<p>Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation.</p><p>For this thesis, a comprehensive study of the cell cycle in the model archaeon <i>Sulfolobus acidocaldarius</i> was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries.</p><p><i>Sulfolobus</i> species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes.</p><p>A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon <i>Methanothermobacter thermautotrophicus</i> was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.</p>

Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation. For this thesis, a comprehensive study of the cell cycle in the model archaeon Sulfolobus acidocaldarius was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries. Sulfolobus species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes. A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon Methanothermobacter thermautotrophicus was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.

The miraculous birth of a new life starts by the formation of an embryo. The process by which an embryo is formed, embryogenesis, has been studied and shown to consist of three types of processes: mitosis, cell differentiation and morphogenetic movements. Scientists and medical doctors are still at a loss to explain the fundamental forces driving embryo development and the causes of birth defects remain largely unknown. Recent efforts by the Embryo Biomechanics Lab at the University of Waterloo have shown a relationship between morphogenetic movements that occur during embryo formation and the frequency and orientation of mitosis. To further study this relationship a means of automatically identifying the frequency and orientation of mitosis on time-lapse images of embryo epithelia is needed. Past efforts at identifying mitosis have been limited to the study of cell cultures and stained tissue segments. Two methods for identifying mitosis in contiguous sheets of cells are developed. The first method is based on local motion analysis and the second method is based on intensity analysis. These algorithms were tested on images of early and late stage embryos of the axolotl (Ambystoma mexicanum), a type of amphibian. The performance of the algorithms were measured using the F-Measure. The F-Measure determines the performance of the algorithm as the true mitosis detection rate penalized by the false mitosis detection rate. The motion based algorithm had performance rates of 68.2% on an early stage image set and 66.7% on a late stage image set, whereas the intensity based algorithm had a performance rates of 73.9% on early stage image set and 90.0% on late stage image set. The mitosis orientation errors for the motion based algorithm were 27.3 degrees average error with a standard deviation (std.) of 19.8 degrees for early stage set and 34.8 degrees average error with a std. of 23.5 degrees for the late stage set. For the intensity based algorithm the orientation errors were 39.8 degrees average with std. of 28.9 degrees for the early stage image set and 15.7 degrees average with std. of 18.9 degrees for the late stage image set. The intensity based algorithm had the best performance of the two algorithms presented, and the intensity based algorithm performs best on high-magnification images. Its performance is limited by mitoses in adjacent cells and by the presence of natural cell pigment variations. The algorithms presented here offer a powerful new set of tools for evaluating the role of mitoses in embryo morphogenesis.

The miraculous birth of a new life starts by the formation of an embryo. The process by which an embryo is formed, embryogenesis, has been studied and shown to consist of three types of processes: mitosis, cell differentiation and morphogenetic movements. Scientists and medical doctors are still at a loss to explain the fundamental forces driving embryo development and the causes of birth defects remain largely unknown. Recent efforts by the Embryo Biomechanics Lab at the University of Waterloo have shown a relationship between morphogenetic movements that occur during embryo formation and the frequency and orientation of mitosis. To further study this relationship a means of automatically identifying the frequency and orientation of mitosis on time-lapse images of embryo epithelia is needed. Past efforts at identifying mitosis have been limited to the study of cell cultures and stained tissue segments. Two methods for identifying mitosis in contiguous sheets of cells are developed. The first method is based on local motion analysis and the second method is based on intensity analysis. These algorithms were tested on images of early and late stage embryos of the axolotl (Ambystoma mexicanum), a type of amphibian. The performance of the algorithms were measured using the F-Measure. The F-Measure determines the performance of the algorithm as the true mitosis detection rate penalized by the false mitosis detection rate. The motion based algorithm had performance rates of 68.2% on an early stage image set and 66.7% on a late stage image set, whereas the intensity based algorithm had a performance rates of 73.9% on early stage image set and 90.0% on late stage image set. The mitosis orientation errors for the motion based algorithm were 27.3 degrees average error with a standard deviation (std.) of 19.8 degrees for early stage set and 34.8 degrees average error with a std. of 23.5 degrees for the late stage set. For the intensity based algorithm the orientation errors were 39.8 degrees average with std. of 28.9 degrees for the early stage image set and 15.7 degrees average with std. of 18.9 degrees for the late stage image set. The intensity based algorithm had the best performance of the two algorithms presented, and the intensity based algorithm performs best on high-magnification images. Its performance is limited by mitoses in adjacent cells and by the presence of natural cell pigment variations. The algorithms presented here offer a powerful new set of tools for evaluating the role of mitoses in embryo morphogenesis.

Hepatocarcinoma cancer (HCC) is one the most common visceral malignancies in Taiwan, which has a very high incidence and a devastatingly poor prognosis. BMP4, belonging to the TGF-£] super-family of proteins is a multifunctional cytokine, known to exert its biological effects through SMAD and non-SMAD dependent pathways and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. In this study, we first demonstrate that BMP4 and its receptor, BMPR1A, are over-expressed in a majority of primary HCC and promote the growth and migration of HCC cell lines in vitro. We also further identify that BMP4 can induce HCC CDK1 and cyclinB1 up-regulation to accelerate cell cycle progression. Our study indicates that the induction of HCC cell proliferation is independent on the SMAD signaling pathway, since Smad4 knockdown of BMP4 induced HCC cell lines still leads to the up-regulation of CDK1 and cyclinB1 expression in HCC. Using MEK kinase selective inhibitors, the induction of CDK1 and cyclinB1 mRNA and protein were shown to be dependent on the activation of MEK/ERK signaling. In vivo xenograft studies confirmed that the BMPR1A- knockdown cells were significantly less tumorigenic than control groups. Taken together, our findings show that the up-regulation of BMP4 and BMPR1A in HCC promote the proliferation and metastasis of HCC cells and that CDK1 and cyclinB1 are important, SMAD-independent molecular targets in BMP4 signaling pathways during the HCC tumorigenesis. We propose here that BMP4 signaling pathways may have potential as new therapeutic targets, in HCC treatment.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 136-153).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 242-266). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 89-99).

Alzheimer's disease is characterized by the presence of amyloid plaques, made up primarily of Aϐ peptides, and neurofibrillary tangles, containing hyperphosphorylated tau. Aϐ is generated by sequential proteolysis of the amyloid precursor protein (APP) by beta and gamma secretases. The leading hypothesis of Alzheimer's disease pathogenesis is the amyloid cascade hypothesis, which suggests that amyloid is central to the disease process. However, tau pathology correlates more closely with cognitive dysfunction and follows a predictable anatomical course through the brain. We hypothesize that if Aϐ is upstream of tau pathology and tau pathology follows this predictable course through the brain, Aϐ production may also propagate through the brain in an anatomical fashion. In order to investigate this possibility, we examined two broad cellular processes induced in cells when exposed to Aϐ, p53-dependent apoptosis and cell cycle activation. We report that p53-dependent apoptosis is associated with a decrease in the Aϐ and sAPP-alpha and an increase in an alternative, caspase-cleaved fragment of APP, resulting from an apparent cleavage in the near extracellular domain of APP. Mitosis is associated with the phosphorylation of both tau and APP, and increased production of Aϐ. Our results indicate that while p53-dependent apoptosis is not associated with increased amyloidogenesis, cell cycle activation increases Aϐ production and may play a role in disease propagation. Together, these findings suggest various treatment approaches, including cell cycle inhibition and disruption of APP endocytosis, which may decrease amyloidogenic processing. Continued research into these potential approaches, coupled with earlier detection of the disease process, could lead to promising treatments for Alzheimer's disease.

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells. / text