BBSRC Research Studentships

Aging is associated with increased cardiovascular risk and vascular dysfunction. Age-dependent changes in the permeability of intestinal tissue through decreased intestinal mucosal thickness or increased reactive species may contribute to altered intestinal drug absorption. Also, impaired blood flow, ischemic changes and the increased use of NSAIDs naturally contribute to an impaired epithelial barrier in elderly patients.

Recent studies have suggested that increased gut permeability promoting systemic inflammation can contribute to atherosclerosis. We hypothesise that increased translocation of gut microbiota associated with aging, may contribute to the decrease in vascular function also associated with aging.

Through a collaborative effort with Royal Lancaster Infirmary and the School of Graduate Entry Medicine and Health, Derby, we propose to test our hypothesis by measuring vascular function in mesenteric arteries from patients (average age 72 yrs) undergoing colon resections by myography and investigating if there is any relationship between these responses and markers of gut inflammation and permeability. Vascular function will be assessed in small resistance arteries (diameter 500-1000 µM) by measuring vasoconstrictor responses to adrenaline, angiotensin, endothelin and vasopressin. Vasorelaxant function will be tested by measuring responses to acetylcholine, bradykinin, prostacyclin and sodium nitroprusside. Markers of gut permeability will be serum levels of LPS and other endotoxins measured within the same patients and compared to different age groups of patients identified in GI clinics and healthy volunteers.

Exposure to environmental ultraviolet radiation (UV) can cause a number of short- and long-term negative effects in human skin, including erythema (burning), skin cancer and ageing. These harmful effects can be mitigated by the application of sunscreens, with the effectiveness of these being expressed in terms of a sun protection factor (SPF) related to the UV dose required for erythema.

Longer wavelength UV (UVA; 315-400 nm) was until recently comparatively neglected in terms of its health impact and it is still the case that many sunscreens do not provide significant protection against this type of radiation. UVA exposure does not result in erythema but is able to cause skin cancer and is also considered to be the major player in UV-induced skin ageing. The standard method for assaying the effectiveness of sunscreens therefore does not adequately evaluate the protection offered against UVA. Moreover, ongoing research in our laboratories and others have shown that the cellular response to UVA is significantly different from the response to shorter wavelength UVB, and that established biomarkers of UV exposure (e.g. p53 activation) may underestimate the damage caused by UVA.

The aim of the project will be to evaluate biomarkers for UVA exposure in a biologically relevant in vitro test system to be developed as part of the project. Work carried out by Prof McMillan and Dr Allinson and also in collaboration with Boots has identified several potential short- and long-term biomarkers for UVA damage. These will be evaluated for robustness using different skin cell culture models.

Scanning probe microscopy (SPM) is one of the cornerstones of modern day nanotechnology and is used to obtain high information content images of biological structures often impossible to obtain using alternative techniques, ultimately with molecular (or even atomic) resolution.

The present project will explore and compare the use of three different SPM methods for studies into the mechanics, dynamics and stages of the assembly of polypeptides into small oligomers (early aggregates, 5-6 nm diameter) and amyloid fibres (~10 nm diameter). Currently, more than 30 different polypeptides are known to form such structures when incubated in aqueous solution in vitro. We will focus on three polypeptides (Aβ, amylin and α synuclein) that are already under investigation in Prof. Allsop's laboratory. We will also examine the effects of key external factors (e.g. the presence of metal ions) on oligomer and fibre formation. We envisage that the project will establish artefact-free techniques for studying mechanics and self-assembly of polypeptides, and will elucidate nanoscale mechanisms of formation of amyloid fibres.

SPM techniques to be employed will be: (i) non-contact mode, (ii) ultrasonic force mode (UFM), and (iii) underliquid imaging mode with ultrasound. The major focus of the project will be on the development and application of SPM ultrasonic methods including UFM, since this is a novel technique with potential improvements in resolution and contrast that has been pioneered by Dr. Kolosov and has not been used previously for this type of study. This approach allows direct imaging of completely novel aspect of protein nanostructure - namely, the nanomechanical interaction between the nanometre scale protein subunits and fibres. Our preliminary results indicate that this would allow us to obtain novel information on the assembly of amyloid fibres, as well as to establish a novel approach applicable more widely to investigation of biomacromolecular structures.

It is certain that the actions of some genes promote the process of ageing or allow it to occur. While some gene expression increases or decreases throughout adult life, we don't know which genes affect ageing. In the model organism Drosophila, expression changes over time in some genes have been correlated with actual biological age, suggesting the genes are involved in the ageing process, either causally or consequentially. Some which have ageing-relevant human homologues associated with age-related diseases and we have acquired transgenic strains to study their effects on ageing.

Other ageing-relevant genes will be sought by interrogating a large dataset of gene expression during ageing in the fly using statistical methods designed specifically for analysis of time-course microarray experiments. This will be combined with a range of bioinformatics sources from flies to humans, to search for patterns indicating higher level control genes, or groups of genes, which may substantially modulate the ageing process. Once identified, these genes will be investigated by creating and testing specific transgenic fly strains.

Using a genetic tool called Geneswitch we can alter gene expression levels at times of our choosing; in this case during adulthood only, thus mimicking drug treatment. We will restore expression of a selection of these genes to young adult levels, aiming to slow ageing without negative impacts on general fitness. Genes so identified may indicate mechanisms important to the ageing process in humans, further the understanding of causes of the negative effects of ageing, and suggest possible drug targets.

Recently, collagen cross-linking induced by combined riboflavin/UV has proven effective in the treatment of some corneal diseases such as keratoconus. This treatment strengthens the collagen fibrils in the cornea by increased cross-linking between collagen molecules. These cross-links are believed to be approximately 1.4 nanometers in size. Preliminary results also show that cross linking may slow down the spread of bacterial infections possibly by increasing resistance to bacterial enzymatic degradation. However the mechanism by which this occurs is poorly understood. This proposal would involve a basic study of the formation of UV induced crosslinks using photosensitisers.

It would involve looking at the most efficient delivery of photosensitisers such as riboflavin and β-Nitro alcohols into the collagen fibrils. Including the use of nano-carriers to facilitate delivery

The optimum wavelength cross-linking agent and UV dosage to obtain optimum cross-linking with minimal cell toxicity would also be determined. UV dosage and transmission will be monitored using Lancaster's well equipped UV dark room, which has been successfully used for UV research with a range of micro-organisms and human cell lines. These include a range of equipment for providing defined treatments in terms of irradiance and wavelength, for example temperature controlled lamp arrays from broad-band UV-A or UV-B treatments. Measurement will use our two double monochromator scanning spectroradiometers which give the ability to quantify spectral irradiance between 240 and 800nm with bandwidth of 2nm and a resolution of 0. 25nm.

Measurement of cross-links within the collagen fibrils will be assessed using FTIR and Raman spectroscopy. Electron microscopy would be used to monitor crosslink density along individual fibrils. The mechanical properties of the collagen fibrils would be determine using force curve atomic force microscopy. The project would also make use of the in vitro organ culture system. The effect of UV cross-linking on bacterial spread in the cornea would be investigated.

This is a fundamental study of the manipulation of collagen cross-linking with a UV optical field. The results from this would have ultimate applications in the tissue engineering and nanotechnology fields.