Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-119).

No description available.

Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied strain to regulate cell morphology, signal transduction, and cell function. The purpose of this dissertation is to elucidate the effects of mechanical stretch on cell mechanobiology via the following three aims. First, a sarcomeric model of SFs was developed to describe the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to various modes of stretch. Using model parameters extracted from literature, this model described the dependence of cyclic stretch-induced SF alignment on a two-dimensional (2-D) surface on positive perturbations in SF tension caused by the rate of lengthening, which was consistent with experimental findings. Second, the sarcomeric model was used to predict how stretch-induced pro-inflammatory mechanotransduction depends on the mode of strain application. Together with experimental data, the results indicated that stretch-induced stress fiber alignment, MAPK activations and downstream pro-inflammatory gene expressions are dependent on SF strain rate (and related changes in SF tension) rather than SF turnover. Third, to produce biocompatible materials that are both mechanically resilient under (physiological) load and also mechanosensitive, a novel hybrid engineered tissue was developed that transmits strain stimuli to cells residing in three-dimensional (3-D) collagen microspheres. However, the macroscopic stress is largely borne by a more resilient acellular polyethylene glycol diacrylate (PEGDA) hydrogel supporting the microspheres. Careful analysis indicated that cell alignment occurs prior to significant collagen fibril alignment.

A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-113).

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 79-89). Also available on the World Wide Web.

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

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

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T11:22:31Z (GMT). No. of bitstreams: 1 Duarte_AdrianadaSilvaSantos_D.pdf: 10849571 bytes, checksum: 5933b39c78d15eee6a1a067f3e9cd55b (MD5) Previous issue date: 2009 / Resumo: A identificação e caracterização estrutural e funcional de genes diferencialmente expressos entre tecidos tumorais e normais constituem etapas fundamentais para permitir a compreensão do processo neoplásico e o desenvolvimento de novas estratégias antitumorais. Ankyrin Repeat Single KH Domain containing 1 (ANKHD1) foi inicialmente identificada em células de adenocarcinoma de próstata humano (LNCaP), no ano de 2003. Entretanto, seu padrão de expressão e sua função ainda não haviam sido caracterizados. A ANKHD1 é uma proteína ortóloga à Multiple Ankyrin repeat and single KH domain (Mask) da Drosophila melanogaster. Mask foi identificada através de um rastreamento genético utilizado para detectar novas proteínas associadas à proteína tirosina fosfatase Corkscrew (CSW), homóloga à Src Homology-2 domain-containing protein tyrosine Phosphatase-2 (SHP2) humana. SHP2 é uma fosfatase de tirosina citoplasmática codificada pelo gene PTPN11 e exerce papel fundamental no desenvolvimento da hematopoese normal e leucêmica. Os objetivos gerais do presente estudo foram caracterizar o padrão de expressão gênica de ANKHD1 em células hematopoéticas normais e leucêmicas e verificar sua função nos processos celulares. Neste estudo foi demonstrado que o gene ANKHD1localiza-se no cromossomo 5, possui vários transcritos variantes possivelmente gerados por mecanismos de clivagem alternativa e codifica proteínas com domínios de repetições de anquirina. A região promotora desse gene possui vários elementos regulatórios importantes como sítios de ligação ao fator de transcrição GATA-1 e sequências ricas em dinucleotídeos CG, as ilhas CpG. A expressão do gene ANKHD1 e de algumas de suas variantes em tecidos normais e em linhagens de células neoplásicas foi detectada em intensidades variáveis. Em modelos de diferenciação e proliferação celular foi demonstrado o aumento da expressão desse gene ao longo desses processos. No entanto durante o processo de apoptose observou-se diminuição na expressão de ANKHD1 e transcritos variantes. Em células de pacientes diagnosticados com Síndrome Mielodisplásica (SMD) foi constatada baixa expressão de ANKHD1. Durante a diferenciação eritróide de células CD34+ obtidas de medula óssea desses pacientes não foi observado o aumento da expressão do gene ANKHD1 e do fator de transcrição GATA-1 como era esperado. As células mononucleares de pacientes com SMD tratadas com decitabina, um agente desmetilante, apresentaram aumento na expressão do gene ANKHD1 em comparação às células não tratadas. O mesmo foi observado em células CD34+ tratadas durante a diferenciação eritróide. No entanto em células de pacientes com Leucemia Mielóide Aguda (LMA) e com Mieloma Múltiplo (MM), caracterizadas pela proliferação e resistência aos mecanismos de apoptose, foi demonstrada a alta expressão do gene ANKHD1 e de seus transcritos variantes. A associação da ANKHD1 com SHP2 foi identificada através de Western Blotting, em células da linhagem de MM denominada RPMI 8226. Foi observada a diminuição da expressão do gene ANKHD1 nessas células quando induzidas ao processo de apoptose por dexametasona. Em conclusão o presente estudo identificou ANKHD1 e alguns de seus transcritos variantes como um novo gene com perfis de expressão variados em células hematopoéticas normais e neoplásicas, demonstrou seu envolvimento em processos celulares básicos à manutenção da homeostase e a sua associação com SHP2 em Mieloma Múltiplo. ANKHD1 pode estar envolvida com o fenótipo anormal da célula neoplásica através de uma possível função na via da apoptose. Os achados aqui descritos sugerem que ANKHD1 pode ser uma molécula alvo para a terapia de neoplasias, e permitirão direcionar novos estudos com o objetivo de melhor elucidar as funções específicas de ANKHD1 em diferentes células hematopoéticas normais e neoplásicas. / Abstract: The identification and the structural and functional characterization of genes differentially expressed between tumors and normal tissues are fundamental steps towards the understanding of the neoplastic process and the development of new anti-cancer strategies. The Ankyrin Repeat Single KH Domain containing 1 (ANKHD1) was first described in humans in a prostate carcinoma cell line LNCaP, in 2003; however, the expression pattern and function of ANKHD1 have not yet been described. ANKHD1 is an orthologous protein of the Drosophila melanogaster, MASK (Multiple Ankyrin repeat and single KH domain), where it was first identified using a genetic screen designed to discover proteins that interact with the protein tyrosine phosphatase Corkscrew (CSW), which is a homolog to the SH2-containing protein tyrosine phosphatase (SHP2) in humans. SHP2 is a cytoplasmic protein-tyrosine phosphatase, coded by the PTPN11 gene and plays an important role in the development of normal hematopoiese and leukemogenesis. The aim of the present study was to characterize the gene expression pattern of ANKHD1 in normal and leukemic hematopoietic cells and to determine their function in cellular process.This study has demonstrated that the ANKHD1 gene is located on chromosome 5, this gene has several possible variant transcripts generated by splicing alternative mechanisms and encodes proteins with domains of ankyrin repeats. The promoter region of this gene has several regulatory elements such as the transcription factor GATA-1 binding sites and rich sequences in dinucleotide CG, CpG islands.The expression of the ANKHD1 gene and some of the gene's variants in normal tissues and neoplastic cell lines was detected in different intensities. The increase in the expression of this gene was demonstrated using cellular differentiation and proliferation models. However, during the process of apoptosis, a decrease in the expression of ANKHD1 transcripts variants was observed. In the cells of patients with myelodysplastic syndrome (MDS), a low expression of ANKHD1 was observed. During erythroid differentiation of CD34+ cells obtained from the bone marrow of these patients, no increase in the expression of ANKHD1 gene and transcription factor GATA-1 was observed, as expected. The mononuclear cells of MDS patients were treated with decitabine, a demethylation agent, and showed an increase in the ANKHD1 gene expression compared to untreated cells. The same was observed in CD34+ cells treated during erythroid differentiation. However in cells of acute myeloid leukemia (AML) or Multiple Myeloma (MM) patients, characterized by proliferation and resistance mechanisms of apoptosis, the high expression of gene transcripts ANKHD1 and its variants was demonstrated. The association of SHP2 with ANKHD1 was identified by Western Blot in RPMI 8226 MM cell line. A decrease in the ANKHD1 gene expression in these cells when the process of apoptosis was induced by dexamethasone was observed. In conclusion, this study identified ANKHD1 and some of gene's transcripts variants as a new gene with a variable expression profile in normal and neoplastic hematopoietic cells. The study has also demonstrated the involvement of the ANKHD1 in basic cellular processes, which maintain homeostasis and the association of ANKHD1 with SHP2 in multiple myeloma. ANKHD1 may be involved with the abnormal phenotype of tumor cells through a possible role in the apoptosis pathway. The findings herein described suggest that ANKHD1 could be a molecular target for neoplasic disease therapy and could guide further studies towards a better elucidation of the specific functions of ANKHD1 in normal and neoplasic hematopoietic cells. / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

The Salvador/Warts/Hippo (Hippo) pathway defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Hippo pathway was initially utilised in D. melanogaster, where the Expanded protein acts in the Hippo signalling cascade to control organ size. Willin is the proposed human orthologue of Expanded and the aim of this thesis is to investigate whether willin can activate the mammalian Hippo signalling pathway. Ectopic willin expression causes an increase in phosphorylation of the core Hippo signalling pathway components MST1/2, LATS1 and YAP, an effect which can be antagonised by ezrin. In MCF10A cells, willin over-expression antagonises a YAP-induced epithelial-to-mesenchymal transition via the N- terminal FERM (Four-point-one Ezrin Radixin Moesin) domain of willin. Preliminary results show that willin is expressed within the sciatic nerve of rat and mice, and within the neuromast cells in the zebrafish; suggesting that willin and the Hippo pathway may play a vital role in the developmental regulation within the peripheral nervous system. To conclude, willin influences Hippo signalling activity by activating the core Hippo pathway kinase cassette in mammalian cells.