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A balancing act between the 'Src-Stat3' and 'p53-caldesmon' pathways dictates the outcome of Src-induced invasive phenotypesMooney, Patrick 11 January 2010 (has links)
Cell migration and invasion are essential physiological processes required for the growth and development of all multicellular organisms. However, they have also been implicated in the pathogenesis of certain vascular system diseases and invasive cancers. In this study, we investigate two proteins involved in cell proliferation and survival signaling, p53 and Stat3, which have been found misregulated in atherosclerosis and cancer, to establish what effect they have on the development of Src-induced invasive phenotypes in aortic vascular smooth muscle cells (VSMC) and NIH 3T3 fibroblasts.
In the first stage of this experiment, we investigated the tumor suppressor p53. Once believed to act primarily as a regulator of the cell cycle, DNA repair, senescence and apoptosis, current evidence suggests that p53 can also regulate cell migration and invasion. For our study, we stably transduced VSMC and NIH 3T3 fibroblasts with constitutively active Src (SrcY527F) to generate invasive cell lines with pronounced podosome and rosette formation. We established for the first time that p53 suppresses Src-induced podosome and rosette formation, extracellular matrix degradation, cell migration and invasion in these cells. We also present novel data showing that p53 suppresses these invasive phenotypes, at least in part, by up-regulating the expression of caldesmon, an actin binding protein which stabilizes stress fibers and inhibits podosome and rosette formation.
In the second part of this study, we show that Stat3, a pro-survival and pro-metastatic transcription factor, is required downstream of Src for the promotion of invasive phenotypes in VSMC and NIH 3T3 fibroblasts. Interestingly we have also shown for the first time that Stat3 can localize to podosomes and rosettes in these cells. The exact physiological reasoning for this localization, however, remains to be determined.
This study provides strong evidence suggesting that mutual antagonism between the anti-invasive ‘p53-caldesmon’ and pro-invasive ‘Src-Stat3’ pathways dictates the outcome of Src-induced invasive phenotypes in VSMC and NIH 3T3 fibroblasts. / Thesis (Master, Biochemistry) -- Queen's University, 2010-01-09 21:57:30.056
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Regulation of mechanics and dynamics of actin filaments and networks by actin-binding proteinsJensen, Mikkel Herholdt 24 September 2015 (has links)
Actin is a highly ubiquitous and evolutionarily conserved protein capable of polymerizing and forming filamentous polymers which play a central role in cell mechanics and motility. Here, we study the in vitro regulation of actin mechanics and dynamics by calponin and caldesmon, two actin binding proteins believed to be involved in regulating cytoskeletal mechanics and structure through mechanisms not currently well understood.
Chapters 1 and 2 introduce the reader to actin and its roles in the cell, as well as to the methods and theoretical foundations used in this work.
In Chapter 3, we use total internal reflection and confocal fluorescence microscopy to investigate the polymerization dynamics of actin in the presence of a caldesmon C terminal fragment, H32K. We show that H32K stabilizes a nascent structural state of actin without altering the polymerization dynamics of the filament. We also show that H32K stabilized nascent actin has increased affinity for the actin branching protein complex Arp2/3 involved in driving membrane protrusions during cell motility, and propose the nascent state of actin as a possible transient differentiator targeting certain actin binding proteins to actin in vivo. This is to our knowledge the first reported direct functional effect of nascent actin.
In Chapter 4, we use fluorescence microscopy to quantify actin bending mechanics in the presence of the binding protein calponin and show that calponin reduces the persistence length of actin. We compare our results to the literature and compare the mechanical change to electron microscopy reconstructions, which suggest that calponin affects actin intermonomer contacts through interactions with actin subdomain 2.
In Chapter 5, we expand on the results from Chapter 4 using bulk rheology and show that calponin increases the tensile strength of reconstituted actin networks, similar to the effect seen in whole cells and tissues. We discuss these data within an affine network model and show that the results can be entirely explained in terms of the reduced actin persistence length. We use this to propose a novel physical mechanism for calponin function in vivo.
This work elucidates the physical mechanisms of calponin and caldesmon function and their role in regulating the cellular cytoskeleton. / 2031-01-01T00:00:00Z
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p53 Regulates the Formation of Lamellipodia and Circular Dorsal Ruffles Through Caldesmon and PTENVANDENBERG, Laura Joanna 14 June 2011 (has links)
Vascular smooth muscle cell migration is a significant contributor to many aspects of heart disease, and specifically atherosclerosis. Tissue damage in the arteries can result in the formation of a fatty streak. Smooth muscle cells (SMC) can then migrate to this site to form a fibrous cap, stabilizing the fatty plaque. Since cardiovascular disease is the leading cause of death in developed countries, this function of SMC is an essential area of study.
The formation of lamellipodia and circular dorsal ruffles were studied in this project as indicators that cell migration is occurring. The roles of the proteins p53, Rac, caldesmon and PTEN were investigated with regards to these actin-based structures.
The tumour suppressor p53 is often reported to cause apoptosis, senescence or cell cycle arrest when stress is placed on a cell, but has recently been shown to regulate cell migration as well. It was determined in this project that p53 could inhibit the formation of both lamellipodia and circular dorsal ruffles. It was also shown that this could occur directly through an inhibition of the GTPase Rac. Previous studies have shown that p53 can upregulate caldesmon, a protein which is known to bind to and stabilize actin filaments while inhibiting Arp2/3-mediated branching. It was confirmed that p53 could upregulate caldesmon, and that caldesmon could inhibit the formation of lamellipodia and circular dorsal ruffles. The phosphorylation of caldesmon by p21-associated kinase (PAK) or extracellular signal-related kinase (Erk) was shown to effectively reverse the ability of caldesmon to inhibit these structures. The role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was also studied with regards to this signalling pathway. PTEN was shown to inhibit lamellipodia and circular dorsal ruffles through its lipid phosphatase activity.
It was concluded that p53 can inhibit the formation of lamellipodia and circular dorsal ruffles in vascular SMC, and that this occurs through Rac, caldesmon and PTEN. / Thesis (Master, Biochemistry) -- Queen's University, 2011-06-10 13:15:37.081
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Protokollutveckling för caldesmon samt en jämförelse med SMMS-1 av dess effektivitet som en myoepitelial markör på fibroadenomBrentel, Sahra January 2017 (has links)
Immunohistokemi (IHK) är en metod som används för att undersöka närvaron av specifika biomarkörer i vävnad. Metoden utnyttjar antikroppars affinitet till antigen och dess närvaro visualiseras. Caldesmon är ett protein associerat med cytoskelettet som förekommer i glatta muskelceller och i icke-muskelceller. Det används inom IHK som en glatt muskelmarkör men även som en markör för myoepiteliet. Syftet med studien var att utveckla ett nytt protokoll för caldesmon och att jämföra den med SMMS-1, även den en markör för glatt muskulatur och myoepiteliet. Jämförelsen utfördes på fibroadenom vävnad, en bröstcancerform som utvecklats ur epitelialvävnad och stroma, då den innehåller mycket myoepitelial celler. För att effektivisera processen användes tissue microarray tekniken, där vävnad från flera olika preparat (klotsar) stansades ut och sammanfördes till en ny klots. Instrumentet Ventana Benchmark Ultra användes för att utveckla ett nytt protokoll för caldesmon. Det nya protokollet användes sedan vid jämförelsen med SMMS-1. Resultatet visade att SMMS-1 gav en tydligare infärgning av det myoepiteliala lagret då det enbart färgade in det, medan caldesmon även färgade in stroma. Då fibroadenom vävnad innehöll mycket stroma var det svårare att urskilja det myoepiteliala lagret från dess omgivning med caldesmon infärgningen. / Immunohistochemistry (IHC), is a method used for examining the presence of specific biomarkers in tissue. It uses antibodies affinity for antigens and their presence can then be visualized. Caldesmon is a protein that is associated with the cytoskeleton in smooth muscle and non-smooth muscle cells. It is used within IHC as a marker for smooth muscle but also as a marker for the myoepithelium. The intent of this study was to develop a new protocol for caldesmon and to compare it with SMMS-1, also a marker for smooth muscle and the myoepithelium. The comparison was performed on tissue from fibroadenoma, a type of breast cancer that evolved from epithelial tissue and stroma, due to its high content of myoepithelial cells. For optimal effectiveness, the tissue microarray technique was used, where tissue from several blocks are extracted and brought together into a single new block. A Ventana Benchmark Ultra was used to develop a new protocol for the new antibody, caldesmon. The new protocol was used in the comparison with SMMS-1 on fibroadenoma tissue. The results were that SMMS-1 had a clearer and sharper staining of the myoepithelial layer, due to the fact that it only stained the layer. While caldesmon also stained the stroma in the tissue. Because fibroadenom tissue contained so much stroma the staining done with caldesmon made it harder to separate the myoepithelial layer from its surroundings.
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Charakterisierung von Leupaxin und seiner Interaktionspartner in Karzinomzellen / Charakterisation of Leupaxin and its interaction partners in carcinoma cellsHardenberg, Sandra Gräfin von 29 September 2010 (has links)
No description available.
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Proteomic Analysis and Long Term Live Cell Imaging of Primary Human Cells in CultureMurray, Erica January 2011 (has links)
Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages.
This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells.
This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.
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Analyses on the mechanisms underlying the leupaxin-mediated progression of prostate cancerDierks, Sascha 17 February 2015 (has links)
No description available.
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Proteomic Analysis and Long Term Live Cell Imaging of Primary Human Cells in CultureMurray, Erica January 2011 (has links)
Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages.
This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells.
This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.
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Placental vascular smooth muscle cell differentiation in pregnancies complicated by obesity and gestational diabetesWhittle, Saxon January 2016 (has links)
The increasing demand on healthcare from pregnancies complicated by gestational diabetes (GDM) and obesity is caused in large part by fetal macrosomia (FM). Alterations to the vasculature of the placenta leading to changes to nutrient flux may be more frequent when GDM and obesity occur concomitantly. However, the impact of obesity as an independent comorbidity is poorly understood. The current study sought to characterise structural and functional changes in placenta from pregnancies complicated by GDM and/or obesity and examine the involvement of miRs in this phenomenon, as the phenotype of vascular smooth muscle (VSM) has been documented to be influenced by microRNA (miR) expression. Patients were stratified according to the presence or absence of GDM and/or obesity, which resulted in four groups. Morphometric analysis of CD31 immuno-stained placentas showed that pregnancies complicated by GDM or obesity both had a higher mean sum ratio of the area of the lumen compared to the endothelium. No relationship was found with FM. The ratio increased with maternal body mass index (BMI) in all pregnancies. Immunohistochemistry with a panel of VSM markers suggested an altered phenotype of VSM in pregnancies complicated by GDM and/or obesity. RT-QPCR and immunoblotting showed a higher expression of smooth muscle myosin (SM-MHC), h-caldesmon (HC) and alpha smooth muscle actin (ASMA) in pregnancies complicated by obesity, consistent with a greater contractile capacity. This was most marked when obesity occurred without GDM.Studies were conducted on two miRs, miR-145, which is associated with VSM in many vascular tissues, and the snoRNA-derived species miR-664a-3p, which microarray studies had shown to be higher in placentas from pregnancies complicated by GDM. Dicer and dyskerin, components of the snoRNA-derived miR biogenesis pathway, were increased and reduced respectively in GDM placenta. However, studies in cultured placental villous explants suggested that neither miR species was regulated by glucose, insulin or IGF-I. Placental mesenchymal cells are the developmental precursors of VSM. In primary culture, these cells expressed both miRs. To determine the function of miR-664a-3p, a nucleofection protocol was developed in a fetal mesenchymal cell line, WI38, and applied to first-trimester placental mesenchymal cells. Preliminary proteomic analysis after nucleofection-mediated knockdown of miR-664a-3p suggested a series of novel candidate target proteins for this uncharacterised miR species. Blood vessel structure and VSM phenotype are both altered in pregnancies complicated by GDM and/or obesity. The significance of apparently higher level of contractile proteins with wider vessel lumens in obesity requires further investigation. Translational regulation by miRs including miR-145 and miR-664a-3p is implicated in these alterations. In future, targeted therapies that alter miR levels in the placenta may be useful in control of fetal overgrowth such as FM.
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