Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4250. Adviser: Carol Gross. Includes bibliographical references. Also available online.

Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3241858."

Thesis (Ph. D.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

<p>Biological processes such as cell division and synaptic plasticity are regulated by concentration gradients of signaling molecules. A number of biochemical mechanisms can result in intracellular signaling gradients. For example, restriction of diffusional flux of a chemical from one compartment to another will result in a transient gradient. A sustained gradient can be generated by opposite reactions such as phosphorylation and dephosphorylation of a signaling substrate taking place at different locations in the cell. More sophisticated mechanisms for non-uniform spatial signaling profiles include Turing type patterning and wave-pinning. It is becoming apparent that cell shape can regulate concentration gradients and modulate the downstream processes. In Chapter 1 we review how cell geometry can regulate intracellular signaling gradients in the context of the aforementioned gradient-generating mechanisms. The works reviewed make heavy use of mathematical modeling in order to investigate how reaction and diffusion taking place in complex cell geometries can modulate concentration gradients. That is a motivation for Chapter 2 where we implement a computational method to simulate reaction and diffusion on curved surfaces representing the cell membrane coupled with reaction and diffusion in the enclosed volume (representing the cell cytosol). To solve the reaction-diffusion equations on the surface we use the closest point method, a finite-difference technique that embeds the equations in the surrounding space. Such method is coupled with an embedded boundary technique to solve the equations in the enclosed volume with boundary conditions accounting for material exchange between surface and volume. The method is second-order convergent in the grid spacing despite a simple accuracy analysis predicts first-order errors. In Chapter 3 we use mathematical modeling in order to propose mechanisms accounting for the spatiotemporal dynamics of Rho-GTPase signaling at dendritic spines during synaptic plasticity. Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines (structural plasticity) through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation following synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Since Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased remains unclear. We model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine and interestingly, retention is enhanced by high diffusivity of Cdc42. These findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.</p> / Dissertation

<p> Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is caused by the loss of laminin-211 and laminin-221 heterotrimers which are most abundant in skeletal and cardiac muscle basal lamina; mutations in the LAMA2 gene cause the loss of these laminin isoforms. This absence of laminin-211/221 in MDC1A reduces the capacity for myofiber adhesion, loss of sarcolemmal integrity and subsequently the ability of the skeletal muscle syncytium to generate force in a coordinated and efficient manner. Patients experience progressive muscle wasting which confines them to a wheelchair at an early age and respiratory failure that leads to their untimely death. Currently, there is no effective treatment or cure for this devastating disease. Previous studies have shown that laminin-111, an embryonic form of laminin, delivered before disease onset can reduce muscle pathology and improve viability in the dyW-/- mouse model of MDC1A. These studies suggested that laminin-111 may act to strengthen and reinforce the sarcolemma and provide a protective niche for muscle repair. Since most patients are diagnosed with MDC1A after disease onset, we determined if laminin-111 could be beneficial after disease onset. Our studies suggest dyW-/- mice treated with laminin-111 after disease onset show improvement in muscle function and histology. Results from this study along with an understanding of laminin-111 pharmacokinetics will help pave the way in developing this protein as an exciting potential therapeutic for MDC1A patients. Duchenne Muscular Dystrophy (DMD) is the most common X-linked disease affecting 1 in 3,300 live male births. Patients with DMD suffer from severe, progressive muscle wasting and weakness with clinical symptoms first detected between 2 to 5 years of age; as the disease progresses patients are confined to a wheelchair in their teens and die in their early 20s mainly due to cardiopulmonary complications. DMD is caused by the loss of the sarcolemmal protein dystrophin (427kDa) due to mutations in the dystophin gene. When present, dystrophin acts as a scaffold linking the cell cytoskeleton to the extracellular matrix. This loss of dystrophin in DMD results in patients experiencing greater susceptibility to muscle damage via reduced structural and functional integrity of their muscle. One potential therapeutic avenue that needs to be explored involves increasing the levels of the ?7?1 integrin in order to compensate for the loss of dystrophin. To test this hypothesis, a muscle cell-based assay was developed in order to report ?7 integrin promoter activity with the intent of identifying molecules that promote ?7 integrin expression. Laminin-111 was identified as an enhancer of ?7 integrin expression. Theoretically, the identification of ?7 integrin enhancing compounds that help boost ?7?1 integrin expression as part of drug-based therapies may lead to a novel therapeutic approach for the treatment of this disease. Systemic laminin-111 treatment significantly reduces myofiber degeneration in both forms of MDC1A and DMD muscular dystrophy. This dissertation reinforces the potential of laminin-111 as a systemic protein therapy, capable of restoring sarcolemmal integrity thus reducing muscle disease progression. The importance of ?7 integrin in skeletal and cardiac muscle was highlighted here through the generation of the ?7-/-:: laminin-?2-/- double knockout mouse model. This mouse has never been studied before and could prove to be another important mouse model needed to explore therapeutic avenues for muscular dystrophy.</p>

<p> Understanding how protein structures evolve is essential for deciphering relationships between homologous proteins, which can inform structure classification and function annotation and aid in protein modeling and design methods. The observation that structure is more conserved than sequence, over the course of evolution, implies a model of evolution where sequences diverge within a discrete set of well-defined folds, which suggests that homology does not exist across fold definitions. However, as more structures have been experimentally solved and the coverage of the universe of folds has increased, the original view of a discrete fold space has been revised to include a more nuanced view of a continuous space defined by regions in which structural similarities can connect globally disparate topologies.</p><p> Structural, functional and evolutionary relationships are known to, in some cases, span fold definitions. A hallmark of relationships connecting disparate topologies is the conservation of local structure motifs within globally different folds. In order to systematically identify and analyze these relationships, a new approach to structure comparisons and structure classification is required. The goal of this work is to systematically identify evolutionary relationships between folds and to generate a classification of the fold universe that can accurately represent even the relationships that exist across disparate topologies. </p><p> An exhaustive library of supersecondary-structure motifs (Smotif), defined as two secondary structures connected by a loop, is established and characterized. A novel Smotif-based, superposition-independent structure comparison method (SmotifCOMP) is developed that quantitatively measures the Smotif-based similarity of compared structures in order to identify evolutionary relationships. SmotifCOMP is able to provide a quantitative and robust measure of similarity between disparate topologies since it does not rely on a global superposition. The comparison method is used to perform a systematic analysis of the SCOP Superfamilies and generate a non-hierarchical, network-based representation of the fold universe.</p><p> This thesis describes the development of a novel method of comparing structures and an improved representation of the relationships within the fold space. This work provides insight into the existence of evolutionary relationships between folds and strengthens the view of a connected and continuous fold universe.</p>

The exciting and intriguing biological effects associated with amelanocyte stimulating hormone, α-MSH, have initiated a variety of studies to identify the biologically important conformations of the backbone and sidechains structures. To further investigate the topology of the critical "message" residues, several bicyclic (sidechain-to-sidechain) and x¹ restricted peptides have been synthesized and biologically examined, and selected analogues have been studied by NMR techniques and by theoretical molecular modeling studies. Bicyclic melanotropic peptides possessing the central sequence, Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹, exhibited nearly identical nanomolar biological potencies in the lizard skin bioassay. Molecular modeling studies suggested the hypothesis of incomplete receptor binding by either the His or Trp residue as being responsible for the decrease in potencies relative to a-MSH, and the parent monocyclic peptides from which these bicyclic peptides were designed. This hypothesis is further supported by twodimensional NMR studies of a representative bicyclic peptide, Ac-Cys⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Trp⁹-Cys¹⁰-Lys¹¹-NH₂. Peptides synthesized to probe the topographical space of the tryptophan residue at position nine provided extraordinary results regarding the biological phenomena of prolongation. These peptides were based on the template, Ac-Nle⁴-Asp⁵-His⁶-DPhe⁷-Arg⁸-Xaa⁹-Lys¹⁰-NH₂, where Xaa consists of the four isomers of β-MeTrp, DTrp , and L- or D-TCA residues. Some of these peptides were selected for studies on the cloned hMC1 melanocortin receptor. These studies resulted in a partial hypothesis accounting for the prolonged biological activities observed in other physiological assay systems. Conformational analysis by solution 20 NMR techniques revealed similar peptide backbone secondary structure features with main differences of structure occurring in the sidechain x¹ space. The implications and results are discussed. Homology-based molecular modeling of the hMC1 melanocortin receptor was also undertaken and provided evidence for ligand-receptor interactions which are being tested by receptor mutagenesis studies. This three-dimensional computer model provides an opportunity to probe detail chemical ligand-receptor interactions and further study differences in biological activities and biological mechanisms.

<p> Coral reef ecosystems worldwide are facing increasing degradation due to disease, anthropogenic damage, and climate change, particularly in the Tropical Western Atlantic. Mesophotic coral ecosystems (MCEs) have been recently gaining attention through increased characterization as continuations of shallow reefs below traditional SCUBA depths (>30 m). As MCEs appear to be sheltered from many stressors affecting shallow reefs, MCEs may act as a coral refuge and provide larvae to nearby shallow reefs. The Deep Reef Refugia Hypothesis (DRRH) posits that shallow and mesophotic reefs may be genetically connected and that some coral species are equally compatible in both habitats. The research presented here addresses key questions that underlie this theory and advances our knowledge of coral connectivity and MCE ecology using the depth-generalist coral <i>Montastraea cavernosa</i>. Chapter 1 presents an overview of the DRRH, a description of MCEs in the Gulf of Mexico (GOM), and the framework of research questions within existing reef management infrastructure in the GOM. Through microsatellite genotyping, Chapter 2 identifies high connectivity among shallow and mesophotic reefs in the northwest GOM and evidence for relative isolation between depth zones in Belize and the southeast GOM. Historical migration and vertical connectivity models estimate Gulf-wide population panmixia. Chapter 3 focuses on population structure within the northwest GOM, identifying a lack of significant population structure. Dominant migration patterns estimate population panmixia, suggesting mesophotic populations currently considered for National Marine Sanctuary protection benefit the Flower Garden Banks. Chapter 4 quantifies the level of morphological variation between shallow and mesophotic <i>M. cavernosa</i>, revealing two distinct morphotypes possibly representing adaptive tradeoffs. Chapter 5 examines the transcriptomic mechanisms behind coral plasticity between depth zones, discovering a consistent response to mesophotic conditions across regions. Additionally, variable plasticity of mesophotic corals resulting from transplantation to shallow depths and potential differences in bleaching resilience between shallow and mesophotic corals are identified. The dissertation concludes with a synthesis of the results as they pertain to connectivity of shallow and mesophotic corals in the Gulf of Mexico and suggests future research that will aid in further understanding of MCE ecology and connectivity.</p><p>