Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Characterization and cytotoxicity studies of thiol-modified polystyrene microbeads doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I)

Halide octahedral molybdenum clusters [(Mo6X8)L6]n- possess luminescence properties that are highly promising for biological applications. These properties are rather dependent on the nature of both the inner ligands X (i.e. Cl, Br, or I) and the apical organic or inorganic ligands L. Herein, the luminescence properties and the toxicity of thiol-modified polystyrene microbeads (PS-SH) doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I) were studied and evaluated using human epidermoid larynx carcinoma (Hep2) cell cultures. According to our data, the photoluminescence quantum yield of (Mo6I8)@PS-SH is significantly higher (0.04) than that of (Mo6Cl8)@PS-SH (6Br8)@PS-SH (6X8)@PS-SH showed that all three types of doped microbeads had no significant effect on the viability and proliferation of the cells.

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Reactions of copper macrocycles with antioxidants and HOCl:potential for biological redox sensing

A series of simple copper N(2)S(2) macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.

Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper.

Characterization of Transglutaminase 2 in macrophage clearance of apoptotic cells

Removal of dead or diseased cells is crucial feature of apoptosis for managing many biological processes such as tissue remodelling, tissue homeostasis and resolution and control of immune responses throughout life. Tissue transglutaminase (TG2) is a protein crosslinking enzyme that has been implicated in apoptotic cell clearance but also mediates many important cell functions including cell adhesion, migration and monocyte-macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4, ß1 and ß3 integrin. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise extracellular role of TG2 in apoptotic cell clearance remains ill-defined. This thesis addresses macrophage TG2 in cell corpse clearance. TG2 expression (cytosolic and cell surface) in human macrophages was revealed and data demonstrate that loss of TG2 activity through the use of inhibitors of function, including cellimpermeable inhibitors significantly inhibit the ability of macrophages to clear apoptotic cells (AC). This includes reduced macrophage recruitment to and binding of apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus it defines for the first time a role for TG2 activity at the cell surface of human macrophages in multiple stages of AC clearance and proposed that TG2, in association with heparan sulphates, may exert its effect on AC clearance via crosslinking of CD44.

Isopeptidase activity of human transglutaminase 2:disconnection from transamidation and characterization by kinetic parameters

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 -driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

Chemical characterization of soil organic matter in an oligotrophic, subtropical, freshwater wetland system: Sources, diagenesis and preservation

Freshwater wetland soils of the Everglades were studied in order to assess present environmental conditions and paleo-environmental changes using organic geochemistry techniques. Organic matter in dominant vegetation, peat and marl soils was characterized by geochemical means. Samples were selected along nutrient and hydrology gradients with the objective to determine the historical sources of organic matter as well as the extent of its preservation. Effective molecular proxies were developed to differentiate the relative input of organic matter from different biological sources to wetland soils. Thus historical vegetation shifts and hydroperiods were reconstructed using those proxies. The data show good correlations with historical water management practices starting at the turn of the century and during the mid 1900's. Overall, significant shortening of hydroperiods during this period was observed. The soil organic matter (SOM) preservation was assessed through elemental analysis and molecular characterizations of bulk 13C stable isotopes, solid state 13C NMR spectroscopy, UV-Vis spectroscopy, and tetramethyl ammonium hydroxide (TMAH) thermochemolysis-GC/MS. The relationship of the environmental conditions and degradation status of the soil organic matter (SOM) among the sites suggested that both high nutrient levels and long hydroperiod favor organic matter degradation in the soils. This is probably the result of an increase in the microbial activity in the soils which have higher nutrient levels, while longer hydroperiods may enhance physical/chemical degradation processes. The most significant transformations of biomass litter in this environment are controlled by very early physical/chemical processes and once the OM is incorporated into surface soils, the diagenetic change, even over extended periods of time is comparatively minimal, and SOM is relatively well preserved regardless of hydroperiod or nutrient levels. SOM accumulated in peat soils is more prone to continued degradation than the SOM in the marl soils. The latter is presumably stabilized early on through direct air exposure (oxidation) and thus, it is more refractory to further diagenetic transformations such as humification and aromatization reactions.

Characterization of L-Serine Deaminase and its Potential Role in the Pathogenicity of Pseudomonas aeruginosa

All pathogens require high energetic influxes to counterattack the host immune system and without this energy bacterial infections are easily cleared. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. Recent evidence has linked L-SD directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. Genome sequence analysis revealed the presence of two L-SD homo logs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains of sdaA (PAOsdaA) and sdaB (PAOsdaB) with their isogenic parent P. aeruginosa P AO 1. We demonstrated that P. aeruginosa is unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine and this glycine-dependent induction of L-SD activity requires the inducer serine. The amino acid leucine was shown to inhibit L-SD activity from both SdaA and SdaB and the net contribution to L-serine deamination by SdaA and SdaB was ascertained at 34% and 66 %, respectively. These results suggest that P. aeruginosa LSD is quite different from the characterized E. coli L-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as a sole carbon source were also isolated and in addition, suicide vectors were constructed which allow for selective mutation of the sdaA and sdaB genes on any P. aeruginosa strain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

Characterization of Atlantic Cod Spawning Habitat and Behavior in Icelandic Coastal Waters

The physical habitat used during spawning may potentially be an important factor affecting reproductive output of broadcast spawning marine fishes, particularly for species with complex, substrate-oriented mating systems and behaviors, such as Atlantic cod Gadus morhua. We characterized the habitat use and behavior of spawning Atlantic cod at two locations off the coast of southwestern Iceland during a 2-d research cruise (15–16 April 2009). We simultaneously operated two different active hydroacoustic gear types, a split beam echosounder and a dual frequency imaging sonar (DIDSON), as well as a remotely operated underwater vehicle (ROV). A total of five fish species were identified through ROV surveys: including cusk Brosme brosme, Atlantic cod, haddock Melanogrammus aeglefinus, lemon sole Microstomus kitt, and Atlantic redfish Sebastes spp. Of the three habitats identified in the acoustic surveys, the transitional habitat between boulder/lava field and sand habitats was characterized by greater fish density and acoustic target strength compared to that of sand or boulder/lava field habitats independently. Atlantic cod were observed behaving in a manner consistent with published descriptions of spawning. Individuals were observed ascending 1–5 m into the water column from the bottom at an average vertical swimming speed of 0.20–0.25 m s−1 and maintained an average spacing of 1.0–1.4 m between individuals. Our results suggest that cod do not choose spawning locations indiscriminately despite the fact that it is a broadcast spawning fish with planktonic eggs that are released well above the seafloor.

Characterization of a Novel Mouse Phenotype with Marked Hydrocephalus

In multigenic diseases, disorders where mutations in multiple genes affect the expressivity of the disease, genetic interactions play a major role in prevalence and phenotypic severity. While studying the genetic interactions between Pax3 and EdnrB in the melanocyte lineage, a new phenotype was noted in 80% of Pax3 mutants that we believe to be a novel murine model for hydrocephalus. Hydrocephalus, an accumulation of cerebrospinal fluid in the cranial cavity due to obstruction of flow in and out of the cavity, is one of the most common birth defects surpassing Down syndrome. Characteristic to hydrocephalus is a "domed" head appearance, expansion of the ventricles of the brain, and loss of neurons with hyperproliferation of glial cell types all three of which were seen in the mutant mice. The phenotype also consisted of craniofacial deformities coupled with skeletal defects including, but not limited to kyphosis, lordosis, and an apparent shortening of the some limbs. For the cellular analysis of the hydrocephalus phenotype, brains were removed and stained with two antibodies: Glial Fibrillary Acidic Protein (GFAP) and Neurofilament (NF), which are astrocyte- and neuron- specific respectively. A higher number of cells expressing GF AP and a lower number of cells expressing NF were seen in the mutant brain, when compared to control. For skeletal deformity analysis, affected mice skeletons were stained with Alizarin Red and Alcian Blue showing no apparent difference in ossification. Future genetic analysis of these mutant mice has the potential to identify novel gene modifiers involved in the promotion of this particular phenotype.

Characterization of the Anti-Quorum Sensing Activity Exhibited by Marine Macro algae of South Florida

Quorum sensing is a communication mechanism employed by many bacteria. The bacteria secrete signal molecules known as acyl homoseriene lactones (AHLs) that cue to population size/density. Bacteria can be alerted of this optimum population by the concentration of these signal molecules. When the concentration of AHLs exceed a threshold valve, they enter the bacterial cell and causes the transcription of genes encoding virulence factors necessary for their colonization and survival. The marine algae Delise a pulchra, found off the coast of Australia is thought to produce compounds that inhibit the activity of the AHLs. The algae employ these compounds, known as furanones, as an anti-fouling agent. We postulated that marine algae of South Florida might contain similar activity; we screened 30 different algal species and found 22 species had the activity. Algal extracts were made from Halimeda incrassata using hexane, chloroform, ethyl acetate and methanol as solvents. The extracts were assayed for anti-quorum sensing activity. The results showed many of the South Florida green algae to possess anti-quorum sensing activity, however extracts of H incrassata did not show quorum sensing inhibition.

Characterization of dynamic speckle sequences using principal component analysis and image descriptors

Speckle is being used as a characterization tool for the analysis of the dynamic of slow varying phenomena occurring in biological and industrial samples. The retrieved data takes the form of a sequence of speckle images. The analysis of these images should reveal the inner dynamic of the biological or physical process taking place in the sample. Very recently, it has been shown that principal component analysis is able to split the original data set in a collection of classes. These classes can be related with the dynamic of the observed phenomena. At the same time, statistical descriptors of biospeckle images have been used to retrieve information on the characteristics of the sample. These statistical descriptors can be calculated in almost real time and provide a fast monitoring of the sample. On the other hand, principal component analysis requires longer computation time but the results contain more information related with spatial-temporal pattern that can be identified with physical process. This contribution merges both descriptions and uses principal component analysis as a pre-processing tool to obtain a collection of filtered images where a simpler statistical descriptor can be calculated. The method has been applied to slow-varying biological and industrial processes

Isolation and Characterization of Microsatellite DNA Markers in the Deep-Sea Amphipod Paralicella tenuipes by Illumina MiSeq Sequencing

Funding This work was supported by the HADEEP projects, funded by the Nippon Foundation, Japan (2009765188), the Natural Environmental Research Council, UK (NE/E007171/1) and the Total Foundation, France. We acknowledge additional support from the Marine Alliance for Science and Technology for Scotland (MASTS) funded by the Scottish Funding Council (Ref: HR09011) and contributing institutions. We also acknowledge support from the Leverhulme Trust to SBP. Additional sea time was supported by NIWA’s ‘Impact of Resource Use on Vulnerable Deep-Sea Communities’ project (CO1_0906)

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel.