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Abstract: The essential trace metals iron, zinc, and copper play important roles both in retinal physiology and disease. They are involved in various retinal functions such as phototransduction, the visual cycle, and the process of neurotransmission, being tightly bound to proteins and other molecules to regulate their structure and/or function or as unbound free metal ions. Elevated levels of “free” or loosely bound metal ions can exert toxic effects, and in order to maintain homeostatic levels to protect retinal cells from their toxicity, appropriate mechanisms exist such as metal transporters, chaperones, and the presence of certain storage molecules that tightly bind metals to form nontoxic products. The pathways to maintain homeostatic levels of metals are closely interlinked, with various metabolic pathways directly and/or indirectly affecting their concentrations, compartmentalization, and oxidation/reduction states. Retinal deficiency or excess of these metals can result from systemic depletion and/or overload or from mutations in genes involved in maintaining retinal metal homeostasis, and this is associated with retinal dysfunction and pathology. Iron accumulation in the retina, a characteristic of aging, may be involved in the pathogenesis of retinal diseases such as age-related macular degeneration (AMD). Zinc deficiency is associated with poor dark adaptation. Zinc levels in the human retina and RPE decrease with age in AMD. Copper deficiency is associated with optic neuropathy, but retinal function is maintained. The changes in iron and zinc homeostasis in AMD have led to the speculation that iron chelation and/or zinc supplements may help in its treatment.

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Abstract: It has been proposed that increased tissue iron concentration, which has been observed in certain regions of the brain in individuals with Alzheimer’s Disease (AD), could provide a marker for diagnosis through detection with MRI. This is investigated in this thesis using high field MRI to examine post mortem human brain tissue. It is shown here that by using data from multiple brain regions discriminant analysis can successfully differentiate between AD and control samples, even when no statistically significant differences are observed in individual brain regions. A unique set of complementary techniques was used to investigate iron content, R2 and R2* of tissue samples from the caudate nucleus, putamen, globus pallidus substantia nigra, amygdala and pons, from a set of three control and AD cases. The particulate iron content of the samples was investigated by SQUID magnetometry and was followed by iron quantification. A trend of increased particulate and total iron concentration was observed in the AD tissue compared to control, however this did not reach statistical significance in any brain region. High resolution MRI relaxometry at 9.4 T was carried out on tissue from the caudate nucleus, putamen, globus pallidus and substantia nigra using a custom design Bruker micro-imaging MicWB40 probe. As part of the work towards this PhD the probe was tested, and MRI relaxometry protocols optimised for high resolution (86 x 86 μm in plane, 150 μm slice thickness) mapping of tissue samples with high iron concentration. Again, no statistically significant differences were observed between AD and control tissue. However, discriminant analysis of these data (particulate or total iron or R2 or R2*) from multiple brain regions achieved differentiation between control and AD cases with 100% sensitivity and specificity for this small sample set. This demonstrates the potential clinical usefulness of MRI of measurements of non-haem brain iron to aid in disease diagnosis. Synchrotron X-ray fluorescence (SXRF) mapping of 30 μm thick sections, cut from the MRI samples, showed the relative concentration distribution of iron, copper and zinc in one AD and control sample from each brain region. Each metal was shown to have a distinct distribution. In particular, the inhomogeneity of iron concentration within individual brain regions, such as the putamen, was demonstrated. This may explain the wide variation in iron concentration reported in the literature for the same brain regions, and highlights the importance of close anatomical matching of samples when making comparisons. The ability of high resolution SXRF mapping to investigate the metal content within individual cells was demonstrated and used to show an increase in iron in individual AD neurons, in addition to the surrounding grey and white matter tissue. Spatially matched SXRF and MRI maps were used to demonstrate a strong, statistically significant linear relationship between tissue iron concentration and R2, R2* and R2’ at 9.4 T. The gradient of the linear relationship between iron and R2, agrees extremely well with the predicted gradient at this field, where the prediction was made by Vymazal et al. (1996) using MRI relaxometry at 0.05 to 1.5 T. To the best of my knowledge, this is the first time that this relationship has been quantified at 9.4 T, or at any field above 7 T. MRI at 14.1 T was carried out on low iron concentration regions (the pons and amygdala). Matched SXRF and R2* maps did not show a strong linear relationship between iron and R2*. The iron concentration in these regions is less than 50 μg/g and it was concluded that in tissue with low iron content, other tissue properties - such as water content - are dominating the value of R2*. This result was replicated with data measured at 9.4 T, when only tissue with an iron concentration of less than 50 μg/g was considered.

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Abstract: Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss. These data suggest that regions affected by α-synuclein pathology may display enhanced vulnerability and cell loss if Cu-dependent protective mechanisms are compromised. Additional investigation of copper pathology in PD may identify novel targets for the development of protective therapies for this disorder.

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Abstract: This review gives a brief description of the theory and application of X-ray absorption spectroscopy (XAS) comprising both X-ray absorption near-edge structure and extended X-ray absorption fine structure, especially focusing onto the use in inorganic medicinal chemistry. The advantages and limitations of the methods are described and showcased through examples from studies on anticancer metal compounds. The strength of XAS and its relevance for the structural chemical characterization of metal-based compounds in vivo in native tissue samples is exemplified. The common data reduction and analysis strategies are presented and recent advances in the field and perspectives for the near future are given.

Open Access

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Abstract: In experiments preliminary to the design of an x-ray excited optical luminescence (XEOL) based chemical mapping tool we have used x-ray micro (4.5 „¿m „ªƒn5.2 „¿m) and macro (1 mm „ªƒn6 mm) beams with similar total fluxes to assess the effects of a high flux density beam of xrays at energies close to an absorption edge on inorganic surfaces in air. The near surface composition of corroded cupreous alloys was analysed using parallel x-ray and optical photoemission channels to collect x-ray absorption near-edge structure (XANES) data at the Cu-K edge.