The molecular basis of modern marker chemistry

This thesis focuses on empirical investigations and refinements of immunohistochemical marker chemistry to gain insights into the design of novel markers for light and electron microscopy. In Chapter 2, incorporation of d-block metals into polymerised biphenyl-3,3′,4,4′-tetramine (polyDAB) identified complexes of Ni(II), Pt(II), Pt(IV) and Au(III) to be powerful catalysts of silver reduction from physical developers. Na2S(aq) treatment increased the range and activity of catalytic complexes, allowing previously invisible immunohistochemical deposits of polyDAB to be clearly seen in diagnostically relevant samples. Chapter 3 refined this technique by manipulating reagent concentrations whilst suppressing tissue argyrophilia, increasing immunohistochemical sensitivity by an order of magnitude. Marker deposition and thus amplification, was dependent on conjugate quality and coupling method. In Chapter 4, scanning and transmission electron microscopy identified 8 d-block metals that increased the electron opacity of polyDAB, including W(VI), Os(VIII), Pt(II) and Au(III). The majority were detectable by energy dispersive X-ray analysis (EDX), but were present in insufficient quantities for use in analytical electron microscopical tomography (AEMT). In Chapter 5, immunohistochemical polymerisation of halogenated aromatic diamines and bis-diamines as AEMT markers was investigated. The 16 compounds studied produced deposits of varying properties and compositions, morphological criteria identifying those of 1,2-diamino-4-bromobenzene and 1,2-diamino-4,5-diiodobenzene as suitable candidates; EDX indicated that the latter might be applicable to AEMT. Chapter 6 investigated silver deposition from a physical developer by photoconversion. Photo-excitation of immunofluorescently-stained tissue sections in the presence of physical developer caused selective silver deposition at immunopositive sites, a novel method that might find application in AEMT. In Chapter 7, characterisation of polyDAB revealed a molecular weight range of 600 to over 100,000; IR spectra were consistent with an indamine- or phenazine-like polymer. Poor solubility restricted further characterisation. In Chapter 8, additional applications of halogenated compounds were investigated and results suggested potential applications in biological research and diagnosis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The molecular basis of modern marker chemistry

This thesis focuses on empirical investigations and refinements of immunohistochemical marker chemistry to gain insights into the design of novel markers for light and electron microscopy. In Chapter 2, incorporation of d-block metals into polymerised biphenyl-3,3′,4,4′-tetramine (polyDAB) identified complexes of Ni(II), Pt(II), Pt(IV) and Au(III) to be powerful catalysts of silver reduction from physical developers. Na2S(aq) treatment increased the range and activity of catalytic complexes, allowing previously invisible immunohistochemical deposits of polyDAB to be clearly seen in diagnostically relevant samples. Chapter 3 refined this technique by manipulating reagent concentrations whilst suppressing tissue argyrophilia, increasing immunohistochemical sensitivity by an order of magnitude. Marker deposition and thus amplification, was dependent on conjugate quality and coupling method. In Chapter 4, scanning and transmission electron microscopy identified 8 d-block metals that increased the electron opacity of polyDAB, including W(VI), Os(VIII), Pt(II) and Au(III). The majority were detectable by energy dispersive X-ray analysis (EDX), but were present in insufficient quantities for use in analytical electron microscopical tomography (AEMT). In Chapter 5, immunohistochemical polymerisation of halogenated aromatic diamines and bis-diamines as AEMT markers was investigated. The 16 compounds studied produced deposits of varying properties and compositions, morphological criteria identifying those of 1,2-diamino-4-bromobenzene and 1,2-diamino-4,5-diiodobenzene as suitable candidates; EDX indicated that the latter might be applicable to AEMT. Chapter 6 investigated silver deposition from a physical developer by photoconversion. Photo-excitation of immunofluorescently-stained tissue sections in the presence of physical developer caused selective silver deposition at immunopositive sites, a novel method that might find application in AEMT. In Chapter 7, characterisation of polyDAB revealed a molecular weight range of 600 to over 100,000; IR spectra were consistent with an indamine- or phenazine-like polymer. Poor solubility restricted further characterisation. In Chapter 8, additional applications of halogenated compounds were investigated and results suggested potential applications in biological research and diagnosis

LR White sections as slot grid support films for transmission electron microscopy

The utility of LR White sections as slot grid support films for the examination of thin resin-embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar-carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar-carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coatin

The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach

The bacterial nitroreductase NfnB has been the focus of a great deal of research for its use in directed enzyme prodrug therapy in combination with the nitroreductase prodrug CB1954 with this combination of enzyme and prodrug even entering clinical trials. Despite some promising results, there are major limitations to this research, such as the fact that the lowest reported Km for this enzyme far exceeds the maximum dosage of CB1954. Due to these limitations, new enzymes are now being investigated for their potential use in directed enzyme prodrug therapy. One such enzyme that has proved promising is the YfkO nitroreductase from Bacillus Licheniformis. Upon investigation, the YfkO nitroreductase was shown to have a much lower Km (below the maximum dosage) than that of NfnB as well as the fact that when reacting with the prodrug it produces a much more favourable ratio of enzymatic products than NfnB, forming more of the desired 4-hydroxylamine derivative of CB1954

Microneedle-assisted transfersomes as a transdermal delivery system for aspirin

Transdermal drug delivery systems offer several advantages over conventional oral or hypodermic administration due to the avoidance of first-pass drug metabolism and gastrointestinal degradation as well as patients’ convenience due to a minimally invasive and painless approach. A novel transdermal drug delivery system, comprising a combination of transfersomes with either solid silicon or solid polycarbonate microneedles has been developed for the transdermal delivery of aspirin. Aspirin was encapsulated inside transfersomes using a “thin-film hydration sonication” technique, yielding an encapsulation efficiency of approximately 67.5%. The fabricated transfersomes have been optimised and fully characterised in terms of average size distribution and uniformity, surface charge and stability (shelf-life). Transdermal delivery, enhanced by microneedle penetration, allows the superior permeation of transfersomes into perforated porcine skin and has been extensively characterised using optical coherence tomography (OCT) and transmission electron microscopy (TEM). In vitro permeation studies revealed that transfersomes enhanced the permeability of aspirin by more than four times in comparison to the delivery of unencapsulated “free” aspirin. The microneedle-assisted delivery of transfersomes encapsulating aspirin yielded 13-fold and 10-fold increases in permeation using silicon and polycarbonate microneedles, respectively, in comparison with delivery using only transfersomes. The cytotoxicity of different dose regimens of transfersomes encapsulating aspirin showed that encapsulated aspirin became cytotoxic at concentrations of ≥100 μg/mL. The results presented demonstrate that the transfersomes could resolve the solubility issues of low-water-soluble drugs and enable their slow and controlled release. Microneedles enhance the delivery of transfersomes into deeper skin layers, providing a very effective system for the systemic delivery of drugs. This combined drug delivery system can potentially be utilised for numerous drug treatments

Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis

Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ