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>

abstract: One of the fundamental questions in molecular biology is how genes and the control of their expression give rise to so many diverse phenotypes in nature. The mRNA molecule plays a key role in this process as it directs the spatial and temporal expression of genetic information contained in the DNA molecule to precisely instruct biological processes in living organisms. The region located between the STOP codon and the poly(A)-tail of the mature mRNA, known as the 3′Untranslated Region (3′UTR), is a key modulator of these activities. It contains numerous sequence elements that are targeted by trans-acting factors that dose gene expression, including the repressive small non-coding RNAs, called microRNAs. Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored. This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

abstract: The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators. / Dissertation/Thesis / Masters Thesis Biology 2018

<p> Behavior in higher eukaryotes is a complex process which integrates signals in the environment, the genetic makeup of the organism, and connectivity in the nervous system to produce extremely diverse adaptations to the phenomenon of existence. Unraveling the subcellular components that contribute to behavioral output is important for both understanding how behavior occurs in an unperturbed state, as well as understanding how behavior changes when the underlying systems that generate it are altered. Of the numerous molecular species that make up a cell, the regulation of messenger RNAs (mRNAs), the coding template of all proteins, is of key importance to the proper maintenance and functioning of cells of the brain, and thus the synaptic signals and information integration which underlie behavior. RNA binding proteins, a class of regulatory molecules, associate with mRNAs and facilitate their maturation from pre-spliced nascent transcripts, their stabilization and degradation ensuring appropriate levels are maintained, as well as their translation and subcellular compartmentalization, which ensures that proteins are translated at the appropriate level and in the places where they are required to fulfill their cellular functions. Our laboratory identified polymorphisms in the gene coding for the CUGBP and ELAV-like Factor 6 (CELF6) RNA binding protein to be associated with Autism Spectrum Disorder risk in humans. ASD is a spectrum of disorders of early neurodevelopment which present with lowered sociability and communication skills as well as restricted patterns of interests. When expression of the <i>Celf6</i> gene was ablated in mice, we found that they exhibited reductions to early communication as well as altered aspects of their exploratory behavior. In this dissertation, I explore the communication changes in young mouse pups with loss of CELF6 protein and identify that despite being able to produce vocalization patterns similar to their wild-type littermates, they nevertheless exhibit reduced response to maternal separation. Despite a history of literature on other CELF family proteins, the functions of the CELF6 protein in the brain have not been previously described. I provide characterization of the mRNA binding targets of CELF6 in the brain, and show that they share common UGU-containing sequence motifs which has been noted for other CELF proteins, and that CELF6 binding occurs primarily in the 3' untranslated regions (3' UTR) of mRNA. I hypothesized that this mode of interaction would result in regulation of mRNA degradation or translation efficiency as 3' UTR regions are known for providing binding sites for numerous regulators of such processes. In order to answer this question, I cloned sequence elements from the 3' UTRs of target mRNAs into a massively parallel reporter assay which has enabled me to test the effect of CELF6 expression on hundreds of binding targets simultaneously. When expressed in vitro, I found that CELF6 induced reduction to reporter library levels but exhibited few effects on translation efficiency, and I was able to rescue effects to reporter abundance mutation of binding motifs. Intriguingly, like CELF6, CELF3, CELF4, and CELF5 were all able to produce the same effect. CELF5 and CELF6 both showed similar, intermediate repression of reporter library mRNAs, while CELF3 and CELF4 exerted the strongest levels of repression. The level of repression under these conditions was somewhat predicted by number of motifs present per element, however a large amount of the variance in reporter levels is still unexplained and a mechanism for CELF6's action is unknown. Nevertheless, the work I present in this dissertation shows that CELF6 and other members of its family are key regulators of mRNA abundance levels which has direct implications to downstream consequence in the cell. As several of CELF6 binding target mRNAs are known regulators of neuronal signaling and synaptic function, the information I present is crucial for future experimentation. This work well help lead us to understand how behavior is altered when this protein is absent, along the way uncovering important mechanistic steps connecting the molecular landscape of cells to the behavior of organisms.</p><p>

Mechanisms of antioxidant action of zinc dialkvldithio­ phosphate (ZnDRP) and several structurally related compounds (basic zinc dialkyldithiophosphate (b-ZnDRP), dialkylthio-phosphoryl disulphide (DRDS), dialkyldithiophosphoric acid (DRDPA), dialkylthiophosphoric acid (DRTPA)) were studied in both the presence and absence of a soluble iron catalyst (iron (III) stearate (FeST) ). Oxygen absorption studies in two hydrocarbon substrates, white mineral oil and decalin, show that each of the above thiophosphoryl compounds is an effective inhibitor of oxidation at 130°C in the absence of FeST. In the presence of FeST, however, their effectiveness is severely reduced and a higher concentration of antioxidant is required to provide stabilisation against oxidation. The initial presence of hydroperoxide is shohl1 to have a marked effect on the antioxidant activity of each of the above thiophosphoryl compounds in both the presence and absence of FeST. All the above compounds are shown to be capable of decomposing cumene hydroperoxide (CHP) in both the presence and absence of FeST at 110°C. The long term stabilisation against hydrocarbon oxidation provided by the above thio-phosphoryl compounds in the presence of added CHP may be attributed to ionic hydroperoxide decomposition, promoted by sulphur containing acids formed as a result of the oxidation of the parent additive molecule by CHP. Although reaction with FeST may lead to the removal of some of the sulphur acids, hydroperoxide decomposition is shown to still be occurring in the presence of soluble iron. Of the above thiophosphoryl compounds, only DRDPA gives iron (III) dialkyldithiophosphate (FeDRP) on reaction with FeST. In the absence of CHP, FeDRP was shown to be an inhibitor of hydrocarbon oxidation. In the presence of CHP, however, FeDRP becomes inactive due to free radical decomposition of the hydroperoxide by the iron complex. FeDRP is shown to be an effective hydroperoxide decomposer even when present in small amounts, the mechanism of decomposition depending on the initial CHP:FeDRP ratio. The initial homolytic decomposition of hydroperoxide is caused by the FeDRP itself, but the subsequent ionic reactions are associated with the oxidation product(s) of the iron complex.