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> 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>

Ovorubin, apoprotein and ovorubin reconstituted from apoprotein and carotenoid were prepared from the eggs of Pomacea canaliculata australis, by adsorption on alumina CB or on carboxymethyl cellulose. Electrophoresis indicated an homogeneous preparation, but solubility tests indicated two components, very similar except for spectral differences. Paper chromatography revealed the presence of all the commonly occurring amino acids. 20% of the ovorubin molecule consisted of carbohydrate (glucosamine, galactose, mannose and fucose). The carotenoid component is in the trans form. Ovorubin was shown to possess a strong antitryptic activity, comparable with that of ovomucoid, which was not lost on removal of the carotenoid. The apoprotein was not more readily denatured than ovorubin, except after several precipitations with acetone. No direct correlation between changes in spectrum and inhibitory activity was demonstrated, neither was the spectrum modified by reaction with trypsin, Acetylation of amino groups of the apoprotein did not prevent inhibition, but did prevent recombination with the carotenoid. Ovorubin did not inhibit acetylated trypsin. A protein with antitryptic activity was isolated from eggs of Pila ovata gordoni, but was not extensively investigated. Trypsin slowly attacked ovorubin; pepsin produced a quite rapid fall in inhibition of trypsin and, after addition of trypsin and Chymotrypsin, the degradation product was isolated and shown to retain some antitryptic activity although the protein moiety was smaller. Ovorubin was shown to inhibit chymotrypsin and a protease from Aspergillus oryzae. Indications were obtained that an enzyme produced by B. subtilis was also inhibited. The nature of the second component in ovorubin preparations was discussed. The properties of ovorubin were compared with the proteins of hens' eggs; in composition and antitryptic action ovorubin is very similar to ovomucoid. A possible bacteriostatic role was suggested for ovorubin.

Crustacyanin, the blue carotenoprotein of the lobster carapace was obtained in a state homogeneous in cellulose acetate electrophoresis. It was crystallised and shown to be a globulin of large particle size, free from lipid and carbohydrate. The minimum molecular weight of the protein calculated on the carotenoid content, was found to be one-tenth of that expected from its size. Reversible changes in spectrum occurring on removal of salt were shown to be attended by dissociation into subunit of the size expected from the minimum molecular weight. Apocrustacyanin was shown to be of the subunit size and heterogeneous in cellulose acetate electrophoresis. Native cnustacyanin was reconstituted from the apoprotein and astaxanthin. Possible modes of binding of the carotenoid are suggested to account for the spectral changes occurring in urea. Electrophoretic similarities between crustacyanin treated with urea and apocrustacyanin are discussed in relation to the number of different subunits. It is suggested that crustacyanin is composed of a specific geometrical association of several small subunits, possibly different, and that the carotenoid stabilises the subunits in a configuration essential for the association and possibly also assisting in the binding. Ovoverdin, the green carotenoprotain of lobster ovary, was obtained homogeneous in cellulose acetate electrophoresis and shown to be a glycolipoprotein, precipitating at low ionic strength out soluble in distilled water. The possibility of two molecules of carotenoid being associated with each molecule of protein has been investigated. Astaxanthin-proteins were purified from the carapaces of Eriphia spinifrons, Carcinus maenas, Astacus astacus and Palinurus vulgaris. Their absorption spectra and properties arc compared with those of crustacyanin and ovoverdin. Glycolipoproteins containing a number of carotenoids have been purified from the ovaries of Carcinus maenas, Pecten maximus and Palinurus vulgaris, and from the eggs of Palinurns vulgaris. Their properties have been investigated and the ode of association of the carotenoids is discussed.

Since their first clinical application in the 1980’s recombinant proteins have become an increasingly larger section of the drug market, growing into a multi-billion dollar global market. The pursuit of improving the design, production, and application of recombinant proteins for biotherapeutic uses is a key driver in industry and academia. The majority of the recombinant biotherapeutic proteins used in the clinic are produced in mammalian cell expression systems due to their capability to undertake human-like complex post translational modifications. The currently ‘gold standard’ mammalian cell expression system for the production of recombinant proteins is the Chinese hamster ovary (CHO) cell line. However, even with advances in mammalian cell expression technology, there is still a high cost and a long development period required for a recombinant protein therapeutic to go from design to market. As such, bottlenecks include the time taken for mammalian cells to grow and divide with slow doubling times compared to microbial systems and limited capacity to synthesise and secrete recombinant proteins. One of the cellular processes that underpins both cell growth and recombinant protein production is the translation of mRNA. Translation consists of three distinct steps: initiation, elongation and termination. One major cell signalling pathway that is considered a master regulator of both initiation and elongation of translation is mTOR, also involved in regulating ribosome biogenesis and cell proliferation. During the process of polypeptide elongation (mRNA translation), elongation factor 2 (eEF2) is a key control point that regulates protein synthesis via its de/phosphorylation. Phosphorylation of eEF2 results in its inactivation; slowing or halting elongation resulting in the attenuating of protein synthesis. This study set out to establish if manipulation of the mTOR signalling pathway and/or manipulation of phosphorylation of elongation factor 2 and the kinase that inactivates eEF2, eEF2K, in CHO cells impacts upon CHO cell growth and recombinant protein production yields. Transient expression of wild type and a Thr56Ala eEF2 mutant in CHOK1 cells affected the short term (24-48 hour) phosphorylation of eEF2 but did not appear to have an effect upon intracellular recombinant protein production and cellular growth in culture over 96 hours. Stable over expression of the wild type eEF2 construct in CHO cells resulted in a 2-fold increase in expression of eEF2 and a decrease in phosphorylation of eEF2 at the protein level; but the there was no change in the levels of total eEF2 mRNA expression. Stable expression of the Thr56Ala and Thr56Glu eEF2 mutants had a greater effect upon eEF2 expression resulting in a 3-5 fold increase in total eEF2 expression, however the phosphorylation of eEF2 was almost unchanged in an Ala56 eEF2 cell line, whereas it was reduced in the Glu56 eEF2 mutant cell 20 line. Growth of the CHO cells lines expressing the eEF2 mutants show that over expression of any of the eEF2 mutants resulted in a change in growth, but the Ala56 eEF2 mutant showed the largest change in cellular growth. Short term transient expression of recombinant firefly luciferase in the stable eEF2 cell lines revealed that the Thr56Ala mutant greatly increases the CHO cells total recombinant protein production. Further, mutation of eEF2 to Ala56 or Glu56 had little effect upon the mis-incorporation of amino acids during translation. The transient knockdown of eEF2K was achieved and this was shown to prevent eEF2 phosphorylation. However, CHO cells do not appear to tolerate the knockdown of eEF2K stably or at only very low levels. This suggests that a sustained, high level of eEF2K knockdown is lethal to the cell; which would result in the loss of eEF2 regulation for an extended period of time. Transient expression of the eEF2K shRNA into CHOK1D6 cells stably expressing firefly luciferase, a nonsecreted protein, resulted in a 5-fold increase in luciferase expression showing that knockdown of eEF2K increased the short term productivity of these cells. Interestingly, the study of CHO cell lines with varying recombinant monoclonal antibody protein production capacities revealed that levels of total and phosphorylated eEF2 did not appear to change in correlation with the mAb titre. These data suggest that eEF2 activity is tightly regulated across cell lines and is not directly related to recombinant protein secretion in these industrially used CHO cell lines The addition of 2 µM of PMA, an activator of mTOR signalling, increased antibody production in a low producer cell line but had no effect upon the antibody production from a high producing cell line. Together the data presented here shows that manipulation of eEF2 and eEF2K activity can enhance cellular growth and recombinant protein production from CHO cells. As such, new engineering approaches that allow the manipulation of both elongation and polypeptide synthesis combined with the secretory capacity of the cell are likely to yie ld new CHO host cells with more predictable recombinant protein capacity and further advance our understanding of the role of mRNA translation in controlling cell proliferation, and both global and recombinant protein synthesis.

Chromosome copy number errors (or aneuploidy) of gametes and embryos occurs in humans more frequently than in any other studied species, with a spectrum of manifestations from implantation failure to affected live births. It is predominantly problem arising in maternal meiosis with at least 20% of oocytes being aneuploid, a proportion that increases dramatically with advancing maternal age. Currently the only intervention to reduce the chances of transmitting aneuploidy is by invasive embryo biopsy procedures in high-risk groups (mainly patients with advanced maternal age) undergoing in-vitro fertilisation. Despite the severity of this problem, aneuploidy of the human preimplantation embryo is relatively poorly understood. With this in mind the purpose of this thesis is to explore the premise underpinning the use of preimplantation genetic screening (PGS) in human embryos and investigate its clinical applications and current methodologies. A series of published works demonstrate what I believe to be a significant contribution to the development of applications for studying human preimplantation aneuploidy, also providing insight into its origins and mechanisms at the earliest stages of human development. Specifically, I present a novel standard set of protocols as a general reference work from practitioners in the fields of embryo biopsy and array comparative genomic hybridisation (CGH - the current ‘gold standard’ for preimplantation aneuploidy screening). I present a summary of work encapsulated in three published clinical papers using a linkage based analysis of Single Nucleotide Polymorphism (SNP) karyotypes (Karyomapping). Karyomapping was designed as a near-universal approach for the simultaneous detection of chromosomal and monogenic disorders in a PGS setting and these results demonstrate the utility of the technique in three separate scenarios. In order to study the underlying mechanisms of female meiosis I present my findings on the use of a calcium ionophore to activate human oocytes artificially. An algorithm based on Karyomapping (termed MeioMapping) is demonstrated for the first time specifically to investigate human female meiosis. By recovering all three products of human female meiosis (oocyte, and both polar biopsies – herein termed “Trios”) using calcium ionophore, I present a novel protocol (commissioned by Nature Protocols) to allow exploration of the full extent of meiotic chromosome recombination and segregation that occurs in the female germline. Finally I present a published set of experiments using this protocol to provide new insight into meiotic segregation patterns and recombination in human oocytes. This work uncovers a previously undescribed pattern of meiotic segregation (termed Reverse Segregation), providing an association between recombination rates and chromosome mis-segregation (aneuploidy). This work demonstrates that there is selection for higher recombination rates in the female germline and that there is a role for meiotic drive for recombinant chromatids at meiosis II in human female meiosis. The work presented in this thesis provides deeper understanding of meiotically derived maternal aneuploidy and recombination. More importantly it provides a vehicle within an ethical framework to continue to expand our knowledge and uncover new insights into the basis of meiotic errors that may aid future reproductive therapies.

No description available.

Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT-2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis.