OAR@UM Collection:

An analysis of phenotype - genotype features of a maltese patient cohort with crohn's disease and ulcerative colitis

Fri, 01 Jan 2016 00:00:00 GMT

Title: An analysis of phenotype - genotype features of a maltese patient cohort with crohn's disease and ulcerative colitis Abstract: Crohn's disease (CD) and Ulcerative colitis (UC) are the two major fonns of inflammato1y bowel diseases (IBD) with a multifactorial aetiology. Genetic studies have identified 163 susceptibility loci for IBD, mostly shared between CD and UC. We undertook a study in our local IBD population. The aims of our study were: • To determine if the phenotype of the Maltese IBD patients is similar to that of the European cohorts. • To determine the frequency of the NOD2/CARD 15 polymorphisms (Arg702Trp, Gly908Arg and Leu1007insC mutations) in our CD patients as compared to the European Population. • To detennine if the genetic architecture of the Maltese population is similar to the European population. • To determine if the prevalence of common IBD polymorphisms (rs35261698, rs2172252, rs3197999, rs4151651, rs3129891, rs9268832, rs482044, rs2066844, rs2066847, rs6887695, rs4833095, rs187238, rs1004819, rs11209026 and rs2241880) are present in the Maltese IBD patients when compared to a Maltese Control population. • To determine if there is any correlation between the phenotype and the genotypes of both patients with UC and CD. Methods: Patients with IBD who fulfilled the Copenhagen criteria (Langholz, 1999) for its diagnosis were recruited from Mater Dei Hospital, Malta. The patients' clinical case notes were reviewed and the patients were interviewed. Informed consent was obtained. Their phenotypic and clinical features were entered into a database. Blood was withdrawn from the patients. A control group was recruited. DNA extraction was then done at the Department of Physiology, University of Malta. Analysis for the NOD2/CARD 15 polymorphisms was done at the University of Malta. Further analysis on the extracted DNA samples was done at the University of Kiel, Germany. The extracted DNA was sent over and the genetic analysis was done using the Immunochip version 2. Results: 194 patients with CD, 109 patients with UC and 216 healthy volunteer controls, age and gender matched were randomly recruited. Their phenotypic features were similar to that described in other European populations. Analysis of the DNA in CD patients for the NOD2/CARD 15 polymorphisms revealed a mutation rate of 12%, which is statistically much lower than that found in European Populations (p= <0.005). The genetic architecture of the Maltese population is similar to that of the European population, especially of those people from Tuscany, Italy. Data from the Immunochip v2 was compared using a Manhattan plot. This demonstrated the absence of statistically significant SNPs in our IBD population when compared to the European IBD population. [...] Description: PH.D.

Vaccine prevention of capsular group C meningococcal disease in childhood

Fri, 01 Jan 2016 00:00:00 GMT

Title: Vaccine prevention of capsular group C meningococcal disease in childhood Abstract: Invasive capsular group C meningococcal (MenC) disease remains a prevalent cause of death and disability in children that may be prevented by vaccination. The introduction of MenC conjugate (MenCC) vaccines in immunisation programmes has increased the burden of vaccine injections administered in childhood. Consequently, the possibility of decreasing the number of Men CC vaccine injections needs to be investigated, as has been studied in this thesis. The immunogenicity of a novel combined Haemophilus irifhtenzae type b (Hib )-MenC conjugate vaccine (HibMenC-TT), investigated in a Phase 3 trial, was found to be non-inferior to a licensed MenC-CRM197 vaccine following a prime and boost schedule, after which 94.8% of participants had MenC serum bactericidal antibody titres, using rabbit complement, (rSBA) 2:1: 128 irrespective of the vaccine formulation used for priming. Subsequently, an investigation of the immunogenicity of reduced MenCC prime and boost schedules in a Phase 4 trial, demonstrated that, after a 12 month Hib-MenC-TT boost, a single infant MenC-CRM197 vaccine priming schedule was non-inferior to two infant dose MenC-CRM197 priming (MenC rSBA geometric mean titres 660 vs 295, difference in mean log10 MenC rSBA of 0.35). After Hib-MenC-TT boosting, only a single infant dose MenC-TT priming schedule was found to induce robust MenC rSBA titres that persisted up till 24 months of age. Data from these trials supported the introduction of the Hih-MenC-TT booster dose and the subsequent reduction of the number of MenCC infant priming doses in the UK immunisation schedule. Furthermore, these results could be used to guide the implementation of a MenCC immunisation programme in Malta, where an epidemiological study performed as part of this thesis demonstrated that the overall, and specifically the infant, incidence rates of MenC disease are much higher than those in Europe. I Description: PH.D

Biomarker-driven therapeutic groups sensitive to PP2A activators in cancer patients

Fri, 01 Jan 2016 00:00:00 GMT

Title: Biomarker-driven therapeutic groups sensitive to PP2A activators in cancer patients Abstract: Deregulation of the feedback mechanism of protein phosphatase 2 A (PP2A) is implicated in tumorigenesis directly through reduced expression of functional catalytic subunit of PP2A (PP2A-C) or through overexpression of its inhibitors a4, SET, SETBP1 and CIP2A. PP2A controls proliferation, growth and apoptosis, attenuating various signalling pathways. The cBIO Portal for Cancer Genomics (cBIOPortal) shows that the PP2A is deregulated in 63% of Triple Negative Breast Cancer (TNBC) patients. TNBC patients derive little benefit from traditional target-specific therapies due to lack of the favourable prognostic markers ER, PR and HER2. In the breast cancer cell line models, sensitivity to a PP2A activator (FTY720) was observed solely in TNBC cell lines. The significant overexpression of CIP2A in the sensitive cell line MDAMB231, exemplifies PP2A activity regulation. Results showed that PP2A core complex and regulatory subunit expression at transcript and protein level could not directly predict FTY720 sensitivity, hence the need for PP2A activity biomarkers. A candidate set of PP2A activity biomarkers was derived from in silica analysis of The Cancer Genome Atlas (TCGA) expression data from the invasive breast cancer cohort. PP2A activity biomarkers were differentially expressed in cases with potential PP2A deregulation, as defined by low expression of PP2A core subunits and overexpression of PP2A regulators. A biological link to PP2A was established through pathway analysis of a short-list of candidate genes, identifying 5 potential biomarkers. The expression of PP2A core components and regulators, breast cancer signature genes and the 5 biomarker candidates were assessed in a Maltese cohort of breast cancer patients (N = 90) using a specific technique for RNA analysis in formalin fixed paraffin embedded (FFPE) tissue (Quantigene v2.0). lmmunohistochemical analysis of PP2A regulators and PP2A phospho-targets, pAKT and pS6K, was carried out on the same local breast cancer patients and on a cohort of 302 breast cancer cases on tissue microarrays (TMAs) provided by our collaborators at Leeds University. The multi-level approach provided evidence for various mechanisms for PP2A activity regulation: isoform formation of the catalytic subunit a. isoform of PP2A (PPP2CA}; post translational modifications of PPP2CA; transcriptional regulation of PP2A regulators and differential cellular localisation of regulators. The complexity of PP2A regulation warranted the use of biomarkers that reflect PP2A activity or FTY720 sensitivity. Moreover, validation of the 5 candidate PP2A activity markers across in silica data, cellular models and patients, identified AURKA and KIF2C as transcriptional biomarkers of PP2A-dependent growth factor activation (PP2AGFA}. Expression of the PP2AGFA markers is a prerequisite for mitotic entry due to their pivotal role in cell cycle regulation. AURKA and KIF2C are implicated with an aggressive phenotype of malignancies, characterised by increased proliferation and acquired motility. Using AURKA and KIF2C, TCGA and local breast cancer cases were classified into a novel class of tumours (PP2AGFA} that associate with FTY720-sensitive breast cell lines. The PP2AGFA class of tumours were represented by a prevalent overexpression of CIP2A and the TNBC or Basal breast cancer subtypes. The novel class defined by overexpression of AURKA and KIF2C, represents a new therapeutic class of tumours with predicted susceptibility to restoration of PP2A activity. Description: PH.D.

Determining the 'in vitro' effect of Padina pavonica on the oestrogen receptor and oestrogen responsive primary cell lines

Fri, 01 Jan 2016 00:00:00 GMT

Title: Determining the 'in vitro' effect of Padina pavonica on the oestrogen receptor and oestrogen responsive primary cell lines Abstract: Backgound: Padina pavonica appears to improve the bone mineral density at the lumbar spine and at the hip in post-menopausal women (1). The aims of this project are to compare it to other treatments available on the market, for the treatment of post-menopausal osteoporosis and to shed more light on the mechanism of action of Padina pavonica. Aims and Objectives: The aim of this research-based experiment is to compare the ability of osteoblasts treated with the extract of Padina pavonica (EPP) to differentiate and fix calcium, with osteoblasts treated with raloxifene and oestradiol. Raloxifene, a selective oestrogen receptor modulator (SERM) and oestradiol in the form of hormone replacement therapy (HRT) are drugs normally used in the management of post-menopausal osteoporosis. A secondary aim is to determine whether the extract exerts its action by modulating the oestrogen receptor. This would imply that the extract of Padina pavonica has a SERM-like activity and is thus potentially prone to the same adverse effects, as other members of this class. Methodology: This is a research-based experiment in which primary osteoblasts were isolated and cultured from human bone explants. Primary cells obtained using this method were used to objectively assess the extract's effect on the differentiation of progenitor bone cells to terminally mature osteoblasts. Results were compared to bone cells treated with raloxifene and oestradiol. Alkaline phosphatase activity was measured as an early marker of osteoblast differentiation using spectrophotometry. An MTT assay was employed as a measure and marker of cellular viability and proliferation. These tests were performed after seven days of incubation. The alkaline phosphatase: MTT ratio was calculated and used as another end point in order to reflect whether any increases in alkaline phosphatase were due to an increase in cell mass or whether this was due to the formation of more terminally differentiated osteoblasts. Cells were also incubated with the drugs for fifteen days. The Alizarin Red assay was then performed. The latter was employed as measure of calcium fixation and bone matrix mineralization. Oestrogen receptor mono clonal IgG antibodies were used in order to try and assess whether the drugs' activity was oestrogen receptor dependent or independent. The results was analysed usmg multiple-linear regression analysis and the Kruskall-Wallis non-parametric test. Results: Human primary osteoblasts cells can be easily grown in culture and used as a model for the testing of drugs with potential use in the management of postmenopausal osteoporosis. Cells treated with the extract of Padina pavonica expressed alkaline phosphatase activity. This was then found not to be statistically different from oestrogen or raloxifene (p-value: 0.501). A statistically significant difference between drugs was noted in the cell viability assay (MTT) (p-value: 0.002). The highest cell viability was noted in the cells treated with oestradiol. There was no statistically significant difference between cells treated with EPP and cells treated with raloxifene on the cell viability assay (p-value: 0.528). The latter was reflected in the alkaline phosphatase:MTT ratio of the differently treated cultures which revealed a statistically significant difference between groups (p-value 0.034). Cells treated with the extract of Padina pavonica were only slightly inferior to raloxifene in tenns of osteoblast differentiation as supported by the second highest average estimated marginal means of the alkaline phosphatase to MTT ratio. The different drugs did not show any statistically significant difference in the bone matrix mineralization assay (p-value: 0.548). The oestrogen receptor antibody tests did not reveal any statistically significant results, but suggest a SERM-like activity ofEPP. Conclusions: Our data supports previous studies, which show a potential role for the extract of Padina pavonica in the management of post-menopausal osteoporosis. The mechanism of action of this drug remains to be fully understood. This work indicates a possible direct or indirect modulation (by co-factors) of the oestrogen receptors by EPP and suggests a SERM-like activity of this product. Further Quantitative Polymerase Chain reaction (q-PCR) studies supported and confirmed by Western-blot protein analysis are key steps in understanding the impact of the extract of Padina pavonica on the protein component of bone matrix and to be able to relate differential gene expression signatures to established cell signaling in physiological and pathological pathways. Description: M.SC.REPRODUCTIVE HEALTH