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Orientador: Wilson Nadruz Júnior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T15:00:13Z (GMT). No. of bitstreams: 1 Fernandes_MaruskadoRocioNeufert_D.pdf: 2265410 bytes, checksum: 8aea5cb9fd86986cff8999d0e81b40fc (MD5) Previous issue date: 2011 / Resumo: O aumento do estresse circunferencial ou mecânico é um dos principais estímulos responsáveis pela aterogênese induzida por hipertensão arterial, além de ser um determinante para a localização das placas ateroscleróticas na árvore arterial. Neste contexto, moléculas mecano-sensíveis ou responsivas ao estresse mecânico podem exercer um papel fundamental no desenvolvimento do fenótipo pró-aterogênico em células vasculares. A quinase de adesão focal (FAK) tem sido considerada uma proteína central na mecanotransdução em diversos tipos celulares, por seu papel potencial na ativação de vias de sinalização envolvidas no crescimento celular, anti-apoptose e inflamação. Neste trabalho, nós inicialmente caracterizamos a ativação da FAK em linhagem de célula endotelial de aorta de coelho (RAEC) submetida a estiramento mecânico pulsátil e, em seguida, investigamos a influência da inibição desta proteína, por meio de oligodeoxinucletídeo-antisense e pelo inibidor farmacológico PP2, sobre a expressão de moléculas pró-aterogênicas e a adesividade leucocitária neste modelo experimental. Nossos resultados mostraram que a FAK é ativada precocemente por estiramento mecânico e é fundamental para a expressão de TLR2, TLR4, VCAM-1 e E-selectina induzida por sobrecarga mecânica em células endoteliais. A inibição da FAK endotelial com PP2 e oligodeoxinucletídeo-antisense bloqueou a adesão de células monocitóides THP1 às células endoteliais induzida por estiramento in vitro. O próximo passo foi avaliar o impacto da FAK sobre expressão de moléculas pró-aterogênicas induzida por sobrecarga hemodinâmica in vivo, utilizando o modelo de coarctação da aorta em ratos Wistar. Os resultados dos estudos in vivo demonstraram que a FAK é ativada nas primeiras horas após instituição da sobrecarga pressora em segmentos de aorta. Após 7 dias de coarctação, os segmentos aórticos proximais à estenose apresentaram aumento na expressão de TLR2, TLR4, VCAM-1, E-selectina, metaloproteinases de matriz 2 e 9, além de maior adesividade às células THP1. Estes fenômenos foram inibidos por meio de tratamento prévio dos animais com small interference RNA direcionado especificamente contra a FAK. Em conjunto, estes dados indicam que a FAK exerce um papel fundamental na resposta pró-aterogênica de células vasculares ao estresse mecânico in vitro e in vivo / Abstract: The increase in circumferential or mechanical stress is a major stimulus by which hypertension stimulates atherogenesis and a main determinant for the location of atherosclerotic plaques in the arterial tree. Mechano-sensitive molecules can play a key role in the development of pro-atherogenic vascular cell phenotype. Focal Adhesion Kinase (FAK) has been considered a central protein in mechanotransduction, because of its potential role in the activation of cell signaling pathways involved in cell growth, anti-apoptosis, and inflammation. In this work, we initially evaluated the activation of FAK in rabbit aortic endothelial cell (RAEC) lineage subjected to cyclic mechanical stretch and then investigated the impact of FAK inhibition, by transfection with specific oligodeoxynucleotide antisense and pre-treatment with the pharmacological inhibitor PP2, on the expression of pro-atherogenic molecules and leukocyte adhesion in this experimental model. Our results showed that FAK was rapidly activated by mechanical stretch and was critical to stretch-induced expression of TLR2, TLR4, VCAM and E-selectin in endothelial cells. FAK endothelial inhibition also blocked the adhesion of THP1 monocytoid cells to endothelial cells induced by stretch in vitro. The next step was to investigate the role of FAK in load-induced expression of pro-atherogenic molecules in vivo, by subjecting Wistar rats to aortic constriction. The results of in vivo assays revealed an early activation of FAK in aortic segments subjected to pressure overload. After 7 days of aortic constriction, vascular segments subjected to high pressure exhibited increased expression of TLR2, TLR4, VCAM-1, E-selectin, matrix metalloproteinases 2 e 9, and higher adhesion to THP-1 monocytoid cells. These events were inhibited by pre-treatment of rats with small interference RNA designed to silence FAK expression. In general these findings indicate that FAK is critical do stretch-induced expression of pro-atherogenic molecules in vascular cells in vitro and in vivo / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T14:15:11Z (GMT). No. of bitstreams: 1 Consonni_SilvioRoberto_D.pdf: 7954683 bytes, checksum: 766ce32e5bbb5a5c0b8cc78fd2735dc0 (MD5) Previous issue date: 2015 / Resumo: Há um crescente avanço no desenvolvimento das tecnologias que permitam a localização de proteínas em células por microscopias de luz e eletrônica combinadas, com o uso das sondas moleculares miniSOG e Apex2, por exemplo. Adicionalmente busca-se compreender como proteínas são responsáveis pelas funções celulares e teciduais. A Quinase de Adesão Focal (FAK), proteína da cascata de sinalização das integrinas é considerada uma mediadora em potencial do estresse mecânico nos cardiomiócitos. Sabe-se que em cardiomiócitos submetidos a estímulos hipertróficos, ocorre rápida ativação da FAK e sua redistribuição subcelular, contudo são pouco conhecidos os mecanismos envolvidos nesses processos. Outra proteína de grande importância no coração é a Calsarcina-1 (CS1), regulador negativo da via de Calcineurina, crucial no desenvolvimento da hipertrofia cardíaca. Entretanto os mecanismos envolvidos nessa regulação negativa, assim como a distribuição subcelular de CS1 são pouco conhecidos. Visando à interação entre essas áreas para estudo da distribuição espaço-temporal dos componentes celulares e de proteínas, bem como a importância da FAK e CS1 no sarcômero e seu papel na sinalização hipertrófica sob estímulo mecânico, o objetivo geral desse trabalho foi explorar a capacidade das proteínas FAK e CS1 para incorporar geneticamente sondas moleculares que permitissem monitorar por meio de imagens o comportamento dessas moléculas-chave na biologia do disco Z em MVRNs submetida ao estiramento mecânico. Para isso, foram realizados ensaios de localização subcelular com uso de sondas moleculares aplicadas à microscopia correlativa, bem como ensaios bioquímicos, moleculares e de atividade enzimática. Os dados confirmaram a FAK associada às fibras de actina e adesões focais em células H9c2 e foi demonstrado à microscopia de luz que FAK wild-type (wt) translocou-se parcialmente para o compartimento nuclear após estimulação do agonista fenilefrina, enquanto que FAK Y397F (forma mutante inativa) não apresentou mesmo fenótipo. Por outro lado, apesar da padronização e expressão das construções com FAK e miniSOG ou Apex2 em células HEK 293T e H9c2, não foi conclusiva a localização subcelular da FAK, por meio do uso de microscopia eletrônica em MVRN. Provavelmente devido à distribuição difusa da maior parte das moléculas de FAK, não foi identificada uma região elétron-densa conclusiva à microscopia eletrônica de transmissão. No tocante à importância da CS1, observou-se que o estiramento cíclico não induziu o aumento na expressão proteica ou gênica relativa de CS1 e CnA, assim como não houve alteração na atividade fosfatase de CnA. No entanto houve redução da interação de CS1 e CnA, bem como alteração na localização de CS1 em MVRN sob estímulo mecânico. Dados de superexpressão e silenciamento de CS1 corroboram a regulação negativa de CS1 à CnA em MVRN sob estímulo mecânico. Baseando-se em dados estruturais, especulou-se que como o sítio de ligação de NFAT e CS1 à CnA são muito próximos e ao mesmo tempo distante do sítio ativo da fosfatase, é possível que o papel de CS1 na regulação negativa de CnA ocorra por impedimento espacial ao fator de transcrição NFAT. Portanto, esses resultados podem contribuir para uma possível inferência farmacológica, visto que a via de Calcineurina-NFAT é uma das principais mediadoras de hipertrofia em cardiomiócitos, mediante estímulos patológicos / Abstract: There is an increasing move towards the development of technologies that allow the localization of proteins in cells by combined electron and light microscopy, with the use of molecular probes such as miniSOG and APEX2. Additionally we seek to understand how proteins are responsible for the cellular and tissue functions. The Focal Adhesion Kinase (FAK) is protein of integrin signaling cascade considered as a potential mediator of mechanical stress in cardiomyocytes. It is known that in cardiomyocytes subjected to hypertrophic stimuli by rapid activation of FAK and its subcellular redistribution, however the mechanisms involved in these processes are poorly understood. Another very important protein in the heart is Calsarcin-1 (CS1), a negative regulator of the Calcineurin pathway which is crucial in the development of cardiac hypertrophy. However the mechanisms involved in the negative regulation as well as the subcellular distribution CS1 are poorly understood. Aiming at the interaction between these areas to study the spatial-temporal distribution of cellular and protein components, and the importance of FAK and CS1 in the sarcomere and its role in hypertrophic signaling under mechanical stimulation, the aim of this study was to explore the ability of FAK and CS1 to incorporate genetically molecular probes that allow monitoring through images the behavior of these key molecules in the Z disc biology in MVRNs subjected to mechanical stretch. To this end, we performed subcellular localization assays using molecular probes applied to the correlative microscopy, biochemical and molecular assays and enzymatic activity. These data confirm the FAK associated with actin and focal adhesions fibers in H9c2 cells and has been shown by light microscopy that FAK wild-type (wt) is partially translocated to the nuclear compartment after stimulation of the agonist phenylephrine, while FAK Y397F (inactive mutant form) did not show the same phenotype. Moreover, despite standardization and expression of FAK and miniSOG or APEX2 in HEK 293T cells and H9c2, it was inconclusive subcellular localization of FAK, through the use of electron microscopy, in MVRN. Probably due to the diffuse distribution of most FAK molecules, it has no conclusive electron-dense region in transmission electron microscopy. Regarding the importance of CS1, it was observed that the cyclic stretch did not induce an increase in protein expression or gene relative CS1 and CnA, as there was no change in the phosphatase activity of CnA. However there was less interaction CS1 and CnA and change in CS1 location in MVRN under mechanical stimulation. CS1 overexpression and silencing corroborate the negative regulation of the CS1 over CnA in MVRN under mechanical stimulation. Based on structural data, it has been speculated that as the NFAT and CS1 binding sites are very close in CnA and at the same time distant from the active site of the phosphatase, it is possible that the role of CS1 in the negative regulation of CnA occur by steric hindrance to the NFAT transcription factor. Therefore, these results may contribute to a possible pharmacological inference, whereas Calcineurin-NFAT pathway is a major mediator of hypertrophy in cardiac myocytes by pathologic stimuli / Doutorado / Biologia Tecidual / Doutor em Biologia Celular e Estrutural

Indiana University-Purdue University Indianapolis (IUPUI) / Pyk2: Potential Regulator of Post-Menopausal Bone Loss H.W. LARGURA1,2*, P. ELENISTE2, S. HUANG2, S. LIU1, M. ALLEN3, A. BRUZZANITI2. 1Indiana University School of Dentistry Department Orthodontics and Oral Facial Development, 2Indiana University School of Dentistry Department of Oral Biology, 3Indiana University School of Medicine Department of Anatomy and Cell Biology, Indianapolis, Indiana, USA Osteoporosis is a pathologic condition of bone, commonly found in post-menopausal women, which occurs from an imbalance between bone formation and resorption. Following menopause, the bone resorbing activity of osteoclasts exceeds bone formation by osteoblasts, resulting in decreased trabecular and cortical bone and a subsequent decrease in bone mass. Reduced bone mass increases the risk of pathologic fracture of bones. Due to adverse effects associated with current treatment protocols for bone loss, alternative treatment modalities with reduced adverse effects are needed. Estrogen plays a role in maintaining balance in the bone remodeling cycle by controlling remodeling activation, osteoblast and osteoclast numbers, and their respective effectiveness in formation and resorption. With declining estrogen levels, this elegantly balanced interaction is altered and bone resorption exceeds bone formation, resulting in bone loss and increased bone fragility. Pyk2 is a protein tyrosine kinase that plays an important role in regulating bone resorption by osteoclasts, as well as osteoblast proliferation and differentiation. Deletion of the Pyk2 gene in mice leads to an increase in bone mass, in part due to dysfunctional osteoclast and osteoblast activity. In this study, we examined the role of Pyk2 in the effects of estrogen on bone mass. We used wild type (WT) and Pyk2 knock-out (KO) mice that had been ovariectomized (OVX) and treated with or without estrogen (E2)-releasing pellets. Control mice included sham OVX surgery receiving placebo pellet. We found that deletion of Pyk2 conferred increased bone mass in sham, OVX and OVX+E2 mice. In addition, Pyk2 KO mice supplemented with 17estradiol exhibited a marked increase in bone volume/trabecular volume, trabecular number, and trabecular thickness, but not cortical bone parameters compared to WT mice. Results of this study provide evidence for the role of Pyk2 in the effects of estrogen on bone mass. Understanding the role of Pyk2 in bone could lead to the development of new pharmaceutical targets for the treatment of bone loss associated with osteoporosis.

Editorial on the Research Topic. Editorial: Editor’s Pick 2021: Highlights in Cell Adhesion and Migration.

Im Mittelpunkt dieser Arbeit standen Signaltransduktionsprozesse in den Strukturen der Kraftübertragung quergestreifter Muskelzellen, d. h. in den Costameren (Zell-Matrix-Kontakten) und den Glanzstreifen (Zell-Zell-Kontakten der Kardiomyozyten).<br><br>Es ließ sich zeigen, dass sich die Morphologie der Zell-Matrix-Kontakte während der Differenzierung von Skelettmuskelzellen dramatisch ändert, was mit einer veränderten Proteinzusammensetzung einhergeht. Immunfluoreszenz-Analysen von Skelettmuskelzellen verschiedener Differenzierungsstadien implizieren, dass die Signalwege, welche die Dynamik der Fokalkontakte in Nichtmuskelzellen bestimmen, nur für frühe Stadien der Muskeldifferenzierung Relevanz haben können. Ausgehend von diesem Befund wurde begonnen, noch unbekannte Signalwege zu identifizieren, welche die Ausbildung von Costameren kontrollieren: In den Vorläuferstrukturen der Costamere gelang es, eine transiente Interaktion der Proteine Paxillin und Ponsin zu identifizieren. Biochemische Untersuchungen legen nahe, dass Ponsin über eine Skelettmuskel-spezifische Insertion im Carboxyterminus das Adapterprotein Nck2 in diesen Komplex rekrutiert. Es wird vorgeschlagen, dass die drei Proteine einen ternären Signalkomplex bilden, der die Umbauvorgänge der Zell-Matrix-Kontakte kontrolliert und dessen Aktivität von mitogen activated protein kinases (MAPK) reguliert wird.<br><br>Die Anpassungsvorgänge der Strukturen der Kraftübertragung an pathologische Situtation (Kardiomyopathien) in der adulten quergestreiften Muskulatur wurden ausgehend von einem zweiten Protein, dem muscle LIM protein (MLP), untersucht. Es konnte gezeigt werden, dass ein mutiertes MLP-Protein, das im Menschen eine hypertrophe Kardiomyopathie (HCM) auslöst, strukturelle Defekte aufweist und weniger stabil ist. Weiterhin zeigte dieses mutierte Protein eine verringerte Bindungsfähigkeit an die beiden Liganden N-RAP und alpha-Actinin. Die molekulare Grundlage der HCM-verursachenden Mutationen im MLP-Gen könnte folglich eine Veränderung der Homöostase im ternären Komplex MLP &ndash; N-RAP &ndash; alpha-Actinin sein. Die Expressionsdaten eines neu generierten monoklonalen MLP-Antikörpers deuten darauf hin, dass die Funktionen des MLP nicht nur für die Integrität des Myokards, sondern auch für die der Skelettmuskulatur notwendig sind. / The cell-matrix-contacts (costameres) and cell-cell-contacts (intercalated discs of cardiomyocytes) of cross-striated muscle cells transmit mechanical forces to the exterior. On top of this mechanical function, both structures have been implied to be involved in signal transduction processes.<br><br>Dramatic morphological changes in the overall structure of cell-matrix-contacts of skeletal muscle cells were revealed during differentiation. Moreover, this reorganisation was accompanied by alterations in protein composition. Immunofluorescence microscopy indicated that signalling pathways which control the dynamics of focal contacts in non-muscle cells seem to be important only for early differentiation stages of skeletal muscle cells. To explore novel signalling pathways involved in regulating the formation of costameres, signalling molecules engaged were identified. Thus, paxillin and ponsin transiently interact at the precursors of costameres during muscle development. In addition, biochemical data indicate that a skeletal muscle specific module in the carboxyterminal part of ponsin can recruit the adapter protein Nck2 to this complex. Hence, the three proteins might form a ternary signalling complex involved in controlling the reorganisation of cell-matrix-contacts. Apparently, the activity of this signalling complex is regulated by mitogen activated protein kinases (MAPK).<br><br>A second approach has focussed on adaptational processes of the same structures observed in pathological situations. In particular, the role of muscle LIM protein (MLP) in hypertrophic cardiomyopathy (HCM) was investigated. It was shown that a HCM-causing mutant MLP protein fails to fold properly and that the consequent loss of stability is reflected in altered binding properties: the mutant MLP protein shows decreased binding to both N-RAP and alpha-actinin. Hence, the molecular basis for HCM-causing mutations in the MLP gene might be an altered homeostasis of the ternary complex MLP &ndash; N-RAP &ndash; alpha-actinin. Increasing evidence indicates that the functions of MLP are required not only for the integrity of the myocardium. In addition, MLP seems to have regulatory functions in skeletal muscle tissues.

Orientador: William Dias Belangero / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T23:43:39Z (GMT). No. of bitstreams: 1 Faria_LidiaDornelasde_M.pdf: 3066212 bytes, checksum: 6cb5506682740e2fcb2de4b1694998a3 (MD5) Previous issue date: 2015 / Resumo: A Terapia por ondas de choque (TOC) é uma alternativa não invasiva utilizada como método de indução a formação óssea que consiste em pulsos sonoros de alta energia transmitidas de modo focal a um tecido específico. Artigos demonstram aumento de vascularização, que ativação de proteínas como BMP (bone morphogenic protein) e Erk (extracellular signal-regulated kinase) induzindo diferenciação osteogênica após sua utilização no tecido ósseo. O presente estudo visou avaliar os níveis das proteínas Erk e Akt (akutely transforming), envolvidas na cascata protéica responsiva a deformação celular gerada por estímulo mecânico e consequente transformação em estímulo bioquímico induzindo a osteogênese. Os animais selecionados para o estudo foram anestesiados e divididos em dois diferentes grupos, onde no dia 1, o primeiro grupo foi submetido a TOC em sessão única de 500 impulsos gerados por aparelho eletrohidráulico a 0,12mJ/mm²na tíbia intacta e o segundo não recebeu TOC. Na sequência, os animais foram divididos em 3 subgrupos para cada tempo de segmento de 7, 14 e 21 dias A determinação dos níveis das proteínas propostas foi realizada por meio de immunoblotting. A fosforilação das proteínas Erk e Akt dos tecidos ósseos das tíbias extraídas dos ratos aumentou nos grupos submetidos a TOC após 7, 14 e se manteve elevado até o 21° dia quando comparado ao controle / Abstract: Extracorporeal shock wave therapy (ESWT) is a non-invasive alternative used as a method for inducing bone formation that consists of high-energy acoustic pulses transmitted in a focal way to a specific tissue. Studies show increase in vascularization which activate proteins such as BMP and Erk inducing osteogenic differentiation after its use in the bone tissue. The present study aimed at evaluating the levels of Erk and AKT proteins involved in the protein cascade responsive to cell deformation in biochemical stimulus inducing osteogenesis. The animals selected for the study were under anesthesia and divided in two different groups where on day 1 the first group was submitted to ESWT in one 500 pulse-session generated by an electrohydraulic device at 0,12mJ/mm² in intact tibia and fibula and the second did not receive ESWT. Then the animals were divided into 3 sub-groups, one for each segment times of 7, 14 and 21 days. Immunoblotting analysis was performed to determine the levels of the proposed proteins. The Erk and Akt protein phosphorylation of the bone tissues of extracted tibia from the animals increased in the groups submitted to ESWT and kept elevated until the 21st day when compared to the control group / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências

Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering.

Les myopathies centro-nucléaires (CNM) sont un groupe de maladies musculaires sévères caractérisées par une faiblesse musculaire générale. La forme la plus sévère est la CNM liée à l’X (XLCNM), causée par des mutations de la Myotubularine (MTM1). D’autres formes autosomales existent et sont causées par des mutations de l’Amphiphysine2 (BIN1) et de la Dynamine2 (DNM2). Les mécanismes pathologiques menant aux CNMs restent à éclaircir et à ce jour aucune thérapie n’est disponible pour traiter les patients. Nous avons modulé les niveaux de protéines de MTM1, BIN1 et DNM2 dans des modèles murins de CNMs. Nous avons découvert que la sous-régulation de DNM2 sauvait le modèle murin de XLCNM et que la sur-expression de la protéine BIN1 humaine sauvait le modèle murin XLCNM ainsi que la forme autosomale causée par les mutations DNM2. Nous avons montré que MTM1 contrôlait l’adhésion cellulaire et le recyclage de l’intégrine dans les cellules musculaires. Nous avons observé que la sur-expression de BIN1 sauvait la dérégulation du recyclage de l’intégrine dans le modèle murin de XLCNM, ce qui suggère un lien fonctionnel entre BIN1 et MTM1 nécessaire pour l’adhésion focale au niveaux musculaire. Notre étude montre que MTM1, BIN1 et DNM2 participe à une voie de signalisation commune et que BIN1 et DNM2 représentent de nouvelles cibles thérapeutiques pour le traitement des CNM. / Centronuclear myopathies (CNM) are a group of severe muscle disorder characterized by general muscle weakness. The most severe form is the X-linked CNM (XLCNM), caused by mutations in Myotubularin (MTM1). Others autosomal forms are caused by mutations in Amphiphysin 2 (BIN1) and Dynamin 2 (DNM2). The CNM pathomechanisms are still unclear and to date there are no therapies available to the disease. To investigate the pathways dysregulated in CNM and to identify new therapeutic strategies, we modulated MTM1, BIN1 and DNM2 protein levels in the CNM mouse models. We discovered that DNM2 downregulation rescued the XLCNM mouse model and that the overexpression of human BIN1 rescued the XLCNM and the autosomal dominant CNM form due to DNM2 mutations. We have also showed that MTM1 controls cell adhesion and integrin recycling in mammalian skeletal muscle and BIN1 overexpression rescued the integrin recycling alteration in XLCNM mouse model suggesting that MTM1 and BIN1 are functionally linked and necessary for focal adhesions in muscle. Therefore, our studies highlight that MTM1, BIN1 and DNM2 are in a common pathway and, BIN1 and DNM2 could be new therapeutic targets to treat the different CNM forms.

Indiana University-Purdue University Indianapolis (IUPUI). / The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.