published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Ovarian cancer is a highly metastatic disease having a poor prognosis (<25%). The factors and underlying mechanisms that regulate ovarian cancer metastasis, however, are still incompletely understood. p70 S6 kinase (p70S6K), a serine/threonine kinase, is frequently activated in high-grade malignant human ovarian cancer. The aim of this study is to investigate the molecular mechanisms by which p70S6K may promote ovarian cancer metastasis. The results show that p70S6K is a critical regulator of the actin cytoskeleton, peritoneal adhesion and dissemination, and multicellular aggregates/spheroids formation in the acquisition of the metastatic phenotype. The regulation of p70S6K on the actin cytoskeleton is through two important functions: as an actin cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70S6K induced a marked reorganization of the actin cytoskeleton and directional migration of ovarian cancer cells. Actin binding and immunofluorescence studies showed that p70S6K had a direct interaction with the actin filaments with no other proteins involved. This interaction did not affect actin polymerization kinetics but cross-linked the actin filaments to inhibit cofilin-induced actin depolymerization. In addition, p70S6K mediated the activation of Rac1 and Cdc42 GTPases and their downstream effector p21-activated kinase 1, but not RhoA. Peritoneal adhesion and dissemination is regulated by p70S6K through integrin expression. Expression of p70S6K siRNA efficiently inhibited ovarian cancer cell adhesion to fibronectin and laminin among different peritoneal extracellular matrix components, as well as to human primary peritoneal mesothelial cells. These effects were associated with the expression of alpha5 and beta1 integrin. Studies into the mechanisms suggest that p70S6K may upregulate alpha5 integrin by a transcriptional mechanism whereas beta1 integrin is regulated at a post-transcriptional level. Enhanced expression of alpha5 and beta1 integrin by active p70S6K mediated the subsequent peritoneal adhesion. In ovarian cancer xenografts, p70S6K and beta1 integrin interference significantly inhibited peritoneal dissemination through reduction in the number and weight of tumors. Multicellular spheroids are present in the malignant ascites of ovarian cancer patients. Using a 3-dimensional culture system, expression of p70S6K siRNA resulted in inhibition of multicellular spheroid formation, which was mediated by N-cadherin but not E- or P-cadherin. In addition to spheroid formation, inhibition of p70S6K was associated with reduced growth of spheroids and disaggregation capabilities on different extracellular matrix components. Taken together, these findings indicate that p70S6K plays an important role in the biology of ovarian cancer metastasis through regulation of several critical steps in dissemination and migration, suggesting that p70S6K could be explored as a potential therapeutic target in ovarian cancer. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

<p>Clubfoot is the most common congenital structural deformity that leads to physical impairments in children in many poor developing countries. Inadequately treated or neglected clubfoot has been found to be a common cause of ohysical disability globally among children and young growing adults. Many children are referred to the clinics for treatment but some parents do not comply with the treatment regimen whcih requires attending for consecutive treatment sessions. The purpose of this study was to investigate barriers to treatment attendance parents/caregivers of children with clubfoot encounter in complying with clubfoot treatment during the plaster csting phase in Uganda.</p>

DNA fingerprinting was applied for the molecular elucidation of taxonomic relationships within a genus of orchids which have previously been based on morphological characteristics. Phragmipediwn consists of 15-20 species native to Central and South America. This research project included two studies. In the first study DNA was isolated from 11 samples (including two unidentified ones). These individuals, which were mostly hybrids, were found in the Wheeler Orchid Collection and Species Bank at Ball State University. In order to position Phragmipediwn within the orchid family fingerprinting was also performed on individuals in the sister taxa, Cypripedium and Paphiopedium, which are members of the same subfamily, and on a member of the outgroup taxon Vanda. The polymerase chain reaction (PCR) was employed to yield fingerprints resulting from the use of random primers. Fifty nine random amplified polymorphic DNA (RAPD) bands were obtained using 5 different primers to yield 107 polymorphic bands. As many as 75% of genetic loci were found to be shared between hybrids that resulted from a cross of more than one individual in the same section. However the percentage dropped to 35-65 % when only one parent was shared in the cross. Furthermore, the sister group taxa Cypripedium and Paphilopedium shared from 12 % -35 % of their polymorphic loci with the members of the genus Phragmipedium. The outgroup taxon Vanda shared 17% of its polymorphic loci with the rest of the samples.In a second study DNA was isolated from one member of each of the five sections of the genus Phragmipedium, and RAPD-PCR fingerprinting was used to compare their genetic similarities to that of the two sister taxa and the outgroup taxon. It was found that individuals in different genera shared 25% or less of their polymorphic bands. Between sections of the same genus 20-50% of genetic loci were shared. Two sections, Platypetalwn and Phragmipedium showed the highest degree of genetic relatedness (41-53%). Again the outgoup taxon shared less than 20% Phragmipediwn samples on the phenograms produced but the percentage was again insignificant. However, genetic analyses of the members of the section Lorifolia gave conflicting results: 46% genetic identity was observed in the first trial and 20% in the second.In conclusion, RAPD-PCR fingerprinting results appeared to be effective in the positioning of sections within a genus indicating the degree of similarity of closely related taxa. Also RAPD-PCR was able to place an unknown individual within a specific section of the genus. However, it could not be employed to determine the identity of unknown species due to the high degree of genetic diversity observed between even closely related individuals. / Department of Biology

To study resistance (R) genes that are expressed when Sophrolaeliacattleya Ginny Champion 'Riverbend' orchid tissue was infected with the tobacco mosaic virus (TMV0), a subtraction library of cDNA clones was previously constructed using mRNA isolated before and after infection (Shuck, unpublished). From 200 clones collected, 5 clones were randomly selected, DNA was isolated, and the cDNA insert was sequenced. These sequences were imported into BLAST to search for homology to other R genes. This search revealed clone 4A to have an 84% homology to a 54 nucleotide region from the Arabidopsis thaliana oligouridylate binding protein which is highly expressed and known to bind RNA Polymerase III transcripts and adenovirus associated RNAs. Further bioinformatics analysis was performed utilizing databases and analysis packages available on the Internet, software such as Vector NTI (Informax, Bethesda, MD), and manual searches. However, no additional domains or motifs indicative of pathogen resistance genes were located in any of the 5 clones. Subsequently, total proteins expressed at various time points following infection were examined on denaturing 5-20% gradient polyacrylamide gels stained with the ProteoSilver Plus TM silver stain kit (Sigma, St. Louis, MO) in order to examine the timing and duration of expression of proteins involved in TMV-O resistance. One protein of-18 kDa was highly expressed at 4 hr after infection that was not seen in the negative control. By 8 hr the band was no longer expressed, it was expressed again from 30 - 48 hr, but was not seen again in later time points. Finally, total mRNA isolated from pooled time points and subjected to in vitro translation indicated a reduction in translation products after infection, providing evidence of posttranscriptional gene silencing (PTGS) following TMV-O infection. / Department of Biology