Biologic scaffolds composed of extracellular matrix (ECM) have been used to promote site-specific, functional remodeling of tissue in both preclinical animal models and human clinical applications. Although the mechanisms of action of ECM scaffolds are not completely understood, proteolytic degradation of the ECM scaffold and subsequent progenitor cell recruitment are thought to be important mediators of the constructive remodeling process. Proteolytic degradation of the ECM scaffolds results in the generation and release of cryptic peptides with novel bioactive properties not associated with their parent molecules such as angiogenic, antimicrobial, mitogenic, and chemotactic properties. While previous studies have suggested that degradation products of ECM scaffolds are chemotactic for progenitor cells in vitro, the present thesis expands upon these findings in vivo. In a non-regenerating model of mid-second phalanx digit amputation, treatment with ECM degradation rpodcuts resulted in the accumulation of a heterogeneous population of cells with in vitro differentiation potential along osteogenic, adipogenic, and neuroectodermal lineages. Focusing specifically on the Sox2+ population of cells found at the site of injury, work in the present thesis showed that Sox2+ cells co-express bone marrow and periosteal stem cell markers CD90 and Sca1, but not dermal stem cell marker CD133 or circulating stem cell marker c-kit (CD117). Additionally, bone marrow chimeric studies utilizing wild type C57/BL6 and Sox2 eGFP/+ mice showed that the Sox2+ cells are not derived from the bone marrow, but more likely from a local tissue source such as the periosteum. Fractionation of the ECM degradation products resulted in the identification of a highly conserved cryptic peptide derived from the C-terminal telopeptide of the collagen type IIIα molecule with chemotactic activity for multiple progenitor cells in vitro, IAGVGGEKSGGF. Administration of the cryptic peptide in a model of digit amputation resulted in the accumulation of Sox2+, Sca1+, Lin- cells at the site of amputation. Peptide treatment also resulted in the formation of a bone nodule at the site that coincided with the spatial location of Sox2+ cells. In vitro, the peptide accelerated osteogenesis of mesenchymal stem cells and increased the expression of osteogenic and chondrogenic genes. The result of this body of work shows that degradation products of ECM scaffolds contain cryptic peptides with the ability to influence chemotaxis and differentiation of progenitor cells in vitro and in vivo. The ability to influence stem cell phenotype and fate may be useful in designing new therapies for regenerative medicine approaches to complex, composite tissue reconstruction. Additionally, the findings of the present thesis may serve as the basis for future studies investigating the importance of ECM degradation in the downstream constructive remodeling events at a site of ECM implantation in soft tissue models of injury.

It is a time consuming task to generate textures for large 3D terrain surfaces in computer games, flight simulations and computer animations. This work explores the use of cellular automata in the automatic generation of textures for large surfaces. I propose a method for generating textures for 3D terrains using various approaches - in particular, a hybrid approach that integrates the concepts of cellular automata, probabilistic distribution according to height and Wang tiles. I also look at other hybrid combinations using cellular automata to generate textures for 3D terrains. Work for this thesis includes development of a tool called "Texullar" that allows users to generate textures for 3D terrain surfaces by configuring various input parameters and choosing cellular automata rules. I evaluate the effectiveness of the approach by conducting a user survey to compare the results obtained by using different inputs and analyzing the results. The findings show that incorporating concepts of cellular automata in texture generation for terrains can lead to better results than random generation of textures. The analysis also reveals that incorporating height information along with cellular automata yields better results than using cellular automata alone. Results from the user survey indicate that a hybrid approach incorporating height information along with cellular automata and Wang tiles is better than incorporating height information along with cellular automata in the context of texture generation for 3D meshes. The survey did not yield enough evidence to suggest whether the use of Wang tiles in combination with cellular automata and probabilistic distribution according to height results in a higher mean score than the use of only cellular automata and probabilistic distribution. However, this outcome could have been influenced by the fact that the survey respondents did not have information about the parameters used to generate the final image - such as probabilistic distributions, the population configurations and rules of the cellular automata.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 85-112).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains x, 92 p.: ill. Includes abstract and vita. Advisor: Clay B. Marsh, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 84-92).

Hedgehog (Hh) is an important morphogen that dictates tissue patterning during embryonic development. Recent studies showed that mutation in Indian Hedgehog(IHH)resulted in Brachydactyly type A1(BDA1);however, the disease pathogenesis in patients without IHH mutation remained unknown. PDZD2 is a multi-PDZ domain-containing protein of unknown function in early development. Human PDZD2 is mapped to chromosome 5p13.2, which co-localizes with the disease-associated gene in a family of BDA1 patients, suggesting involvement of PDZD2 in limb development. In situ hybridization revealed that Pdzd2 was expressed in the distal mesenchyme partially overlapping with Shh expressionin mouse limb bud. During digit patterning, Pdzd2 was expressed in the interzone regions that flanked the Ihh/Gli1-expressing phalanx condensation. Moreover,Pdzd2 was expressed in the paraxial mesoderm adjacent to the differentiating neural tube. Pdzd2expression in various Hh-active tissues in mouse and chicken suggested an evolutionary conserved role of PDZD2 in modulating general Hh signaling during early development. Interestingly, PDZD2 protein was detected at the neural tube away from its site of synthesis, suggesting a non-cell autonomous role of PDZD2 possibly via its secreted form, sPDZD2. Functional studies showed that overexpression of sPDZD2 in the chicken neural tube leads to down-regulation ofNKX2.2andOLIG2expression.sPDZD2 was shown to counteract the ectopic NKX2.2 expression induced by long-range signaling of ectopic HH. Consistently, sPDZD2exhibited an inhibitory effect on SHH-induced reporter activity in a Gli-luciferase cell line. For in vivo analysis, a transgenic mouse line carrying a floxed Pdzd2 allele (Pdzd2fl) was generated to ablate Pdzd2 expression.Pdzd2+/fl mice were crossed with Protamine-cre to generate the null allele (Pdzd2-). However, heterozygous intercross yielded no homozygous mutant as early as E9.5, suggesting early embryonic lethality. Thus, conditional Pdzd2 knock-out in the limb was pursuedusingPrx1-cre.However, no significant perturbation of Hh signaling was observed in Pdzd2fl/fl:Prx1-cre limb buds, which might be due to incomplete knock-out of Pdzd2, or functional redundancy among Hh modulators. To study the relevance of Pdzd2in the development of BDA1, Pdzd2-/fl mouse was crossed with the BDA1 mouseIhhE95K/E95Kto study the effect of reducing Pdzd2expression under defective Hh signaling. Preliminary analysis of the Pdzd2+/-, Ihh+/E95K compound mutants showed more severe phenotypes comparing with IhhE95K/E95K. These included delayed limb development and further diffusion ofGli1expression in the digits, suggestive of a direct involvement of Pdzd2in BDA1. It was speculated that Pdzd2negatively modulated Ihh signaling and restricted Hh signals from entering the interzone, which was required for normal digit patterning. Depletion of Pdzd2might result in an expansion of Ihh signaling into the interzone, leading to the BDA1phenotypessimilar to the current BDA1 disease model proposed forIhhE95Kmutation. Taken together, my study revealed the novel expression of Pdzd2in close proximity to multiple Hh-active tissues during early development and provided the first evidence that PDZD2/sPDZD2 is a negative modulator of general Hh signaling. These data strongly supported PDZD2as a disease-associated locus in the family of BDA1 patients that do not carry IHHmutations. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Purpose: The purpose of the article is to determine whether there are any differences between high and low-income group students in their selection of a cellular phone brand or network operator. Design / Methodology / Approach: Four hypotheses are set to determine if there are any significant differences between the two income groups in current decision-making. It is established that there exist no significant difference between high and low-income students in their selection of cellular phones and network operators. The levels of agreement or disagreement on various statements do, however, give an indication of the importance that students place on aspects that they view as important when acquiring a cellular phone or network operator. Findings: In the article, it is established that no significant differences exist between the two income groups. The levels of agreement or disagreement indicate the importance that subscription method, social value, service quality and branding has on student decision-making. Implications: The article provides a better understanding of the influence that income plays in student’s decision-making in acquiring cellular products and services. Possible future research in student cellular usage can be guided through the information obtained in this article. Originality / Value: The article provides information to cellular network operators, service providers and cellular phone manufactures regarding the influence of income on students’ acquisition of cellular products and services. Information from the article can assist in the establishment of marketing plans for the student market by these role players.

BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2, \(BCL-X_L\), and MCL-1 have been implicated in the initiation, progression, and chemoresistance of human cancer. Small molecules and peptides have successfully targeted the anti-apoptotic BCL-2/\(BCL-X_L\) groove that binds and sequesters pro-apoptotic BH3 death helices. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. Notably, selective antagonists such as ABT-737 are highly effective at inducing apoptosis in BCL-2/\(BCL-X_L\)-dependent cancers but are rendered inactive by overexpression of MCL-1, a formidable chemoresistance protein that lies outside the molecule's binding spectrum. By screening a library of stabilized alpha-helices of BCL-2 domains (SAHBs), we previously discovered that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. Here, we deployed this chemically-constrained peptidic inhibitor of MCL-1, MCL-1 SAHB, in a competitive binding screen to identify selective small molecule inhibitors of MCL-1. Rigorous in vitro binding and functional assays were used to validate the compounds and their mechanisms of action, and most notably, MCL-1 inhibitor molecule 1 (MIM1) displayed exquisite selectivity in these assays. NMR analysis documented that MIM1 engages the canonical BH3-binding pocket of MCL-1. Importantly, MIM1 selectively triggers caspase 3/7 activation and apoptosis of a cancer cell line that is dependent on induced overexpression of MCL-1 but showed no activity in the isogenic cell line that is driven instead by overexpressed \(BCL-X_L\). Thus, a selective stapled peptide inhibitor of MCL-1 was successfully applied to identify a high fidelity small molecule inhibitor of MCL-1 that exhibits anti-cancer activity in the specific context of MCL-1 dependence.

Metazoans use Hedgehog signaling to direct many stages of embryonic development, and deregulation of this pathway is implicated in many types of cancer. I investigated several steps of Hedgehog pathway transduction that were poorly understood in mechanistic terms. The mature Hedgehog ligand is produced by a self-proteolysis reaction that covalently attaches a cholesterol molecule to the signaling half of the protein. I showed that the catalytic cysteine forms a disulfide bridge that is essential for the folding and function of the C-terminal tail of Hedgehog, and identified two protein disulfide isomerases that remodel this bridge to free the catalytic thiol group after folding is complete. Using pulse chase assays to follow Hedgehog processing, I demonstrated that the self-proteolysis reaction takes place in the endoplasmic reticulum, that the cleaved C-terminal tail of Hedgehog is degraded before moving to the Golgi, and that Hedgehog mutants defective in processing get degraded in their entirety by the same route. Lipidated Hedgehog ligand requires the transmembrane protein Dispatched for secretion. I devised a system to test Dispatched function in cultured cells, and showed that some inactive Dispatched mutants fail to bind Hedgehog, while others bind more tightly than the wild type protein. Scube2 was implicated as a Hedgehog pathway component in zebrafish genetic studies. I showed that Scube2 is a secreted protein that binds Hedgehog via its cholesterol adduct and solubilizes it in aqueous media. Dispatched and Scube2 bind Hedgehog on opposing faces, and they function synergistically to release it from the membrane. Vertebrate Hedgehog signaling relies on intraflagellar transport through an antenna-like organelle called the primary cilium. The Hedgehog receptor Patched and transducer protein Smoothened localize to primary cilia in a mutually exclusive pattern, depending on Hedgehog ligand presence. I showed that cytoplasmic components of the pathway Suppressor of Fused (SuFu, a pathway inhibitor) and Glioma-associated oncogene transcription factors (the Gli family, the effectors of the pathway) localize to primary cilia and accumulate there when Smoothened is activated. SuFu and Gli form a complex that dissociates when the pathway is turned on, and this dissociation depends on trafficking through the cilium.

Adipose Fatty Acid Binding Protein 4 (FABP4) or aP2, plays an important role in several immunometabolic pathologies such as type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Long considered to be a cytosolic protein, aP2 has recently been detected in conditioned media of adipocytes. Interestingly, there is a growing body of literature showing association of increased circulating levels of aP2 with cardiovascular disease and metabolic syndrome. Our lab has discovered a role for aP2 secreted from adipocytes in regulating liver glucose output and blood glucose levels in diabetes. The emerging biology of this novel adipokine makes it critical to understand the route and mechanisms that lead to its secretion.