The centrosome is the main microtubule-organising centre in animal cells; important to assemble a bipolar mitotic spindle ensuring proper chromosome segregation and genomic stability. Whereas correct centrosome number (1-2) is tightly maintained in normal cells, cancer cells usually have an increased number of centrosomes (>2), termed centrosome ampli cation. Centrosome ampli cation has been correlated with aneuploidy, increased tumour grade, chemoresistance and overall poor prognosis. Cancer cells primarily adapt to supernumerary centrosomes by clustering them into two poles resulting in a `normal' pseudo-bipolar mitosis. Undermining centrosome clustering is a potential target for cancer-speci c treatment. Indeed, depleting the kinesin HSET has already been shown to speci cally kill cancer cells by impairing the centrosome clustering mechanism. However, it is unclear whether this process requires adaptation or it is inherent to all cell types. Using a panel of non-transformed cell lines, we observed that cells expressing Ecadherin have ine cient clustering mechanisms compared to cell lines without E-cadherin. Loss of E-cadherin (siRNA/CRISPR) promotes centrosome clustering and survival of epithelial cells with multiple centrosomes. In addition, loss of DDR1, involved in regulating cortical contractility downstream of E-cadherin, increases centrosome clustering in epithelial cells. Using Atomic Force Microscopy we con rmed that indeed loss of E-cadherin leads to increased cortical contractility in mitotic cells. Inhibition of actomyosin contractility prevents e cient clustering in cells that do not express E-cadherin, further suggesting that it is important for this process. Loss of E-cadherin and DDR1 is strongly correlated with high levels of centrosome ampli cation in breast cancer cell lines suggesting that these changes are an important adaptation mechanism to centrosome amplification.

Corneal degenerative diseases and opacity are leading causes of corneal impairment and blindness worldwide. Like many epithelial tissues, the constant renewal of transparent corneal epithelial cells is essential for a lifelong healthy cornea and optimal vision. The limbus (the boundary between the cornea and the conjunctiva) is believed to be the site that harbours adult stem cells responsible for corneal maintenance and repair after injury, referred to as limbal epithelial stem cells (LESCs). In the basal limbal epithelium, an active LESC subset divides to yield progenitor cells that migrate centripetally into the corneal epithelium for cell renewal. This asymmetric division however, is assumed to be regulated by a balance between cell renewal and loss of cells from the corneal surface. The search for specific LESC molecular markers has been difficult and to date there are few if any candidate markers that unambiguously identify LESCs but not their immediate progeny. Consequently, LESC clonality, activity and proliferative dynamics have remained poorly understood. In addition, the nature of the regulatory molecular pathways involved during LESC activity is still an open key question. In this research project, we identified stem cells on the ocular surface of the eye, assayed their activity and demonstrated quantitively for the first time how the cornea responds to damage. The retention of DNA labelling reagents in control and wounded corneas was combined with clonal analyses of cell division and migration using mice mosaic for reporter LacZ expression. Corneal transplant in LacZ reporter transgenic mice showed migration of LacZ-positive cells into the donor corneal button, with long-term disruption of patterns of migration. Corneal epithelial scrape wounds at the periphery also showed long– term disruption. Label retention suggested a small but statistically significant proliferation response of LESCs to injury, but this was attenuated or absent in aging mice and Pax6 mutants. The Hippo signalling pathway has been shown to have promising results in regulating stem cell activity and proliferation in many organs, however, its effect on LESC proliferation is unknown. Here, we investigated the regulatory role of the Hippo−YAP signalling pathway during cell proliferation, and determined whether the label retention assay in a uniform population of dividing cells is a real indicator of slow-cycling cells in vivo. Cell-cycling kinetics, Abstract v proliferation rate, and label retention were determined in a spontaneous human corneal epithelial (HCE-S) cell line, using double-labelling IdU and EdU thymidine analogues. During homeostasis, HCE-S cells underwent approximately one cell cycle per day, however, cells in which YAP-dependent signalling was activated by 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (Hsp90), showed slower proliferation rate and longer cell-cycle time. In vitro label-retention assay in confluent cultures estimated number of ~3-4 cell cycles needed to dilute out the label from slow-cycling cells in vivo. The data are suggestive that the Hippo pathway has a potential regulatory role in proliferative corneal epithelium, and that label-retention assay is a real indicator of slow-cycling cells. Furthermore, this research observed the proliferative dynamics of conjunctival stem cells. Clonal analysis of patterns of cell growth in the conjunctiva were analysed following tamoxifen-induction of LacZ transgene activity. The long−term presence of coherent patches of LacZ-positive cells suggested the presence of long-lived conjunctival stem cells but that the turnover time for the bulbar conjunctival epithelium may be more than 16 weeks. The key results of this research are that the limbus is the niche for stem cells, that there is a unidirectional migration of cells from the limbus to the corneal epithelium during homeostasis, but this is disrupted, perhaps permanently, by wounding. We find only a mild response of limbal epithelial stem cells to acute corneal injury, which is reduced to no response with age and is dependent on genetic background.

F. tularensis is an intracellular pathogen, and is the causative agent of tularemia in humans. The ability of F. tularensis to parasitize host cells is largely dependent upon genes within a pathogenicity island (FPI), including those in the iglABCD operon. Specific mechanisms and gene products involved in regulation of the FPI are not well understood. I initiate the study of this regulatory system by creating an efficient Tn5-based mutagenesis system optimized for use in F. tularensis, and utilize this system to construct a lacZ reporter library. I identify genes differentially regulated in response to growth on two different media, including those in the iglABCD and fslABCD operons, and identify iron availability as a factor contributing to the differential regulation. One of these reporter strains, carrying a chromosomal iglB-lacZ fusion, is used as the basis for a secondary transposon mutagenesis to identify mutations that affect iglABCD expression. One such mutation is in FTL_1542 (migR), a hypothetical protein, and reduces expression of the iglABCD approximately 8-fold. The effect of this mutation on igl expression is likely through its effect on another known virulence regulator, fevR, as demonstrated by data from RT-PCR experiments. I compare the phenotypes of LVS fevR and migR mutant strains in primary macrophage and epithelial cell lines and in neutrophils. The mutation in migR effects growth and intracellular trafficking in macrophages but not epithelial cells, and reverses the ability of wild type F. tularensis to block the respiratory burst in neutrophils. When similar mutations were examined in the human virulent F. tularensis strain Schu S4, migR retained its regulatory role, but did not impair replication in macrophages. The migR mutation in Schu S4 did however have an attenuating effect when administered to mice intranasally. Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.

Abstract not available

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-122).

Virtually all signaling pathways that play key roles in development such as the transfroming growth factor (TGF)-beta, notch, and wnt pathways also influence tumor formation, implying that cancer is in a sense development gone awry. Therefore, identification and elucidation of developmental pathways has great potential for generating new diagnostic tools and molecular therapy targets. Singleminded-2s (SIM2s), a splice variant of the basic helilx-loop-helix / PER-ARNT-SIM (bHLH/PAS) transcriptional repressor Singleminded-2, is lost or repressed in approximately 70% of human breast tumors and has a profound influence on normal mammary development. In order to gain a better understanding of the mechanisms by which SIM2s restricts malignant transformation and progression in breast cancer, we depleted SIM2 RNA in MCF-7 cells using a retroviral shRNA system and examined gene expression and functional abilities of the SIM2-depleted MCF-7 cells (SIM2i) relative to a control MCF line expressing a non-specific “scrambled” shRNA (SCR). Depletion of SIM2 resulted in an epithelial-mesenchymal transition (EMT)-like effect characterized by increased migration and invasion, altered morphology, and loss of epithelial markers concomitant with gain of mesenchymal markers. The root of this effect may be loss of SIM2- mediated repression of the E-cadherin repressor slug, as SIM2 is able to bind and repress transcription from the slug promoter, and slug expression is dramatically elevated in SIM2i MCF-7 cells. Consistent with the previously established role of slug in resistance to various cancer therapies, SIM2i cells are resistant to the radiomimetic doxorubicin and appear to have elevated self-renewal capacity under certain conditions. Intriguingly, SIM2 protein levels are elevated by treatment with DNA damaging agents, and SIM2 interacts with the p53 complex via co-regulation of specific p53- target gene such as p21/WAF1/CIP1. These results provide a plausible mechanism for the tumor suppressor activity of SIM2, and provide insight into a novel tumor suppressive transcriptional circuit that may have utility as a therapeutic target.

The primary function of the lens capsule of the eye unfolds during the process of accommodation; wherein, tension imposed onto its equator is released, allowing the elastic capsule to mold the underlying lens nucleus and cortex into a more quasispherical morphology to change focus from distant to near objects. Given its highly mechanical nature, it is prudent to study the native lens capsule from the perspective of biomechanics for such applications as understanding the mechanism of accommodation. Further, cataract surgery introduces alterations to the geometry of the lens capsule that lead to changes in resident cell behavior from quiescent to contractile and synthetic. Though resultant changes in capsule histology are well documented little has been done to quantify the corresponding altered mechanics, which is important for elucidating related post-surgical pathologies and improving prosthetic lens design. In this study we present the first data on the in situ multiaxial mechanical behavior of the native and hyperglycemic anterior lens capsule in both the porcine and human models. From these data, native stresses in the lens capsule are calculated using a finite element analysis, and alterations in the corresponding strain field are calculated after the introduction of a continuous circular capsulorhexis, which is imposed during cataract surgery. Finally, we quantify both the altered mechanical behavior and contractile loads imposed onto the lens capsule after cataract surgery.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "December 2001." Typescript. Vita. Includes bibliographical references (leaves 95-113). Also available on the Internet.

Thesis supervisor: Maureen D. Donovan. Includes bibliographic references (p. 239-247).

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: tissue engineering; trachea; chondrocyte; epithelium; aerosol. Includes bibliographical references (p. 64-65).