAT811 and of three ester analogues (2.1, 2.3 and 2.4). I also report the development of a more commercial route to CAT811 not involving RCM but using intramolecular nucleophilic cyclisation. This intramolecular nucleophilic cyclisation strategy was attempted for the preparation of a histidine containing macrocyclic ester (4.1a) but was unsuccessful. An alternate strategy involving intramolecular lactamization proved successful for the synthesis of histidine-based macrocyclic esters (4.1a-4.3a). Reduction to the corresponding alcohols (4.1b-4.3b) was successful and oxidation of (4.1b and 4.3b) afforded the corresponding aldehydes (4.1c and 4.3c) for biological assay against ovine calpain 2. Aldehyde 4.3c has an IC50 of 1 μM and the corresponding alcohol 4.3b shows no activity (IC50 > 50 μM) consistent with the modelling which indicated that these two compounds did not adopt a β-strand conformation in the docking studies. Aldehyde 4.1c, on the other hand, shows significant inhibitory activity with an IC50 of 238 nM but as expected the corresponding alcohol 4.1b shows little activity (IC50 = 29 μM). Modelling studies showed that both the aldehyde 4.1c and the alcohol 4.1b on docking can form a β-strand with appropriate H-bonding interactions. The aldehyde is more active than the alcohol due to the reactivity of the aldehyde warhead allowing for the reversible formation of a hemiacetal. A similar difference in reactivity is observed for CAT811 (30 nM) and its alcohol analogue (700 nM). These results demonstrate the value of molecular modelling as a screening mechanism before unproductive synthetic work is considered.

Calpains are a ubiquitous family of calcium-dependent cysteine proteases involved in a wide range of cell regulatory and differentiation processes. In many protozoan organisms, atypical calpains have been discovered that lack the characteristic calcium-binding penta-EF-hand motif of typical vertebrate calpains and most of these novel calpain-like proteins are non-enzymatic homologues of typical calpains. The gene family is particularly expanded in ciliates and kinetoplastids, comprising 25 members in the parasite Trypanosoma brucei. Unique to kinetoplastids, some calpain-like proteins contain N-terminal dual myristoylation/palmitoylation signals, a protein modification involved in protein-membrane associations. We analysed the expression of calpain-like proteins in the insect (procyclic) and bloodstream-stage of T. brucei using quantitative real time PCR and identified the differential expression of some of the calpain genes. We also present a comprehensive analysis of the subcellular localisation of selected members of this protein family in trypanosomes. Here, of particular interest is the role of protein acylation for targeting to the flagellum. We show that, although acylation is important for flagellar targeting, additional signals are required to specify the precise subcellular localisation.

Variation in the calpain-10 gene has been linked to a three-fold increased risk for type 2 diabetes in Pima Indian and some European populations. Furthermore, reduced skeletal muscle expression of calpain-10 is associated with reduced insulin mediated glucose disposal and carbohydrate oxidation. The skeletal muscle specific calpain-3 plays a key role in skeletal muscle integrity and has also been linked to insulin resistance in humans and rodents. The major aims of this thesis were to 1) investigate the hypothesis that alterations in insulin sensitivity in healthy humans would lead to significant changes in the mRNA and protein expression of calpain-10 and -3, 2) investigate the effect of hyperinsulinaemia and lipid availability on calpain-10 and -3 expression, 3) further address the role of genetic variation in the calpain-10 gene on glucose utilisation in humans and finally 4) investigate the expression of calpain-10 in skeletal muscle of type 2 diabetic patients. The studies in this thesis show for the first time that insulin resistance as a result of short term fasting or high fat availability is not associated with changes in calpain-10 and -3 mRNA and protein expression, providing evidence against an adaptive role for these genes in the development of fasting- and lipid-induced insulin resistance.

The mechanism by which β-agonists induce skeletal muscle hypertrophy is believed largely to be through a reduction in protein degradation. These growth promoters are also known to effect the activity of the calcium dependent proteinases (calpains) and their specific endogenous inhibitor calpastatin. The changes in activity appear to be toward a decrease in the calpain system's proteolytic potential. In this study attempts were made to determine whether the altered activity of the enzymes and inhibitor were brought about by induced changes in gene expression as reflected by altered levels of specific mRNAs. Various strategies were employed to generate oligonucleotide and cDNA probes to calpain I and II and calpastatin which would detect their respective mRNAs in L. dorsi total RNA samples originating from a bovine growth trial using the ß-agonist cimaterol. Semi-quantiative measurements of specific mRNAs using Northern blot analysis were related to enzyme and inhibitor activities. In addition ß-agonist-mediated effects on muscle RNA and expression of actin and myosin light chain 2 mRNAs were determined. Using a human calpain cDNA specific hybridization was detected for bovine calpain II mRNA which increased by 34% in the L. dorsi of cimaterol treated animals, similar to the increase in the enzyme activity, 28%. A novel bovine-specific calpastatin cDNA was generated by the polymerase chain reaction and sequence analysis allowed comparison to those already published for other species. Using this PCR cDNA as a probe multiple calpastatin mRNAs were detected in cattle L.dorsi, as had been observed in rabbit. The predominant mRNA increased by 160% in cimaterol treated steers compared to a 76% change in inhibitor activity. There changes were in contrast to the essentially unchanged response of muscle total RNA and actin and myosin light chain 2 specific mRNAs in treated animals. The implications for the calpain system in ß-agonist induced hypertrophy are discussed.

Regulated proteolysis plays an important role in modulating the activities of receptors, cytoskeletal proteins and transcription factors during cell growth and development. A family of calcium-regulated thiol proteases, known as calpains, has been shown to promote this process in mammals, and other eukaryotes. The importance of calpains is highlighted through their involvement in a number of pathologies in humans.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(医学)<br>甲第5555号<br>医博第1525号<br>新制||医||577(附属図書館)<br>UT51-94-C13<br>京都大学大学院医学研究科外科系専攻<br>(主査)教授 畑中 正一, 教授 岡 正典, 教授 山室 隆夫<br>学位規則第4条第1項該当

This investigation involved the synthesis of potential CA clan cysteine inhibitors of m-calpain and cathepsin B. Inhibitors 2.1.3a-j were based on the SJA-6017 construct containing the N-(4-fluorobenzenesulfonyl) moiety at the P₃ address region. The inhibitor 2.1.3k was based on CAT-0059 a novel dipeptide dialdehyde inhibitor containing the 5-formyl pyrrole moiety at the P₃ address region. Chapter 1 introduces proteases in particular m-calpain and cathepsin B implicated in human pathologies cataract and tumour metastasis respectably. Structure, disease processes and known inhibitors for m-calpain and cathepsin B are presented and described. The chapter also describes drug design and rational including the requirement of the β-strand conformation for enzyme substrate binding. Chapter 2 details the synthesis of m-calpain and cathepsin B inhibitors, N-(4-fluorobenzenesulfonyl) peptide aldehyde 2.1.3a-j and the dipeptide dialdehyde 2.1.3k. The synthesis involved the preparation of the N-(4-fluorobenzenesulfonyl) α-amino acids 2.1.8a-f, the N-(4-fluorobenzenesulfonyl) peptide esters of 2.1.10a-g, the peptide alcohols 2.1.11a-k and the peptide aldehydes 2.1.3a-k. Specific coupling reagents for amide bond formation are also discussed. The oxidation of the alcohols 2.1.11a-k with sulfur trioxide and pyridine complex are also addressed. The results from molecular modelling and enzymatic assays of the inhibitors 2.1.3a-k with m-calpain and cathepsin B are presented and discussed.

There have been several reports that cataract development results from unregulated Ca2+ mediated degradation of lens crystallins. The calpain isoform m-calpain, a cysteine protease, is known to be a major player in cataract formation in rodent lenses and recent evidence indicates that over-activation by Ca2+ causes cataractogenesis in other mammals. Molecular modelling studies of seventeen analogues of compound SJA6017 (our lead compound) in a calpain model are compared to measured IC50 values against ovine calpain. The studies validated the potential of the ‘model’, method and defined activity criteria that could be used as a tool to select molecules to synthesize as potential calpain inhibitors. Using this screening methodology and two virtual libraries of potential inhibitory molecules led to the synthesis of several inhibitors including macrocyclic 811. In vitro sheep eye lens culture experiments showed that macrocycle 811 possessed the characteristics to slow cataractogenesis.

Duchenne Muscular Dystrophy (DMD) is a severe X-linked progressive muscle wasting disease resulting from the absence of the membrane-associated protein dystrophin and the secondary components of the dystrophin-glycoprotein complex. Although the genetic basis of the disease has been known for over 15 years, the onset mechanism of the disease is not yet known and no treatment is yet available to significantly increase the lifespan of DMD patients. Increased levels of intracellular calcium have been noted in dystrophic muscle (Turner et al., 1991) and increased intracellular levels of calcium in skeletal muscle lead to increased levels of calcium-dependent proteolysis (Zeman et al., 1985). Increased levels of calpain, a calcium-dependent protease have been reported as early as age 4 weeks in mdx (dystrophin-deficient) mice (Spencer et al., 1995). Increased calpain activity has been demonstrated in mdx myotubes (Alderton et al., 2000a). There is also evidence of a role for calpain in DMD, but the contribution of calpain activity to the onset of DMD has not yet been determined. The purpose of this study was to test the hypothesis that increased calpain activity contributes to the onset of DMD in maturing (birth to weaning) dystrophic skeletal muscles and to determine if increased calpain activity was due to the relative distribution of calpain and calpastatin, calpainâ s endogenous inhibitor. Calpain activity was assessed in quadriceps and diaphragm muscle homogenate supernatant and pellet fractions from C57BL/6 control and mdx mice at ages 7, 14, and 21 days. Total calpain and calpastatin content were determined by Western analysis. In both the quadriceps and diaphragm samples, calpain activity in the supernatant increased with age. There was a significant increase (47.7%; p<0.05) in calciumdependent calpain activity in mdx quadriceps pellet compared to control at age 7 days. In the quadriceps at age 7 days, calpain activity in the pellet in the presence of calcium was significantly greater than at age 14 (61.2%) and 21 days (52.6%; p<0.05). In the diaphragm, there were no significant differences in pellet activity in either the presence or absence of calcium at any age between control and mdx samples. In both control and mdx diaphragms, pellet calpain activity in the absence compared with the presence of calcium was significantly greater at both age 7 (control, 46.4%; mdx, 45.4%) and 14 days (control, 42.4%; mdx, 43.6%; p<0.05). At age 21 days, both control and mdx calpain activities in the diaphragm supernatants in the presence of calcium were significantly greater than those at ages 7 (control, 66.7%; mdx, 72.1%) and 14 days (control, 39.9%; mdx 49.5%; p<0.05). In general, there were no differences in total calpain and calpastatin content that would account for the differences in calpain activity. There were similar patterns of calpain activity and total calpain and calpastatin content in both control and mdx samples, suggesting a similar pattern of development in control and mdx muscle from ages 7-21 days. The increase in calcium-dependent calpain activity in mdx quadriceps pellet compared to control at age 7 days may be due to differences in regulation and/or distribution of the calpain system early in mdx maturation compared to control. From the present study, the role of calpain in the onset of DMD appears to be minor if global calcium-dependent activity is evaluated.<br>Master of Science

Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings: 1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed. 2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age. 3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals). 4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpainâ s role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles. 5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles. 6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles. These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles.<br>Master of Science

Muscular dystrophy is a disease which gradually deteriorates skeletal muscle cells, leading to the eventual death of such cells and the surrounding tissue. Calpains are Ca2+- dependent proteases and together with the Ca2+-dependent specific inhibitor of calpains, calpastatin, are widely distributed in eukaryotic cells. It has been suggested that part of the enhanced deterioration in the dystrophic state is due to enhanced calpain activity; therefore analysis of normal and dystrophic muscle was essential. Conventional techniques for the isolation and characterization of calpain and calpastatin utilize relatively large muscle samples (>100g), whereas biopsy or post-mortem samples are considerably less than this. Thus, the initial and main objective of this project was to develop methods suitable for purification and analysis of the calpain family from limited muscle samples. With these restrictions in mind, techniques were developed for samples ranging from 0.2-1g, a realistic biopsy extraction. The hypothesis to be further evaluated is that some dystrophies are characterized by increased calpain activity, caused either by an increased expression of m- or μ- calpain or decreased inhibition by calpastatin, or both. The procedures are now in place to test this hypothesis further and extensive analyses are required using defined dystrophic types and increased sampling numbers.