Towards novel luminescent probes for monitoring β- galactosidase activity

This thesis describes efforts made towards the synthesis of a biologically stable, luminescent molecular probe, which could be used to investigate in vivo the processing of sugars by β-galactosidases. To this end, a lactose-based probe was designed, featuring a Lanthanide held within a chelate and appended to the glucosyl unit, and a proximal naphthyl moiety, attached to the galactose unit, which would function as a sensitiser for luminescence. A β-galactosidase enzyme from B. circulans was chosen to carry out the investigation. A number of novel methyl glucosides, functionalised with a naphthyl moiety at C6 of the sugar, were prepared. These were then used as glycosyl acceptors to make disaccharides (lactose analogues), with the enzyme (functioning in reverse) catalysing the glycosylation. The enzymatic reaction was optimised by varying the amount of enzyme, the reaction pH, the ratio of glycosyl acceptor to donor, the reaction temperature, concentration and solvent mixture. The optimal conditions were found to be a 0.4 M reaction solution at pH 7.0 with 20% acetonitrile, a 7:1 ratio of glycosyl acceptor to donor, 19.6 U of enzyme per mmol of acceptor, and a reaction temperature of 30°C. The resulting disaccharide products exhibited unusual regioselectivity for the β-galactosidase from B. circulans, with unexpected β(1→3) and β(1→2) glycosidic linkages being formed. In an effort to increase the efficiency of the process of identifying suitable substrates for the enzyme, a dynamic combinatorial chemistry approach was also explored. This used disulfide bonds to attach the naphthyl moiety to the methyl glucoside using linkers of different lengths. From this library, the enzyme successfully processed the novel disulfide GlcOMe-S-S-CH$_2$Np as a glycosyl acceptor with p-nitrophenyl galactose as the glycosyl donor. This resulted in a novel disaccharide featuring a naphthyl group attached via a disulfide bond to the glucosidic residue

A Guide for Policy, Practice and Patients on Wellbeing and Sickle Cell Disorder (SCD)

This guide is based on research examining the shielding experiences of people with sickle cell disorders (SCD) and parents of children with the condition during the COVID-19 pandemic. The aim was to improve NHS services for this population group. Services have duties under the Equality Act 2010 to ensure equity and tackle health inequalities. Since SCD disproportionately affects Black, Asian and Minority Ethnic (BAME) communities, there are also duties not to engage in direct or indirect racist discrimination, nor in harassment or victimization. It is important that anti-racist and anti-bias training is offered in all NHS services and cultural competency encouraged amongst all staff. Additionally, that conditions affecting the BAME population, like SCD, become a mandatory part of all nursing and medical educational and NHS training programmes

Microbial use of low molecular weight DOM in filtered and unfiltered freshwater:Role of ultra-small microorganisms and implications for water quality monitoring

Dissolved organic matter (DOM) plays a central role in regulating productivity and nutrient cycling in freshwaters. It is therefore vital that we can representatively sample and preserve DOM in freshwaters for subsequent analysis. Here we investigated the effect of filtration, temperature (5 and 25 °C) and acidification (HCl) on the persistence of low molecular weight (MW) dissolved organic carbon (DOC), nitrogen (DON) and orthophosphate in oligotrophic and eutrophic freshwater environments. Our results showed the rapid loss of isotopically-labelled glucose and amino acids from both filtered (0.22 and 0.45 μm) and unfiltered waters. We ascribe this substrate depletion in filtered samples to the activity of ultra-small (< 0.45 μm) microorganisms (bacteria and archaea) present in the water. As expected, the rate of C, N and P loss was much greater at higher temperatures and was repressed by the addition of HCl. Based on our results and an evaluation of the protocols used in recently published studies, we conclude that current techniques used to sample water for low MW DOM characterisation are frequently inadequate and lack proper validation. In contrast to the high degree of analytical precision and rigorous statistical analysis of most studies, we argue that insufficient consideration is still given to the presence of ultra-small microorganisms and potential changes that can occur in the low MW fraction of DOM prior to analysis

Microbial uptake kinetics of dissolved organic carbon (DOC) compound groups from river water and sediments

Dissolved organic matter (DOM) represents a key component of carbon (C) cycling in freshwater ecosystems. While the behaviour of bulk dissolved organic carbon (DOC) in aquatic ecosystems is well studied, comparatively little is known about the turnover of specific DOC compounds. The aim of this study was to investigate the persistence of 14C-labelled low molecular weight (LMW) DOC at a wide range of concentrations (0.1 µM to 10 mM), in sediments and waters from oligotrophic and mesotrophic rivers within the same catchment. Overall, rates of DOC loss varied between compound groups (amino acids > sugars = organic acids > phenolics). Sediment-based microbial communities contributed to higher DOC loss from river waters, which was attributed, in part, to its greater microbial biomass. At higher DOC compound concentrations, DOC loss was greater in mesotrophic rivers in comparison to oligotrophic headwaters. A lag-phase in substrate use within sediments provided evidence of microbial growth and adaptation, ascribed here to the lack of inorganic nutrient limitation on microbial C processing in mesotrophic communities. We conclude that the higher microbial biomass and available inorganic nutrients in sediments enables the rapid processing of LMW DOC, particularly during high C enrichment events and in N and P-rich mesotrophic environments

The indignities of shielding during the COVID-19 pandemic for people with sickle cell disorders: an interpretative phenomenological analysis

This article seeks to understand the first-hand experiences of people with sickle cell, a recessively inherited blood disorder, who were identified as clinically extremely vulnerable during the COVID-19 pandemic. Part of a larger sequential mixed-methods study, this article uses a selective sample of eight qualitative semi-structured interviews, which were analysed using interpretative phenomenological analysis (IPA). The first stage of IPA focused on practical concerns participants had correlated to understanding shielding and their feelings about being identified as clinically extremely vulnerable. In a secondary stage of analysis, we examined the emotions that it brought forth and the foundations of those based on discriminations. This article adds to our theoretical understanding of embodiment and temporality with respect to chronicity and early ageing. It explains how people with sickle cell disorders have an embodied ethics of crisis and expertise. It also elucidates how people’s experiences during the pandemic cannot be seen in void but illustrates ableism, racism, and ageism in society writ large

Variation in dissolved organic matter (DOM) stoichiometry in UK freshwaters:Assessing the influence of land cover and soil C:N ratio on DOM composition

Dissolved organic matter (DOM) plays an important role in freshwater biogeochemistry. To investigate the influence of catchment character on the quality and quantity of DOM in freshwaters, forty-five sampling sites draining subcatchments of contrasting soil type, hydrology and land cover within one large upland-dominated and one large lowland-dominated catchment, were sampled over a one-year period. Dominant land cover in each subcatchment included: arable and horticultural, blanket peatland, coniferous woodland, improved-, unimproved-, acid- and calcareous-grasslands. The composition of the C, N, and P pool was determined as a function of the inorganic nutrient species (NO3-, NO2-, NH4+, PO43-) and dissolved organic nutrient (DOC, DON and DOP) concentrations. DOM quality was assessed by calculation of the molar DOC:DON and DOC:DOP ratios and specific ultraviolet absorbance (SUVA254). In catchments with little anthropogenic nutrient inputs, DON and DOP typically comprised >80% of the TDN and TDP concentrations. By contrast, in heavily impacted agricultural catchments DON and DOP typically comprised 5-15% of TDN and 10-25% of TDP concentrations. Significant differences in DOC:DON and DOC:DOP ratios were observed between land cover class with significant correlations observed between both the DOC:DON and DOC:DOP molar ratios and SUVA254 (rs = 0.88 and 0.84, respectively). Analysis also demonstrated a significant correlation between soil C:N ratio and instream DOC:DON/DOP (rs = 0.79 and 0.71 respectively). We infer from this that soil properties, specifically the C:N ratio of the soil organic matter pool, has a significant influence on the composition of DOM in streams draining through these landscapes

Land cover and nutrient enrichment regulates low-molecular weight dissolved organic matter turnover in freshwater ecosystems

Dissolved organic matter (DOM) is a complex mixture of carbon-containing compounds. The low-molecular weight (LMW) fraction constitutes thousands of different compounds and represents a substantial proportion of DOM in aquatic ecosystems. The turnover rates of this LMW DOM can be extremely high. Due to the challenges of measuring this pool at a molecular scale, comparatively little is known of the fate of LMW DOM compounds in lotic systems. This study addresses this knowledge gap, investigating the microbial processing of LMW DOM across 45 sites representing a range of physicochemical gradients and dominant land covers in the United Kingdom. Radioisotope tracers representing LMW dissolved organic carbon (DOC) (glucose), dissolved organic nitrogen (DON) (amino acid mixture), dissolved organic phosphorus (DOP) (glucose-6-phosphate), and soluble reactive phosphorus (SRP, measured as orthophosphate) were used to measure the microbial uptake of different DOM compounds in river waters. The amount of DOM biodegradation varied between different components (DON ≥ DOC > DOP), with the rate of turnover of all three increasing along a gradient of N and P enrichment across the range of sites. Conversely, the uptake of SRP decreased along this same gradient. This was ascribed to preferential utilization of DOP over SRP. Dominant land cover had a significant effect on DOM use as a resource, due to its control of nutrient enrichment within the catchments. We conclude that nutrient enrichment of river waters will lead to further DOM removal from the water column, increased microbial growth, and a decrease in stream oxygen saturation, exacerbating the effects of eutrophication in rivers

Black Sickle Cell Patients’ Lives Matter: Healthcare, long- term shielding and psychological distress during a racialized pandemic in England – a mixed-methods study

Open accessObjective: To understand the psychological and social impact of shielding on people with sickle cell disorders and their carers in the Midlands region of England. This region was badly affected during the pandemic, with the city of Birmingham having some of the highest rates of COVID-19 deaths. Design: A mixed methods project with a quantitative survey on shielding and adapted SF36 v 2 questionnaire which was supplemented by qualitative semi-structured interviews analysed using Interpretive Phenomenological analysis (IPA). Participants: Fifty-one participants who were predominantly of Black Caribbean or Black African heritage anonymously took part in the online survey. We supplemented this with eight in-depth semi-structured interviews with adults with sickle cell disorders using IPA. Results: The adapted SF36 v2 survey indicated worse quality of life and mental health. The open-ended questions from the adapted survey also identified shielding concerns about hospital care, pain management and knowledge of sickle cell by healthcare professionals. From the interviews it emerged that the racialized element of the pandemic caused significant psychological distress for a population group that had to regularly access hospitals. It was noted that psychological health needs both during a pandemic and outside of it were poorly understood and became invisible in services. The psychological impact of experiences of hospital care as well as growing up with an invisible chronic condition were important to understand psychologically