Proper regulation of the Transforming Growth Factor-beta (TGF-b) pathway is important for maintaining homeostasis and development in various tissues across vertebrates and invertebrates. When TGF-b pathway signaling is disrupted it leads to tumor growth, birth defects, and other diseases. The identification and study of the various regulatory methods utilized within TGF-b pathway signaling is important to aid the understanding of disease prognosis and prevention. In the TGF-b pathway in Drosophila, dCORL functions in the dActivin subpathway and acts as a regulator of dSmad2 in the larval brain. dCORL is encoded by a gene on the fourth chromosome, in Drosophila. To learn more about dCORL’s role in the pathway, two fourth chromosomes were created that allow clonal analysis to be conducted. Clonal analysis is needed to determine dCORL’s role in TGF-b regulation in the adult brain. In my first project, both chromosomes were successfully created. Though, the importance of understanding regulatory mechanisms goes past one protein. In my second project, multiple conserved prodomain cysteines were identified in human amino acid alignments of 33 TGF-b family proteins across the three TGF-b subfamilies. Database mining identified conserved prodomain cysteine mutations in 10 proteins and their mutant phenotypes. Common phenotypes for conserved cysteine mutations suggest new heterodimer pairs. The most frequent mutant phenotypes associated with new heterodimers were tumors. Conserved prodomain cysteine mutations were connected to cysteine mutations in known regulatory partner proteins by mutant phenotype, yielding numerous new regulatory interactions. The most frequent mutant phenotypes connecting new regulatory interactions between TGF-b proteins and regulatory partners proteins were tumors. Together, my projects expand knowledge of regulatory mechanisms within the TGF-b pathway in Drosophila and humans, while providing hypotheses for further investigation.

Engineered nanoparticles (NP; 10-9 m) have found use in a variety of consumer goods and medical devices because of the unique changes in material properties that occur when synthesized on the nanoscale. Although many definitions for nanoparticle exist, from the perspective of size, nanoparticle is defined as particles with diameters less than 100 nm in any external dimension. Examples of their use include titanium dioxide added as a pigment in products intended to be ingested by humans, silicon dioxide NPs are used in foods as an anticaking agent, and gold or iron oxide NPs can be used as vectors for drug delivery or contrast agents for specialized medical imaging. Although the intended use of these NPs is often to improve human health, it has come to the attention of investigators that NPs can have unintended or even detrimental effects on the organism. This work describes one such unintended effect of NP exposure from the perspective of exposure via the oral route. First, this Dissertation will explain an event referred to as brush border disruption that occurred after nanoparticles interacted with an in vitro model of the human intestinal epithelium. Second, this Dissertation will identify and characterize several consumer goods that were shown to contain titanium dioxide that are intended to be ingested. Third, this Dissertation shows that sedimentation due to gravity does not artifactually result in disruption of brush borders as a consequence of exposure to food grade titanium dioxide in vitro. Finally, this Dissertation will demonstrate that iron oxide nanoparticles elicited similar effects after exposure to an in vitro brush border expressing model of the human placenta. Together, these data suggest that brush border disruption is not an artifact of the material/cell culture model, but instead represents a bona fide biological response as a result of exposure to nanomaterial.

In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle.