Isolation and characterisation of CML and normal quiescent haemopoietic stem cells

The work contained in this thesis was aimed at isolation of quiescent stem cells from both normal and CML samples and characterisation of the properties of these rare populations. To isolate these specific cell populations, enrichment of the stem cell fraction was achieved by selection methods based on the stem cell marker CD34. Positive or negative CD34 selection yielded up to 99% pure CD34+ cell populations. A fluorescent activated cell-sorting (FACS) strategy was developed to allow isolation of quiescent and cycling stem cells. Using Hoechst (HST) and Pyronin Y (Py) to stain the DNA and RNA respectively and gating on the viable, Propidium lodide- (PI)-, CD34+ population, it was possible to isolate viable stem cells in G0, G1 and S/G2/M phases of the cell cycle. Success of the sorting strategy was demonstrated by PCR analysis of differential cell cycle gene expression in the sorted populations. Further development of FACS methodology allowed the division history and stem cell status, as defined by CD34 expression, to be monitored under a range of conditions. Using this experimental strategy, it was shown that a number of CML stem cells were able to survive exposure to concentrations of ST1 in excess of those achievable in vivo, and that the surviving cells were undivided or quiescent. In an effort to further characterise quiescent stem cells, the HST/Py FACS strategy was used to isolate quiescent and cycling stem cell populations from both normal and CML samples, which were then processed for microarray studies. The results have shown a differential gene expression profile between normal G0 and cycling cells that fully validates the sorting strategy. The most significant different gene expression was in a set of chemokine genes, which were up-regulated in both normal and CML G0 cells compared to cycling cells. This has not been previously reported and represents a novel finding. Additional data analysis is ongoing and may, in time, yield therapeutic targets for CML disease eradication

Neuronal Firing Sensitivity to Morphologic and Active Membrane Parameters

Both the excitability of a neuron's membrane, driven by active ion channels, and dendritic morphology contribute to neuronal firing dynamics, but the relative importance and interactions between these features remain poorly understood. Recent modeling studies have shown that different combinations of active conductances can evoke similar firing patterns, but have neglected how morphology might contribute to homeostasis. Parameterizing the morphology of a cylindrical dendrite, we introduce a novel application of mathematical sensitivity analysis that quantifies how dendritic length, diameter, and surface area influence neuronal firing, and compares these effects directly against those of active parameters. The method was applied to a model of neurons from goldfish Area II. These neurons exhibit, and likely contribute to, persistent activity in eye velocity storage, a simple model of working memory. We introduce sensitivity landscapes, defined by local sensitivity analyses of firing rate and gain to each parameter, performed globally across the parameter space. Principal directions over which sensitivity to all parameters varied most revealed intrinsic currents that most controlled model output. We found domains where different groups of parameters had the highest sensitivities, suggesting that interactions within each group shaped firing behaviors within each specific domain. Application of our method, and its characterization of which models were sensitive to general morphologic features, will lead to advances in understanding how realistic morphology participates in functional homeostasis. Significantly, we can predict which active conductances, and how many of them, will compensate for a given age- or development-related structural change, or will offset a morphologic perturbation resulting from trauma or neurodegenerative disorder, to restore normal function. Our method can be adapted to analyze any computational model. Thus, sensitivity landscapes, and the quantitative predictions they provide, can give new insight into mechanisms of homeostasis in any biological system

Identifying Candida albicans Gene Networks Involved in Pathogenicity

Acknowledgments This is a short text to acknowledge the contributions of specific colleagues, institutions, or agencies that aided the efforts of the authors. Funding RA was generously supported by a Wellcome Trust Institutional Strategic Support Award [WT105618MA], a Microbiology Research Visit Grant [RVG16/18], and a EPSRC/BBSRC Innovation Fellowship [EP/S001352/1]. AB was supported by a programme grant from the UK Medical Research Council [MR/M026663/1] and by the Medical Research Council Centre for Medical Mycology at the University of Aberdeen [MR/N006364/1]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Prenatal development is linked to bronchial reactivity: epidemiological and animal model evidence

Chronic cardiorespiratory disease is associated with low birthweight suggesting the importance of the developmental environment. Prenatal factors affecting fetal growth are believed important, but the underlying mechanisms are unknown. The influence of developmental programming on bronchial hyperreactivity is investigated in an animal model and evidence for comparable associations is sought in humans. Pregnant Wistar rats were fed either control or protein-restricted diets throughout pregnancy. Bronchoconstrictor responses were recorded from offspring bronchial segments. Morphometric analysis of paraffin-embedded lung sections was conducted. In a human mother-child cohort ultrasound measurements of fetal growth were related to bronchial hyperreactivity, measured at age six years using methacholine. Protein-restricted rats' offspring demonstrated greater bronchoconstriction than controls. Airway structure was not altered. Children with lesser abdominal circumference growth during 11-19 weeks' gestation had greater bronchial hyperreactivity than those with more rapid abdominal growth. Imbalanced maternal nutrition during pregnancy results in offspring bronchial hyperreactivity. Prenatal environmental influences might play a comparable role in humans

Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells

Acceptability of Early Infant Male Circumcision as an HIV Prevention Intervention in Zimbabwe: A Qualitative Perspective

Background Early infant male circumcision (EIMC) is simpler, safer and more cost-effective than adult circumcision. In sub-Saharan Africa, there are concerns about acceptability of EIMC which could affect uptake. In 2009 a quantitative survey of 2,746 rural Zimbabweans (aged 18–44) indicated that 60% of women and 58% of men would be willing to have their newborn son circumcised. Willingness was associated with knowledge of HIV and male circumcision. This qualitative study was conducted to better understand this issue. Methods In 2010, 24 group discussions were held across Zimbabwe with participants from seven ethnic groups. Additionally, key informant interviews were held with private paediatricians who offer EIMC (n = 2) plus one traditional leader. Discussions were audio-recorded, transcribed, translated into English (where necessary), coded using NVivo 8 and analysed using grounded theory principles. Results Knowledge of the procedure was poor. Despite this, acceptability of EIMC was high among parents from most ethnic groups. Discussions suggested that fathers would make the ultimate decision regarding EIMC although mothers and extended family can have (often covert) influence. Participants' concerns centred on: safety, motive behind free service provision plus handling and disposal of the discarded foreskin. Older men from the dominant traditionally circumcising population strongly opposed EIMC, arguing that it separates circumcision from adolescent initiation, as well as allowing women (mothers) to nurse the wound, considered taboo. Conclusions EIMC is likely to be an acceptable HIV prevention intervention for most populations in Zimbabwe, if barriers to uptake are appropriately addressed and fathers are specifically targeted by the programme

Aqueous Solution Equilibria and Spectral Features of Copper Complexes with Tripeptides Containing Glycine or Sarcosine and Leucine or Phenylalanine

Copper(II) complexes of glycyl-L-leucyl-L-histidine (GLH), sarcosyl-L-leucyl-L-histidine (Sar-LH), glycyl-L-phenylalanyl-L-histidine (GFH) and sarcosyl-L-phenylalanyl-L-histidine (Sar-FH) have potential anti-inflammatory activity, which can help to alleviate the symptoms associated with rheumatoid arthritis (RA). From pH 2–11, the MLH, ML, MLH-1 and MLH-2 species formed. The combination of species for each ligand was different, except at the physiological pH, where CuLH-2 predominated for all ligands. The prevalence of this species was supported by EPR, ultraviolet-visible spectrophotometry, and mass spectrometry, which suggested a square planar CuN4 coordination. All ligands have the same basicity for the amine and imidazole-N, but the methyl group of sarcosine decreased the stability of MLH and MLH-2 by 0.1–0.34 and 0.46–0.48 log units, respectively. Phenylalanine increased the stability of MLH and MLH-2 by 0.05–0.29 and 1.19–1.21 log units, respectively. For all ligands, 1H NMR identified two coordination modes for MLH, where copper(II) coordinates via the amine-N and neighboring carbonyl-O, as well as via the imidazole-N and carboxyl-O. EPR spectroscopy identified the MLH, ML and MLH-2 species for Cu-Sar-LH and suggested a CuN2O2 chromophore for ML. DFT calculations with water as a solvent confirmed the proposed coordination modes of each species at the B3LYP level combined with 6-31++G**

DNA-binding protein prediction using plant specific support vector machines:validation and application of a new genome annotation tool

There are currently 151 plants with draft genomes available but levels of functional annotation for putative protein products are low. Therefore, accurate computational predictions are essential to annotate genomes in the first instance, and to provide focus for the more costly and time consuming functional assays that follow. DNA-binding proteins are an important class of proteins that require annotation, but current computational methods are not applicable for genome wide predictions in plant species. Here, we explore the use of species and lineage specific models for the prediction of DNA-binding proteins in plants. We show that a species specific support vector machine model based on Arabidopsis sequence data is more accurate (accuracy 81%) than a generic model (74%), and based on this we develop a plant specific model for predicting DNA-binding proteins. We apply this model to the tomato proteome and demonstrate its ability to perform accurate high-throughput prediction of DNA-binding proteins. In doing so, we have annotated 36 currently uncharacterised proteins by assigning a putative DNA-binding function. Our model is publically available and we propose it be used in combination with existing tools to help increase annotation levels of DNA-binding proteins encoded in plant genomes