Accurate chromosome segregation requires a spindle apparatus composed of microtubules that arise from the spindle to attach to the kinetochore, a protein complex assembled at the centromere of each chromosome. Failure to segregate chromosomes accurately may lead to lethal early developmental defects and tumorigenesis. To achieve proper kinetochore binding to microtubules, mammalian cells have evolved elaborate mechanisms to correct attachment errors and stabilize correct ones. Current models suggest that tension between kinetochore pairs (inter-kinetochore stretch) and tension at the kinetochore (intra-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated and microtubule-binding substrates, subsequently reducing their phosphorylations and increasing their microtubule affinity. However, the tension-based models do not explain how the initial microtubule binding at unattached kinetochores occurs, where there is no tension and kinetochore-associated substrates are highly phosphorylated and, hence unable to bind to microtubules. Therefore, there must be a mechanism that explains how the phosphorylation of kinetochore substrates by Aurora B is reduced in the absence of tension. In the first part of this thesis, I examine the structural features of the coiled-coil domain of the kinetochore-associated kinesin motor protein, CENP-E. Using Single-Molecule High-Resolution Colocalization (SHREC) microscopy analysis of kinetochore-associated CENP-E, I show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore. Chemical inhibition of the motor motility or genetic perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Importantly, metaphase chromosome misalignment caused by inhibition of CENP-E can be rescued by chemical inhibition of Aurora B kinase. Therefore, CENP-E regulates the initial kinetochore binding to microtubules and the stabilization of kinetochore-microtubule attachments. Formin-dependent actin assembly is known to play a role in multiple processes, including cytokinesis, filopodia formation, cell polarity, and cell adhesion. Thus, formin malfunction is directly linked to various pathologies, including defects in cell migration and tumor suppression. Although the role of formins in actin polymerization has been well described, the mechanistic processes that regulate the actin assembly function of formins remain poorly understood, especially the interplay among the various sub-families of formins and how they are spatiotemporally regulated. In the second part of this thesis, I show that Aurora B-mediated phosphorylation of the formin, mDia3 regulates actin assembly. Previous studies identified two Aurora B phosphorylation sites in the FH2 domain of mDia3. To this end, phosphomimetic and non-phosphorylatable mutants of a constitutively active form of mDia3 were designed to test whether phosphorylation by Aurora B regulates actin assembly. Using an in vitro actin polymerization kinetic assay and expression of fluorescently-tagged constitutively active mDia3 in cells, I show that phosphorylation of mDia3 by Aurora B induces the actin assembly function of mDia3. Furthermore, using a phospho-specific antibody, I show that mDia3 is phosphorylated by Aurora B. Live-cell analysis shows that perturbations of these phosphorylation sites affect cell migration and cell spreading. Therefore, I illustrate a novel regulatory mechanism for the actin assembly function of mDia3 that is dependent on Aurora B kinase activity.

Cytokinesis is the physical partition of one cell into two. In Chapter 1, I provide a brief introduction to cytokinesis and some of the proteins whose functions I parse out throughout my studies. In Chapter 2, I present work I’ve contributed to elucidate the role of polarity proteins in cytokinesis, as well as a look at the differential requirement for canonically essential cytokinetic proteins in the 4-cell embryo. In Chapter 3, I address a long-standing controversy in the field regarding the relationship between the Rac GAP protein Cyk-4 and the small GTPase Rac, and in particular the inhibitory role of Rac during cell division. My major body of work highlights the necessity not to close the books on the GAP activity of Cyk-4 and its inhibition of Rac. I show that Rac is unable to rescue cytokinesis failure in downstream Rho effectors whose loss weakens the contractile ring, suggesting it is not a promiscuous suppressor of cytokinesis. Additionally, I found that levels of non-muscle myosin-II and the actin binding domain of Utrophin were unchanged with loss of Cyk-4. From this, I infer that Cyk-4 is unlikely to be an activator of the RhoGEF Ect-2. These results emphasize the need to probe further into the cross-talk between these GTPases. In chapter 4, I show inconclusive data addressing the role of cell fate signaling in protection against cytokinesis failure. Overall, this thesis represents my contributions to the field, revealing the complexity involved in assuring successful completion of cytokinesis.

Self-renewal and pluripotency, the hallmarks of human embryonic stem cells (hESC), confer these cells with the capacity to expand indefinitely while maintaining the ability to differentiate into any cell type of the human body; thus, making hESC a valuable source of functional differentiated cells suitable for applications in regenerative medicine, drug discovery, biotechnology, biopharmaceuticals and developmental biology. However, the large-scale production of clinical-grade hESC, required for such applications, has been hampered by the current culture conditions in which hESC still depend on the use of mouse embryonic fibroblast-conditioned medium (MEF-CM) for their efficient growth. Therefore, investigation of the factors provided by MEFs is of the utmost importance to discover which components of MEF-CM allow the long-term expansion of undifferentiated hESC. While considerable progress has been made on the identification of the protein components of MEF-CM, very little is known about the small molecules (metabolites) secreted by MEFs. In this context, an untargeted metabolomics method was developed for the investigation of potential bioactive metabolites present in MEF-CM implicated in the proliferation and/or maintenance of pluripotency of hESC in vitro. A metabolomics method was applied and successfully identified a number of metabolites which were later confirmed in their identities with the use of authentic standards, to be further investigated for their effect on hESC culture. Interestingly, the addition of PGE2, 6-keto-PGF1α, 9, 12, 13-TriHOME, 7-Ketocholesterol and stearidonic acid (the metabolites found in MEF-CM) to the unconditioned medium (UM), a medium incapable of the maintenance of hESC, showed a delay in apoptosis when compared to the negative control UM; thus, suggesting that these metabolites could help with the proliferation of hESC. Increasing evidence that hESC secrete factors into their microenvironment that can also help them to proliferate or to maintain an undifferentiated state prompted the application of the same metabolomics method to the analysis of hESC spent culture media. The results identified lysophospholipids (LPLs) as potential molecules mediating some biological activities; however, the precise role of these LPLs still remains to be determined. Overall, the results of this thesis are expected to impact and add knowledge to the field of stem cell biology providing useful information for the creation and development of more efficient and defined culture conditions for the propagation of hESC with the appropriate quality to realise their widespread application in clinic and other research areas.

No description available.

A novel metal-inducible and amplifiable metallothionein (MT) expression system, pNK, was firstly optimised and characterised for the production of a reporter protein, human growth hormone (hGH) in a suspension CHO cell line grown in a serum-free media. The pNK-based hGH production was demonstrated in cadmium-free condition under various fermentation modes (batch, fed-batch and perfusion) and scales (flask to bench-top bioreactor). Improvement of specific productivity of recombinant protein from pNK was shown to be possible by addition of butyrate or substrate substitution of glutamine by glutamate. Combination of fed-batch and butyrate addition strategies resulted in more than one gram per litre of hGH being obtained from the pNK expression system in a bioreactor. In the second part of the project, based on a statistical approach suggested by Plackett-Burman (P-B), a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support a Super-CHO cell line, C2.8-325, to grow as a single-cell suspension culture with comparable growth rate and viable cell number as observed in a commercial medium containing undefined additives. Using Dulbecco's Modified Eagle's Medium/Ham's F12 1:1 mixture (DMEM/F12) as the basal medium, a P-B design matrix screened 10 nutritional components. Components shown potentially beneficial for cell growth rate and viable cell number were supplemented to DMEM/F12 to formulate the SPF medium. Finally, the pNK expression system and the Super-CHO cell line were applied simultaneously in an attempt to express a humanised anti-CD48 monoclonal antibody (MAb), IgG1-N2A (N2A-MAb). This aimed to test C2.8-SPF grown in newly developed SPF medium for transfection, clone development and recombinant protein production. A stable and N2A-MAb expressing C2.8-SPF cell line was successfully constructed, and N2A MAb expression was subsequently amplified and demonstrated in various cultivation scales (flask and bioreactor). This project demonstrated that the novel metal-inducible and - amplifiable mammalian expression system, pNK, and the novel mammalian host cell-line, Super-CHO C2.8-SPF, capable of growing as a single-cell suspension culture in a chemically-defined protein-free medium, SPF, could be utilised in combination to provide a new, low-cost, and regulatory-compliant recombinant protein expression platform, suitable for the biopharmaceutical industry to use in the manufacture of therapeutic recombinant proteins.

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 112-116). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99326

Thesis (M.S.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Karen Katula; submitted to the Dept. of Biology. Includes bibliographical references (p. 59-61).

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38742

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisors: Josef A. Käs and Tess J. Moon. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Jeffrey Morgan. Includes bibliographical references.