Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Biallelic mutation of the APC tumor suppressor gene occurs in a high percentage of colorectal tumors and is considered the critical event driving tumor initiation in the large intestine. The APC protein performs multiple functions, including negative regulation of the canonical WNT signaling pathway by both cytoplasmic and nuclear mechanisms. As a result, APC suppresses proliferation, cell cycle progression, and genomic instability, while facilitating differentiation, normal directional migration, and apoptosis. The contribution of APC to these phenotypes is not mediated exclusively through its effects on canonical WNT signaling, but also through WNT-independent functions of the APC protein. Intriguing reports that APC interacts with chromatin to repress key WNT-activated targets prompted this study's initial hypothesis that the chromatin-associated fraction of APC regulates gene transcription through multiple mechanisms, both WNT-dependent and WNT-independent. Chromatin immunoprecipitation and next-generation sequencing identified more than 6,000 genomic peaks associated with the APC protein. Target selection was performed by comparison to whole transcriptome sequencing data from APC-deficient and APC wild- type colon cancer cell lines and mouse tumors. 175 transcripts whose expression changes upon APC loss are linked to genomic regions physically associated with the APC protein. Motif analysis of APC-associated genomic peaks found that binding sites for the TCF7L2 and AP-1 transcription factors are overrepresented and occur in many of the same peaks. Luciferase reporter assays indicate that APC antagonizes canonical WNT signaling not only at WNT-activated genes such as PHLDB2 (Pleckstrin Homology Like Domain Family B Member 2), but also at WNT-repressed genes such as MALL (Mal, T-Cell Differentiation Protein-Like). A transcriptional element within the first intron of the MALL gene mediates transcriptional repression by the canonical WNT (open full item for complete abstract)

Committee: Joanna Groden PhD (Advisor); Albert de la Chapelle MD, PhD (Committee Member); Kay Huebner PhD (Committee Member); Mark Parthun PhD (Committee Member); Jeffrey Parvin MD, PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2006, Medicine : Molecular Genetics, Biochemistry, and Microbiology

The APC tumor suppressor is a multifunctional protein involved in cell migration, proliferation, differentiation and apoptosis. It is a component of the Wnt signaling pathway and is best known for its ability to down-regulate beta-catenin and consequent effects on transcriptional regulation. Previous work in our laboratory has demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, suggesting novel tumor suppressor functions of APC. In the present study, the transcription-independent mechanisms of APC function in apoptosis were investigated. We mapped the APC apoptosis-accelerating region to amino acids 1-760 by testing a series of non-overlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the amino-terminal amino acids 1-777. Mutation of the APC aspartic acid residue at position 777 in APC to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase. This mutation also rendered the full length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, and unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an amino-terminal segment are necessary for the transcription-independent mechanism of APC-mediated apoptosis. Additionally, we have elucidated the mechanism of APC-mediated apoptosis by identifying and characterizing a downstream partner of APC in this apoptotic cascade. hTid-1, a homologue of the Drosophila tumor suppressor Tid 56, is an apoptosis modulator predominantly located in mitochondria. We have demonstrated that the amino-terminal segment of APC (APC 1-777) interacts with hTid-1 directly using co-immunoprecipitation, pull-down assays, and immunofluorescence. Immunofluorescence studi (open full item for complete abstract)

Committee: Joanna Groden (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2011, Civil Engineering

A methodology is developed to estimate a route-level probability OD flow matrix for a time-of-day period that takes advantage of the increasing availability of large quantities of boarding and alighting data collected via Automatic Passenger Counter (APC) technologies. Unlike previous transit OD estimation approaches, the problem formulation considers the distribution, rather than only the means, of the APC data. A Heuristic Expectation Maximization (HEM) algorithm is developed to provide an approximate solution to the estimation formulation while achieving computational feasibility for applications to realistically long bus routes. The performance of the HEM algorithm is evaluated numerically and empirically by comparing OD flow estimates to estimates produced by three other methods: the Expectation Maximization (EM) method, the Iterative Proportion Fitting (IPF) method and a Conditional Maximization (CM) method. The EM method provides a solution to the formulation, but it is computationally prohibitive for practical use. The IPF method represents traditional methods, where only the means of the APC data are considered. The CM method is developed in this study as another computationally feasible method that considers the distribution of APC data, but it is seen to extract information on the probability OD flows less efficiently than the HEM method. The numerical study demonstrates that the HEM algorithm provides a good approximation to the solution produced by the EM algorithm. In addition, the numerical and empirical studies demonstrate that the HEM algorithm produces more accurate estimates than the IPF and CM methods when large quantities of APC data are available. Moreover, the numerical and empirical studies quantify the effects of three pertinent factors on the accuracy of OD flow estimates: the number of bus trips with APC data, the sample size of an OD flow survey used with APC data to estimate OD flows, and the magnitude of measurement errors in APC counts (open full item for complete abstract)

Committee: Rabi G. Mishalani PhD (Advisor); Mark R. McCord PhD (Advisor); Prem K. Goel PhD (Other) Subjects: Civil Engineering

Doctor of Philosophy, The Ohio State University, 2008, Molecular Genetics

Published data have revealed that γ-tubulin has essential, but incompletely understood functions in addition to its established role in microtubule nucleation. Our lab has isolated several conditional γ-tubulin alleles in which γ-tubulin localization is normal, microtubules are abundant and mitotic spindle assembly is not inhibited, but growth is strongly inhibited at restrictive temperatures. Additionally, there were defects in the coordination of late mitotic events (Prigozhina et al., 2004), indicating that γ-tubulin plays a role in the regulation of mitosis. Most mitotic regulatory proteins localize at the spindle pole body during mitosis, as does γ-tubulin, and it is possible that interactions between γ-tubulin and mitotic regulatory proteins are important for coordinating the events of mitosis. One focus of my research has, consequently, been to study the distribution of mitotic regulatory proteins in vivo by fusing them to green fluorescent protein. In order to facilitate GFP tagging, I have generated a strain in which the nkuA (A. nidulans homolog of Ku70) gene is deleted. We have found that this deletion, along with the use of heterologous markers from A. fumigatus, increases the correct gene targeting frequency to >90% (Nayak et al., 2006). Using this system, I have made GFP fusions of many mitotic regulatory proteins and studied their distribution over time by time-lapse live cell imaging in control cells and in cells carrying a γ-tubulin mutant, mipAD159. Three critical mitotic regulatory proteins that are mislocalized in mipAD159: Cyclin B, Cdk1 and MspA. Studies on cyclin B localization in mipAD159 indicate that the mitotic defects of γ-tubulin are at least partially caused by misregulation of an important mitotic regulator, the APC/C. In particular, γ-tubulin plays an essential role in the inactivation of the APC/C at the end of mitosis or in G1. I have determined that this role of γ-tubulin in APC/C inactivation is independent of its role in microtub (open full item for complete abstract)

Committee: Berl Oakley PhD (Advisor); Stephen Osmani PhD (Committee Member); Harold Fisk PhD (Committee Member); Hay-Oak Park PhD (Committee Member) Subjects: Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 1995, Electrical Engineering & Computer Science (Engineering and Technology)

Handwritten character recognition is a highly challenging area in the field of pattern recognition. In performing recognition, any single classifier system has its strengths and weaknesses. The objective of this dissertation is to develop multiclassifier systems which utilize the combined strength of several classifiers to make a significant improvement in recognition over the single classifiers. The multiclassifier systems developed were cascaded, vote-to-decide, confidence enhancement, and hierarchical learning systems. In each multiclassifier system, the single classifiers contained their own feature extraction, similarity measure, learning, and classification stages. Feature extraction extracted object features and formed feature representations. Three feature representations were developed, which were the angle sequence, vector contour representation (VCR), and Fourier transform representation (FTR). To evaluate the similarity of objects in different representation forms, measures based on Euclidean distance, vector correlation, string matching cost, and Fourier transform were developed. For learning, two supervised clustering techniques were developed: maximum region clustering (MRC) and accumulated potential clustering (APC). The MRC learning maximized the clustering regions by including as many samples of the same type as possible in each cluster without enclosing any alien sample. In APC learning, the feature space was viewed as an electrostatic field in which each cluster served as a potential generating center. Each object class established the minimum number of cluster centers necessary to protect its members from being attracted to other classes. In the classification stage, the MRC classifiers identified a test sample with the class of its nearest cluster center. The APC classifiers assigned a test sample to the object class which attracted it the most. In a multiclassifier system, the final classification decision was made based on the individual deci (open full item for complete abstract)

Committee: Janusz Starzyk (Advisor) Subjects:

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2007, College of Science

The mainstay of the blood coagulation cascade is the formation of the fibrin clot, catalyzed by the serine protease, thrombin. The prothrombinase complex catalyzes the activation of prothrombin to thrombin and, is composed of the enzyme, factor Xa, and the protein cofactor, factor Va, in the presence of divalent metal ions associated on a membrane surface. The enzyme, factor Xa, alone can activate prothrombin by two sequential proteolytic cleavages at Arg271 and Arg320 resulting in the intermediates, Fragment 1.2 and Prethrombin 2. The overall rate of this reaction is not compatible with survival. On the other hand, the incorporation of an excess of the cofactor, factor Va, into prothrombinase reverses the order of the proteolytic cleavages and increases the catalytic activity of factor Xa by 5 orders of magnitude, making this reaction compatible with survival. The goal of this research is to identify the precise amino acids of the central portion of the factor Va heavy chain involved in its incorporation into the prothrombinase complex and the cofactor function it exerts on the catalytic efficiency of prothrombin activation.

Committee: Dr. Michael Kalafatis (Committee Chair); Dr. Lily Ng (Other); Dr. Amin Zhou (Other) Subjects: