Species accumulation curves and their applications in parasite ecology

Species accumulation curves (SACs) chart the increase in recovery of new species as a function of some measure of sampling effort. Studies of parasite diversity can benefit from the application of SACs, both as empirical tools to guide sampling efforts and predict richness, and because their properties are informative about community patterns and the structure of parasite diversity. SACs can be used to infer interactivity in parasite infra-communities, to partition species richness into contributions from different spatial scales and different levels of the host hierarchy (individuals, populations and communities) or to identify modes of community assembly (niche versus dispersal). A historical tendency to treat individual hosts as statistically equivalent replicates (quadrats) seemingly satisfies the sample-based subgroup of SACs but care is required in this because of the inequality of hosts as sampling units. Knowledge of the true distribution of parasite richness over multiple host-derived and spatial scales is far from complete but SACs can improve the understanding of diversity patterns in parasite assemblages.

Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Writing and characterisation methods for UV-photosensitivity based devices and their applications

The underlying work to this thesis focused on the exploitation and investigation of photosensitivity mechanisms in optical fibres and planar waveguides for the fabrication of advanced integrated optical devices for telecoms and sensing applications. One major scope is the improvement of grating fabrication specifications by introducing new writing techniques and the use of advanced characterisation methods for grating testing. For the first time the polarisation control method for advanced grating fabrication has successfully been converted to apodised planar waveguide fabrication and the development of a holographic method for the inscription of chirped gratings at arbitrary wavelength is presented. The latter resulted in the fabrication of gratings for pulse-width suppression and wavelength selection in diode lasers. In co-operation with research partners a number of samples were tested using optical frequency domain and optical low coherence reflectometry for a better insight into the limitations of grating writing techniques. Using a variety of different fabrication methods, custom apodised and chirped fibre Bragg gratings were written for the use as filter elements for multiplexer-demultiplexer devices, as well as for short pulse generation and wavelength selection in telecommunication transmission systems. Long period grating based devices in standard, speciality and tapered fibres are presented, showing great potential for multi-parameter sensing. One particular scope is the development of vectorial curvature and refractive index sensors with potential for medical, chemical and biological sensing. In addition the design of an optically tunable Mach-Zehnder based multiwavelength filter is introduced. The discovery of a Type IA grating type through overexposure of hydrogen loaded standard and Boron-Germanium co-doped fibres strengthened the assumption of UV-photosensitivity being a highly non-linear process. Gratings of this type show a significantly lower thermal sensitivity compared to standard gratings, which makes them useful for sensing applications. An Oxford Lasers copper-vapour laser operating at 255 nm in pulsed mode was used for their inscription, in contrast to previous work using CW-Argon-Ion lasers and contributing to differences in the processes of the photorefractive index change

The biotechnological applications of transglutaminases

Transglutaminases catalyse a diverse range of reactions leading to the modification of proteins and peptides such that their physical, chemical and biological properties become changed. They are found in many different living organisms and as a consequence display subtle differences in their biochemical and physical properties. it is therefore not surprising that this group of enzymes have been exploited as applied biocatalysts in a wide range of commercial sectors varying from the textile industry to the highly lucrative cosmetic industry. in addition the pathophysiological importance of this group of enzymes has increased significantly over the last decade with their involvement noted in a number of human diseases. As a consequence their identification as therapeutic targets or as monitoring aids for a range of different diseases has caused significant interest from the diagnostics and pharmaceutical industries. This review describes some of the current applications of transglutaminases; together with their potential strategic importance and future uses.

Novel cationic polymers for use at biological interfaces

One of the main problems with the use of synthetic polymers as biomaterials is the invasion of micro-organisms causing infection. A study of the properties of polymeric antibacterial agents, in particular polyhexamethylene biguanide, has revealed that the essential components for the design of a novel polymeric antibacterial are a balance between hydrophilicity and hydrophobicity coupled with sites of cationicity. The effect of cation incorporation on the physical properties of hydrogels has been investigated. Hydrogel systems copolymerised with either N-vinyl imidazole or dimethylaminoethyl methacrylate have been characterised in terms of their water binding, mechanical and surface properties. It has been concluded that the incorporation of these monomers does not adversely affect the properties of such hydrogels and that these materials are potential candidates for further development for use in biomedical applications. It has been reported that hydro gels with ionic character may increase the deposition of biological material onto the hydrogel surface when it is in contact with body fluids. An investigation into the deposition characteristics of hydrogels containing the potentially cationic monomers has been carried out, using specific protein adsorption and in vitro spoilation techniques. The results suggest that at low levels of cationicity, the deposition of positively charged proteins is reduced without adversely affecting the uptake of the other proteins. The gross deposition characteristics were found to be comparable to some commercially available contact lens materials. A preliminary investigation into the development of novel antibacterial polymers has been completed and some novel methods of bacterial inhibition discussed. These methods include development of an hydrogel whose potential application is as a catheter coating.

The development and applications of the combined use of NMR and ultrasound

The objective of the research carried out in this report was to observe the first ever in-situ sonochemical reaction in the NMR Spectrometer in the megahertz region of ultrasound. Several reactions were investigated as potential systems for a sonochemical reaction followed by NMR spectroscopy. The primary problem to resolve when applying ultrasound to a chemical reaction is that of heating. Ultrasound causes the liquid to move and produces 'hot spots' resulting in an increase in sample temperature. The problem was confronted by producing a device that would counteract this effect and so remove the need to account for heating. However, the design of the device limited the length of time during which it would function. Longer reaction times were required to enable observations to be carried out in the NMR spectrometer. The fIrst and most obvious reactions attempted were those of the well-known ultrasonic dosimeter. Such a reaction would, theoretically, enable the author to simultaneously observe a reaction and determine the exact power entering the system for direct comparison of results. Unfortunately, in order to monitor the reactions in the NMR spectrometer the reactant concentrations had to be signifIcantly increased, which resulted in a notable increase in reaction time, making the experiment too lengthy to follow in the time allocated. The Diels-Alder Reaction is probably one of the most highly investigated reaction systems in the field of chemistry and it was this to which the author turned her attention. Previous authors have carried out ultrasonic investigations, with considerable success, for the reaction of anthracene with maleic anhydride. It was this reaction in particular that was next attempted. The first ever sonochemically enhanced reaction using a frequency of ultrasound in the megahertz (MHz) region was successfully carried out as bench experiments. Due to the complexity of the component reactants the product would precipitate from the solution and because the reaction could only be monitored by its formation, it was not possible to observe the reaction in the NMR spectrometer. The solvolysis of 2-chloro-2-methylpropane was examined in various solvent systems; the most suitable of which was determined to be aqueous 2-methylpropan-2-ol. The experiment was successfully enhanced by the application of ultrasound and monitored in-situ in the NMR spectrometer. The increase in product formation of an ultrasonic reaction over that of a traditional thermal reaction occurred. A range of 1.4 to 2.9 fold improvement was noted, dependent upon the reaction conditions investigated. An investigation into the effect of sonication upon a large biological molecule, in this case aqueous lysozyme, was carried out. An easily observed effect upon the sample was noted but no explanation for the observed effects could be established.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

Proteoglycan analogues for ophthalmic and orthopaedic applications

The successful development of biomaterials is increasingly influenced by biomimesis essentially the use of biological structures as design templates. This approach has been used here in the design of injectable systems for the nucleus of the intervertebral disc, corneal inlays and intraocular lenses (IOLs). The strategy is based on the use of C-linked sulphonates to mimic the O-linked sulphate groups that are the hydration drivers in proteoglycans. The elastic modulus of the materials can be tailored for specific applications.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

Solvability and asymptotics of the heat equation with mixed variable lateral conditions and applications in the opening of the exocytotic fusion pore in cells

We investigate a mixed problem with variable lateral conditions for the heat equation that arises in modelling exocytosis, i.e. the opening of a cell boundary in specific biological species for the release of certain molecules to the exterior of the cell. The Dirichlet condition is imposed on a surface patch of the boundary and this patch is occupying a larger part of the boundary as time increases modelling where the cell is opening (the fusion pore), and on the remaining part, a zero Neumann condition is imposed (no molecules can cross this boundary). Uniform concentration is assumed at the initial time. We introduce a weak formulation of this problem and show that there is a unique weak solution. Moreover, we give an asymptotic expansion for the behaviour of the solution near the opening point and for small values in time. We also give an integral equation for the numerical construction of the leading term in this expansion.

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.