Although it was previously reported that Dictyostelium discoideum possessed a single ras gene (Ddras) that was maximally expressed during the pseudoplasmodial stage of development, a second ras gene (DdrasG), has been isolated and characterized. It encodes a protein that is similar to the protein encoded by Ddras and the human ras proteins. However, in contrast to Ddras, the DdrasG gene was only expressed during growth and early development. The two ras proteins may fulfill different functions: the DdrasG protein having a role during cell growth and the Ddras protein having a role in signal transduction during multicellular development. However, the expression of the DdrasG gene throughout development did not appear to have a detrimental effect on differentiation. Although other eukaryotic organisms possess more than one ras gene, D. discoideum is thus far unique in expressing different ras genes at different stages of development. Ras genes are members of a large ras-related multigene family that has been found in a wide variety of organisms. A ras-related gene was isolated from D. discoideum that hybridized to both the Ddras and DdrasG genes under low, but not under high stringency conditions. The predicted amino acid sequence shows a high degree of sequence identity with the human rap proteins and thus has been designated Ddrapl. During vegetative and early development a single 1.1 kb mRNA was present, but by aggregation this transcript was no longer detected and two new transcripts of 1.0 and 1.3 kb were observed and were present throughout the remainder of development. The maximum levels of the Ddrapl specific mRNAs appeared during aggregation and culmination, developmental stages where the levels of DdrasG and Ddras messages were declining. The reciprocal nature of the Ddrapl gene expression with respect to that of the two ras genes suggests the possibility that the ras and rap gene products in D. discoideum have antagonistic roles. Antibodies that are specific for the Ddras, DdrasG and Ddrapl proteins have been generated and can be used to help elucidate the biological functions of the individual proteins. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Diss. (sammanfattning) Göteborg : Univ. , 2009. / Härtill 2 uppsatser.

Lau Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-157). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.2 --- Etiology of HCC --- p.2 / Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3 / Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5 / Chapter 1.1.5 --- Confirmation of candidate genes --- p.6 / Chapter 1.2 --- Ras-Oncogene --- p.8 / Chapter 1.2.1 --- Ras superfamily --- p.8 / Chapter 1.2.1.1 --- Rho family --- p.9 / Chapter 1.2.1.2 --- Rab family --- p.10 / Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11 / Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14 / Chapter 1.3 --- RhoC --- p.16 / Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16 / Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19 / Chapter 1.4 --- Rabl4 --- p.20 / Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20 / Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Cell lines --- p.25 / Chapter 2.1.2 --- Cell culture reagents --- p.26 / Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29 / Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31 / Chapter 2.1.6 --- Vectors for cloning --- p.39 / Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39 / Chapter 2.1.8 --- Restriction digestion reagents --- p.42 / Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42 / Chapter 2.1.10 --- Ligation reagents --- p.44 / Chapter 2.1.11 --- Bacterial culture medium --- p.44 / Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46 / Chapter 2.1.13 --- Reagents for flow cytometry --- p.48 / Chapter 2.1.14 --- Detection of apoptosis --- p.48 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50 / Chapter 2.2.1.1 --- cDNA preparation --- p.50 / Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50 / Chapter (2) --- Total RNA isolation --- p.50 / Chapter (3) --- First-strand cDNA synthesis --- p.51 / Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52 / Chapter 2.2.1.3 --- Western blotting --- p.53 / Chapter (1) --- Cell culture --- p.53 / Chapter (2) --- Protein extraction --- p.53 / Chapter (3) --- Quantification of proteins --- p.53 / Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54 / Chapter (5) --- Western blotting luminol detection --- p.56 / Chapter 2.2.2 --- Cloning protocol --- p.57 / Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57 / Chapter 2.2.2.2 --- Purification of PCR product --- p.58 / Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53 / Chapter 2.2.2.4 --- Insert/vector ligation --- p.59 / Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60 / Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61 / Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61 / Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62 / Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63 / Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64 / Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66 / Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66 / Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66 / Chapter 2.2.3.3 --- DAPI staining --- p.67 / Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68 / Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68 / Chapter 2.2.4 --- Analysis of cell cycle --- p.69 / Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69 / Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69 / Chapter 2.2.5 --- Detection of apoptosis --- p.70 / Chapter 2.2.5.1 --- Transfection --- p.70 / Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70 / Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71 / Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71 / Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71 / Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72 / Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72 / Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72 / Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73 / Chapter 2.2.7.3 --- Cell invasion assay --- p.73 / Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75 / Chapter 2.2.8.1 --- RT-PCR --- p.75 / Chapter 2.2.8.2 --- Western blotting --- p.75 / Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76 / Chapter 2.2.9.1 --- Cloning and transfection --- p.76 / Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76 / Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77 / Chapter 2.2.10 --- Statistics --- p.77 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78 / Chapter 3.1.1 --- RT-PCR --- p.78 / Chapter 3.1.2 --- Western blotting --- p.81 / Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85 / Chapter 3.3 --- Characterization of RhoC --- p.93 / Chapter 3.3.1 --- Cell cycle analysis --- p.93 / Chapter 3.3.2 --- Apoptosis --- p.95 / Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97 / Chapter 3.3.4 --- Cell invasion ability --- p.99 / Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102 / Chapter 3.4 --- Characterization of Rabl4 --- p.107 / Chapter 3.4.1 --- Cell cycle analysis --- p.107 / Chapter 3.4.2 --- Apoptosis --- p.109 / Chapter 3.4.3 --- Roles in intracellular transportation --- p.111 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117 / Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119 / Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122 / Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122 / Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123 / Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124 / Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125 / Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126 / Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132 / Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132 / Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133 / Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134 / Chapter 4.5 --- Conclusion --- p.138 / Chapter 4.6 --- Future prospects --- p.140 / Appendix --- p.143 / References --- p.147

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

Telomerase is a ribonucleoprotein that maintains telomere. It is activated in greater than 85% of human neoplasms. Traditionally, reactivation of telomerase during tumorigenesis was thought to be required solely to impart an indefinite lifespan. Recently, however, several studies have suggested that telomerase may contribute to tumorigenesis via an additional mechanism that is independent of its role in telomere lengthening. We sought to identify the region(s) of hTERT that contribute to this non-classical role of telomerase. We proposed to identify such regions by their ability to impart a tumorigenic phenotype in ALT cells transduced with activated Ras. Also, we attempted to develop methods to demonstrate that this role is not dependant of telomerase localizing to the telomere. The strategies employed and the progress gained toward each goal is presented in this thesis.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 100-111).

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.