Pronounced opsonic activity has been demonstrated in the coelomic fluid of the Oligochaete Eisenia foetida. This humoral factor enhances the uptake of particles by Eisenia phagocytes by 94% in the case of S. cerevisiae. 31.5% inE.coli and by 34.6% in B.megaterium. In order to determine whether agglutinin molecules are responsible for this activity, the 20,000D haemagglutinin was purified. This agglutinin was found to be heat sensitive, (15 min at 60°C destroying activity completely), partially dependent on calcium ions and with an optimal pH range of 5-8. The agglutinin was found to be active towards a range of vertebrate erythrocytes and various fungal and bacterial cells but is not responsible for opsonic activity. Polyclonal antibodies to this purified agglutinin were raised in rabbits and used in immunocytochemical studies to determine the source of this molecule. It was found that the cell membranes of Eisenia phagocytes bear agglutinin or agglutinin like molecules and their possible roles in internal defence mechanisms is discussed. Eisenia coelomocytes were found to release agglutinins in vitro though the site of synthesis of these molecules is yet to be established.

Two molecular forms I and II (separable by gel filtration) from mature Vicia faba seeds have been studied. The extractability of the enzymes and the in vitro conversion of the low MW form, II, to the larger oligomer, I has been examined over a range of salt concentrations. Specific and total activities of the preparations were high when strong salt solutions were used for extraction. It would appear that α-galactosidase I, in comparison with II, is best extracted from the seeds if solutions of high ionic strength are used. The effects of these salt solutions on the relative levels of the two forms have been investigated by gel filtration. Interpretation of the gel elution profiles obtained is, however, complicated by the in vitro conversion of form II to form I, which is favoured by high salt concentrations and some routine procedures in the purification of α-galactosidases, such as ammonium sulphate fractionation. The relationships between the multiple forms have been studied. α-galactosidase II can be resolved into enzymes II1 and II2 by CM-cellulose chromatography, a-Gal actosidases I, II1 and II2 have been highly purified. Form I (MW 160,000) is a tetramer of enzyme II2 as shown by SDS-PAGE. Immunological studies on the three forms have been carried out and the evidence suggests that they are structurally related, although forms I and II2 are more closely related than forms I and II. On hydrolysis,monosaccharides are released from the three purified enzymes, suggesting they are glycoproteins. The three α-galactosidases also agglutinate red blood cells indicating that they possess lectin activity.

Certain aspects of the flagella and flagellar proteins of selected members of the Proteus and Providence groups have been compared. Proteus flagella, negatively stained and examined in the electron microscope, presented both "beaded" and "lined" surface appearance. Structures which possibly represent cross sections of flagella were observed; these show two groups of three sub-units with a slight gap between the triplets. Reaggregation of flagellin occurred in the presence of high concentrations of salt. Purified flagellins of P. vulgaris, P. mirabilis, P. morganii and P. rettgerii and the Providence group gave single bands when electro-phoresed in starch or polyacrylamide gels. Comparison of the mobility of purified flagellins revealed great differences, even between those flagellins isolated from strains of a single accepted species. In addition, tryptic peptide maps of purified flagellins each had a distinctive pattern. Molecular weights of flagellins of the four Proteus species were assessed by electrophoresis jui polyacrylamide gels containing SDS; in all cases values of about 40,000 were obtained. Anri no acid analysis revealed the presence of N-methyl lysine and histidine; neither have been reported conclusively in this group before. N-methyl lysine was restricted to flagellins from certain P. morganii strains while histidine was found in approximately half of the flagellins examined, irrespective of species. The C terminal amino acid of all the flagellins examined is arginine; the C terminal sequence is ser - leu - leu - arg COOH. In every case alanine is the common N terminal amino acid. Seven out of thirty-three of the peptides found on tryptic peptide maps of P. vulgaris NCTG 100 20 flagellin have been completely or partially sequenced. The possibilities of using cyanogen bromide to cleave Proteus flagellin were investigated; citraconylation and maleylation were both extremely effective in keeping the resultant cyanogen bromide fragments in solution. The results are discussed with regard to inter-relationships between the species and the possible uses of these results in the taxonomy of the genus Proteus and the Providence group.

<p> Evolution&mdash;the great tinkerer&mdash;has produced the astounding diversity of form within and between existing species. It is a fundamental goal of evolutionary biology to understand the origin of such diversity. What types of genes underlie evolved changes in morphology? Are certain types of mutations (notably changes within regulatory regions) more likely to be used to produce adaptive changes in form? When distinct populations evolve similar morphological changes, are the underlying genetic bases changes to the same genes, the same genetic pathways, or largely independent? Are changes in form modular, or are their concerted changes to multiple developmentally similar organs? The ever cheapening cost of sequencing, coupled the availability of high-quality reference genomes, allows high-throughput approaches to identifying the loci of evolution. The emergence of a robust genome engineering system, CRISPR/Cas9, allows for efficient and direct testing of a gene's phenotype. Combining both of these techniques with a model system with naturally evolved phenotypic variation, the threespine stickleback, allows for systems-level answers to the many evolutionary questions. </p><p> Chapter one outlines the field of evolutionary developmental biology. It proposes two alternative viewpoints for thinking about the evolution of form. The first is the view of the `Modern Synthesis', linking Mendelian inheritance with Darwinian natural selection, which explains evolution as the change in allele frequencies over time. The second views evolution through the lens of deep homology, focusing on changes to developmental programs over time, even across related organs within the same animal. It then introduces key concepts within evolutionary and developmental biology, including <i> cis</i>-regulation of gene expression, and gene regulatory networks. It then provides examples of evolution reusing similar gene regulatory networks, including <i>Hox</i> genes, <i>Pax6</i> dependent eye initiation, and ectodermal placode development. Teeth use highly conserved signaling pathways, during both their initiation and replacement. Threespine sticklebacks <i>Gasterosteus aculeatus</i> have repeatedly adapted following a shift from marine to freshwater environments, with many independently derived populations sharing common morphological traits, including a gain in tooth number. The following chapters investigate this gain in tooth number in multiple distinct populations of sticklebacks. </p><p> Chapter two describes the discovery and mapping of a spontaneous stickleback albino mutation, named <i>casper</i>. <i>casper</i> is a sex-linked recessive mutation that results in oculocutaneous albinism, defective swim bladders, and blood clotting defects. Bulked segregant mapping of <i> casper</i> mutants revealed a strong genetic signal on chromosome 19, the stickleback X chromosome, proximal to the gene <i>Hps5</i>. <i> casper</i> mutants had a unique insertion of a G in the 6^th exon on <i>Hps5</i>. As mutants in the human orthologue of <i> Hps5</i> resulted in similar albino and blood clotting phenotypes, <i> Hps5</i> is a strong candidate underlying the <i>casper</i> phenotype. Further supporting this model, genome editing of <i>Hps5</i> phenocopied <i>casper</i>. Lastly, we show that <i>casper</i> is an excellent tool for visualizing the activity of fluorescent transgenes at late developmental stages due to the near-translucent nature of the mutant animals. </p><p> Chapter three details the fine mapping of a quantitative trail locus (QTL) on chromosome 21 controlling increases in tooth number in a Canadian freshwater stickleback population. Recombinant mapping reduced the QTL-containing region to an 884kb window. Repeated QTL mapping experiments showed the presence of this QTL on multiple, but not all, wild derived chromosomes from the Canadian population. Comparative genome sequencing revealed the perfect correlation with genetic data of ten variants, spanning 4.4kb, all within the 4<i> th</i> intron of the gene <i>Bmp6</i>. Transgenic analysis of this intronic region uncovered its role as a robust tooth enhancer. TALEN induced mutations in <i>Bmp6</i> revealed required roles for the gene in stickleback tooth development. Finally, comparative RNA-seq between <i> Bmp6</i> wild-type and mutant dental tissue showed a loss of mouse hair stem cell genes in <i>Bmp6</i> mutant fish teeth, suggesting deep homology of the regeneration of these two organs. </p><p> Chapter four investigates the evolved changes in gene expression that accompany evolved increases in tooth number in two distinct freshwater populations. Independently derived stickleback populations from California and Canada have both evolved increases in tooth number, and previous work suggested that these populations used distinct genetic changes during their shared morphological changes. RNA-seq analysis of dental tissue from both freshwater populations compared to marine revealed a gain in critical regulators of tooth development in both freshwater populations. These evolved changes in gene expression can be partitioned in <i>cis</i> changes (mutations within regulatory elements of a gene) and trans changes (changes to the overall regulatory environment) using phased RNA-seq data from marine-freshwater F1 hybrids. Many genes show evidence for stabilizing selection of expression levels, with <i>cis </i> and <i>trans</i> changes in opposing directions (Abstract shortened by ProQuest.). </p><p>

Caleosins were originally described as one of the two major protein components of storage lipid bodies in the seeds of higher plants, the other being oleosins. In contrast with oleosins, caleosins have a single calcium-binding EF-hand domain plus several potential phosphorylation sites and have been hypothesised as playing a role in lipid-body formation and possibly mobilisation in seeds. In Arabidopsis, there are six functional caleosin genes, two of which encode seed-specific proteins while the other isoforms are expressed in a variety of vegetative and reproductive tissues. More recently, seed caleosins have been shown to have a peroxygenase activity but the function of this was uncertain. This study describes the characterisation of a specific membrane-bound caleosin isoform, termed Clo-3 in Arabidopsis and Brassica spp, that appears to be present in all plant tissues and is responsive to a variety of biotic and abiotic stresses. Bioinformatic analysis reveals that similar caleosin-like genes/proteins are present in all vascular plants, as well as in nonvascular plants such as mosses, and even in single-celled algae. Intriguingly, caleosin-like genes are also present in the genomes of most fungi described to date, with the surprising exception of the yeasts. In order to understand the function of caleosins in plants, a detailed structural and functional analysis of this novel class of protein is reported here. Biochemical studies demonstrate that the Clo-3 isoform binds calcium (one atom per molecule), can be phosphorylated most likely, by a casein kinase 2 (CK2) protein kinase, and has putative peroxygenase activity. In addition to biochemical data, microscopy analysis shows that Clo-3 may be located both on the endoplasmic reticulum and chloroplastid envelope membranes. specifically the chloroplast envelope. Biochemical evidence of cell membrane localisation is also presented. Protease digestion experiments show that the membrane bound Clo-3 has a Type I transmembrane orientation, where its N-terminal domain faces the lumen of microsomes while the C-terminal is on the cytosolic face. Such an orientation is common for receptors or proteins that may be activated by signalling molecules. The Clo-3 gene and its encoded protein are each upregulated by salt and drought stresses and by abscisic acid (ABA) treatment. Reverse genetics using RNAi knockdown mutants demonstrate specific transcription factors involved in regulating Clo-3 during different stresses. Peroxygenase activities of Clo-3 enriched microsomes were higher following salt stress. Although the data is representative of potentially many peroxygenases, it does provide indirect evidence that Clo-3 abundance increases and/or catalytic activity is induced during stress. The study also presents evidence of the response of Clo-3 to biotic stress and related signalling molecules. Arabidopsis Clo-3 is highly responsive to the phytohormone salicylic acid, to the salicylic acid synthetic analogue DCINA, the biotic signalling molecule hydrogen peroxide, and to infection by the common fungal pathogen of Brassicas, Leptosphaeria maculans (Phoma), while experiments utilising the non-expressor of pathogenesis related protein 1 (npr1) knockout mutant plant demonstrates Clo-3 response to salicylic acid (SA) is chiefly via npr1 translocation to the nucleus. The type of peroxygenase epitomised by Clo-3 is similar to those involved in the formation of epoxy alcohols from fatty acid hydroperoxides. The latter are a class of oxylipins that are seen in fungal infection, and also play a role in various aspects of fungal spore development including sporulation and a role in cuticle synthesis. As such, Clo-3 in Arabidopsis and possibly similar caleosins in other species might play roles in oxylipin signalling pathways that are involved in a protective role during both biotic and abiotic stress responses.

Various aspects of microtubule nucleation and organization have been investigated m Drosophila and MDCK cells. Drosophila indirect fibrillar flight muscles (IFMs) do not possess centriole-containing centrosomes. The ends of both microtubules and developing myofibrils closely associate with specialized cell junctions called myotendon junctions which are located at the ends of developing muscle cells. Antisera raised against pericentrin, a protein involved in microtubule nucleation, stain myotendon junctions throughout myofibrillogenesis. The antisera also stain the centrosome/spindle poles of Drosophila embryos and cultured Drosophila epithelial cells. Hence, microtubules may be nucleated at the myotendon junctions of IFMs rather than by sites associated with nuclear envelopes as in vertebrate muscle cells. Incipient MDCK daughter cells form an extremely short intercellular bridge. This contains the mid-body and ends of two mid-body associated microtubule bundles. Eventually the two microtubule bundles completely lose contact with the mid-body. These microtubules may have been severed. Consequently, the furrow base can constrict around the mid-body to complete daughter cell separation. A non-affinity purified y-tubulin antiserum stains an antigen closely associated with both sides of the mid-body. This antigen is not y-tubulin. The antigen appears to be associated with a recently discovered mitotic organelle (the telophase disc). Taxol induced microtubule arrays contain 'chains' of microtubules in interphase neuroblast cells of Drosophila third instar larvae. Adjacent microtubules in these 'chains' appear to share several protofilaments. Preliminary results indicate that microtubules of taxol induced asters are composed of 12 rather than the normal 13 protofilaments in MDCK cells. The centrosome may nucleate and release microtubules in interphase MDCK cells. y-Tubulin is only concentrated at the centrosome of control and taxol-incubated interphase MDCK cells. Microtubule ends do not focus at the centrosome in either case. The centrosomally-associated microtubule array increases substantially in size from anaphase to late telophase. At late telophase the array spreads throughout each incipient daughter cell. Subsequently, the array ceases to focus at the centrosome. y-Tubulin and pericentrin initially concentrate at the centre of two of the several taxol-induced microtubule asters in each mitotically arrested MDCK cell. Subsequently, y-tubulin and pericentrin are concentrated at two closely associated discrete spherical sites. These sites are not associated with microtubule asters. This may represent a very clear example of microtubule release from the centrosomes in these cells.

The effect of photolysis of nitroprusside was investigated in both frog ventricular trabeculae and rabbit ear arterial strips. Unphotolysed nitroprusside failed to elicit any effect on frog ventricular twitch tension. However, upon photolysis it had a potent negative inotropic action. The extent of twitch depression was shown to depend on the degree of photolysis. It was postulated that these effects are due to a labile physiologically active photolytic product. This was positively identified as nitric oxide. Preliminary results of the negative inotropic action of thiols and synthesised nitrosothiols are also presented. In contrast to frog ventricle, intact nitroprusside does exert a relaxing effect on precontracted mammalian smooth muscle. This effect is markedly potentiated by photolysis. It is concluded that the mechanism of action of nitroprusside on both tissues involves the release of nitric oxide which is postulated to activate guanylate cyclase. This suggests that mammalian vascular smooth muscle has a mechanism for degrading nitroprusside which is absent in frog ventricle.

<p> Hyper-O-GlcNAcylation of proteins is a subsequent artifact of metabolic disorder and is indicative of many cancers, including cancers of the female reproductive tract. While the incidence of most cancer types has been declining in the U.S., endometrial and cervical cancer remain among the most common cancers diagnosed in women. Diabetic women have a 2-3 fold increased risk of developing endometrial cancer, and tend to have more aggressive cases of cervical cancer, however, the molecular aspects of these risks are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between diabetes and tumorigenicity in cancers of the female reproductive tract. The cervical cancer cell line (SiHa) and the endometrial cancer cell line (Ishikawa) were utilized to study the effect of dysregulation of O-GlcNAcylation on the proliferation, migration, invasion, and related molecular mechanisms. In cervical cancer, O GlcNAcylation was found to be an important regulator of tumorigenicity. Overall, inhibition of O-GlcNAcylation (via the inhibitor, OSMI-1) in SiHa cells impaired cell proliferation (p&lt;0.01) and invasion (p&lt;0.01) yet did not affect cell cycle progression. These effects occurred concomitantly with an alteration of cellular morphology, principally the disruption/decline of K8/18 and &beta;-actin filament expression. The results suggest O-GlcNAcylation regulates several aspects of tumorigenesis in cervical cancer cells, and cytoskeletal proteins are among the targets. Similarly, in endometrial cancer cells, hyper-O-GlcNAcylation (via 1&mu;M Thiamet-G/ThmG or 25mM Glucose) enhanced the expression of EMT-associated genes (WNT5B and FOXC2), and the E-Cadherin suppressor, Snail. Reorganization of actin filaments into stress filaments, consistent with EMT, was also noted in ThmG-treated cells. Interestingly, Hypo-O GlcNAcylation (via 50 &mu;M OSMI-1) also upregulated WNT5B, inferring that any disruption to O-GlcNAc cycling impacts EMT. However, Hypo-O-GlcNAcylation reduced cellular proliferation/migration and the expression of the pro-EMT genes (AHNAK, TGFB2, FGFBP1, CALD1, TFPI2). Finally, Ishikawa cells were used to investigate the effect hyper-O GlcNAcylation on the efficacy of progesterone (P4) in therapy for endometrial cancer proliferation and invasion. Ishikawa cells were exposed to ThmG, to induce hyper-O GlcNAcylation, and 100nM P4. P4 alone, significantly decreased cell proliferation, however, the addition of ThmG, and subsequent hyper-O-GlcNAcylation, negated this affect, returning the cells to control level proliferation (p&lt;0.05). A similar pattern was noted in Matrigel invasion assays, where Hyper-O-GlcNAcylation augmented invasion compared to P4 treatment alone, both with and without progesterone treatment (p&lt;0.05). Progesterone treatment has been shown to induce the expression of p21 and p27, reducing cell growth. In this study, P4 maintained p21 expression and increased p27 expression, however, ThmG decreased p27 expression and the expression of endogenous progesterone receptor B (PR B) despite P4 treatment. These results suggest that hyper-O-GlcNAcylation, common in obese and diabetic patients, may promote tumorigenicity in female cancers and could impair the efficacy of progesterone treatment. O GlcNAcylation has the potential to serve as a biomarker for early diagnosis and could predict treatment success. O-GlcNAc cycling enzyme inhibitors could prove to be useful tools for providers when treating cancer patients with metabolic disorders.</p><p>

<p> Advances in next-generation sequencing and algorithm design have revealed the evolutionarily conserved expression of large numbers of circular RNAs (circRNAs) that are endogenous to eukaryotic cells, many being abundant and in some cases the exclusive output of a given gene. CircRNAs are produced when the pre-mRNA splicing machinery &ldquo;backsplices&rdquo; to join a 3&rsquo; downstream splice donor to a 5&rsquo; upstream splice acceptor. Their circular nature gives them superior resistance to exonucleases and extended half-lives when compared to linear RNAs. CircRNAs are produced in a regulated manner to carry out specific cellular functions, as supported by the identification of alternative splicing factors required for circularization. A generalized biological function for circRNAs has not been unequivocally identified. Aberrantly expressed circRNAs may contribute to tumorigenesis, maintenance or progression of cancer cells. In particular, altered circRNA can contribute to important aspects of melanoma biology, and even harbor prognostic and/or therapeutic value. This work is the first comprehensive, unbiased identification and characterization of circRNAs in melanoma, focusing particularly on circARID1A, a gained circRNA when comparing melanoma to non-transformed melanocytes. Silencing of either linear (protein coding) or circular ARID1A in melanoma cells results in impaired proliferation and distinct transcriptional outputs. SERBP1 was identified as an interacting partner of circARID1A, and as a potential mediator of its function. These studies shed light on the roles of circRNAs in cancer and their mechanisms of action, setting the path for future work on these RNA species in other malignancies.</p><p>

<p> Biophysical and biochemical imbalance of mechanisms relevant to muscle function, can result in morphological changes to the tissue. While the purpose of activities involving exercise is to modify the shape and size of skeletal muscle, and the length of these muscles allows wide ranges of stiffness and stretch to be applied, cardiac tissue is not meant to change much. However, stressful extrinsic factors (poor diet, chemotherapy, etc) or intrinsic factors like inherited mutations in muscle functioning genes can result in a myopathy or a disease of the muscle. In fact, another biological process that requires much compliance of many molecules is embryogenesis. Although the timeline of an embryonic structure is limited, compared to an adult heart and muscle composition, continuous and coordinated movement is essential, but cumulative, prolonged disruptions can be harmful. At the core of muscle biology is the myosin molecule which is a motor protein that hydrolyzes ATP, binds to actin, and the spatial dynamics of its function (contraction-relaxation) alter the length of muscle. Myosin cyclically follows specific steps and undertakes well-defined structural conformations during these events, but mutations can alter the time and stability of any of these aspects. In this thesis I did a comprehensive analysis of the ATPase cycle parameters for both embryonic and cardiac myosin and studied the effects of specific associated or linked mutations have on function. The multiple mutations were in the interest of cataloging common features and defects to identify mechanistic patterns. In a collaborative effort I also used these wet-lab measurements to simulate the cycle using a working kinetic model for the myosin ATPase cycle. We have found distinct differences between three different myopathies that will be discussed in the following chapters.</p><p>