The role of the arginine methyltransferase CARM1 in global transcriptional regulation.

Arginine methylation is a prevalent post-translational modification that is found on many nuclear and cytoplasmic proteins, and has been implicated in the regulation of gene expression. CARM1 is a member of the protein arginine methyltransferase (PRMT) family of proteins, and is a key protein responsible for arginine methylation of a subset of proteins involved in transcription. In this thesis I examine some of the mechanisms through which CARM1 contributes to global transcriptional regulation. Using a ChIP-DSL approach, we show that the p/CIP/CARM1 complex is recruited to 204 proximal promoters following 17β-estradiol (E2) treatment in MCF-7 cells. Many of the target genes have been previously implicated in signaling pathways related to oncogenesis. JAK2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the JAK2 promoter are reflective of a transcriptionally permissive gene, and we observed modest increases in RNA and protein expression. Notably, E2-induced expression of Jak2 was diminished when p/CIP or CARM1 were depleted, suggesting that the p/CIP/CARM1 complex is required for the observed transcriptional response. Collectively, these results suggest that E2-dependent recruitment of the p/CIP/CARM1 complex causes JAK2 to become ‘poised’ for transcription, a finding that may be extendable to other target genes and signalling pathways. Furthermore, bioinformatic examination of p/CIP/CARM1 target promoters suggests that transcription factor crosstalk is the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. Using ChIP-Seq, we identified genomic regions to which CARM1 is recruited. Subsequent characterization of binding events suggest a role for CARM1 in transcriptional elongation, and implicate the transcription factor PAX1 as a novel mechanism through which CARM1 can be recruited to the genome. Identification of CARM1-dependent differentially expressed genes revealed that direct recruitment of CARM1 is not essential for the majority of its transcriptional effects in MEFs. However, CARM1 does play a critical role in cellular growth and proliferation, and in the absence of CARM1, the expression of many cell cycle regulators is dramatically affected. Collectively, this work provides insight into some of the mechanisms through which CARM1 modulates transcription, and highlights its importance in diverse cellular processes

Employer, Industry and Policymaker views on Doctorate Education

As society undergoes green and digital transitions, various policymakers such as the European Commission expect universities to contribute to innovation and progress. As education’s highest achieving graduates, the doctorate holder may be key in this era of innovation and problem-solving. As academic career prospects dwindle, and PhD graduates increasingly enter industry, academic research has highlighted that traditional PhD programmes may not provide the required skills and knowledge for the workforce today. To learn how best to address such issues, we consulted thirteen EU policy documents and industry-led reports; and interviewed thirteen employers to add their voice to the discussion. Findings align with previous reports of a lack of transferable skills, but also introduce new concerns such as the desire for adaptability, experience, and redefining skills with regards to self-presentation. We discuss interdisciplinarity and intersectorality as potential solutions to addressing these needs

Regulation of the BRCA1 gene by an SRC3/53BP1 complex

<p>Abstract</p> <p>Background</p> <p>Steroid Receptor coactivator 3(SRC3) is an oncogene and a member of the SRC family of nuclear receptor coactivator proteins that mediate the transcriptional effects of nuclear hormone receptors as well as other transcription factors.</p> <p>Results</p> <p>We have used protein purification and mass spectrometry to identify the 53BP1 tumour suppressor as a novel SRC3-associated protein. Copurification was demonstrated using multiple antibodies, and was not dependent on DNA damage suggesting that SRC3 is not directly involved in the DNA damage response. However using chromatin immunoprecipitation(ChIP) and siRNA knockdown, we have demonstrated that both SRC3 and 53BP1 co-occupy the same region of the BRCA1 promoter and both are required for BRCA1 expression in HeLa cells.</p> <p>Conclusions</p> <p>Our results suggest that both 53BP1 and SRC3 have a common function that converge at the BRCA1 promoter and possibly other genes important for DNA repair and genomic stability.</p

Increased platelet counts and platelet activation in early symptomatic versus asymptomatic carotid stenosis and relationship with microembolic status: Results from the Platelets And Carotid Stenosis (PACS) Study

Background: Cerebral microembolic signals (MES) may predict increased stroke risk in carotid stenosis. However, the relationship between platelet counts or platelet activation status and MES in symptomatic versus asymptomatic carotid stenosis has not been comprehensively assessed. Setting: University teaching hospitals. Methods: This prospective, pilot observational study assessed platelet counts and platelet activation status, and the relationship between platelet activation and MES in asymptomatic versus early (≤4 weeks after TIA/stroke) and late phase (≥3 months) symptomatic moderate or severe (≥50%) carotid stenosis patients. Full blood count measurements were performed, and whole blood flow cytometry was used to quantify platelet surface activation marker expression (CD62P and CD63) and circulating leucocyte-platelet complexes. Bilateral simultaneous transcranial Doppler ultrasound monitoring of the middle cerebral arteries was performed for 1 hour to classify patients as MES-positive or MES-negative. Results: Data from 31 asymptomatic patients were compared with 46 symptomatic patients in the early phase, and 35 of these patients followed up to the late phase after symptom onset. The median platelet count (211 vs. 200 x 109/L; p=0.03) and the median % lymphocyte-platelet complexes were higher in early symptomatic than asymptomatic patients (2.8 vs. 2.4%, p=0.001). The % lymphocyte-platelet complexes was higher in early symptomatic than asymptomatic patients with ≥70% carotid stenosis (p=0.0005), and in symptomatic patients recruited within 7 days of symptom onset (p=0.028). Complete TCD data were available in 25 asymptomatic and 31 early phase symptomatic, and 27 late phase symptomatic patients. 12% of asymptomatic versus 32% of early phase symptomatic (p=0.02) and 19% of late phase symptomatic patients (p=0.2) were MES-positive. Early symptomatic MES-negative patients had ahigher % lymphocyte-platelet complexes than asymptomatic MES-negative patients (2.8 vs. 2.3%; p=0.0085). Discussion: Recently symptomatic carotid stenosis patients have higher platelet counts (potentially reflecting increased platelet production, mobilisation or reduced clearance) and platelet activation status than asymptomatic patients. MES were more frequently detected in early symptomatic than asymptomatic patients, but the differences between late symptomatic and asymptomatic groups were not significant. Increased lymphocyte-platelet complex formation in recently symptomatic vs. asymptomatic MES-negative patients indicates enhanced platelet activation in this early symptomatic subgroup. Platelet biomarkers, in combination with TCD, have the potential to aid risk-stratification in asymptomatic and symptomatic carotid stenosis patients

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose

Investigating the effects of maximal anaerobic fatigue on dynamic postural control using the Y-Balance Test

ObjectivesThe Y Balance Test is one of the most commonly used dynamic balance assessments, providing an insight into the integration of the sensorimotor subsystems. In recent times, there has been an increase in interest surrounding its use in various clinical populations demonstrating alterations in motor function. Therefore, it is important to examine the effect physiological influences such as fatigue play in dynamic postural control, and establish a timeframe for its recovery.DesignDescriptive laboratory study.MethodsTwenty male and female (age 23.75 4.79 years, height 174.12 8.45 cm, mass 69.32 8.76 kg) partaking in competitive sport, completed the Y Balance Test protocol at 0, 10 and 20 min, prior to a modified 60 s Wingate fatiguing protocol. Post-fatigue assessments were then completed at 0, 10 and 20 min post-fatiguing intervention.ResultsIntraclass correlation coefficients demonstrated excellent intra-session reliability (0.9760.982) across the three pre-fatigue YBT tests. Post-hoc paired sample t-tests demonstrated that all three reach directions demonstrated statistically significant differences between pre-fatigue and the first post-fatigue measurement (anterior; p = 0.019, posteromedial; p = 0.019 & posterolateral; p = 0.003). The anterior reach direction returned to pre-fatigue levels within 10 min (p = 0.632). The posteromedial reach direction returned to pre-fatigue levels within 20 min (p = 0.236), while the posterolateral direction maintained a statistically significant difference at 20 min (p = 0.023).ConclusionsMaximal anaerobic fatigue has a negative effect on normalised Y balance test scores in all three directions. Following the fatiguing protocol, dynamic postural control returns to pre-fatigue levels for the anterior (20 min).Science Foundation Irelan

Memo test: standardisation of computerised assessment of auditory verbal short-term memory

Short-term memory (STM) impairments are prevalent in adults with acquired brain injuries. While there are several published tests to assess these impairments, the majority require speech production, e.g. digit span (Wechsler, 1987). This feature may make them unsuitable for people with aphasia and motor speech disorders because of word finding difficulties and speech demands respectively. If patients perceive the speech demands of the test to be high, the may not engage with testing. Furthermore, existing STM tests are mainly ‘pen-and-paper’ tests, which can jeopardise accuracy. To address these shortcomings, we designed and standardised a novel computerised test that does not require speech output and because of the computerised delivery it would enable clinicians identify STM impairments with greater precision than current tests. The matching listening span tasks, similar to the non-normed PALPA 13 (Kay, Lesser & Coltheart, 1992) is used to test short-term memory for serial order of spoken items. Sequences of digits are presented in pairs. The person hears the first sequence, followed by the second sequence and s/he decides whether the two sequences are the same or different. In the computerised test, the sequences are presented in live voice recordings on a portable computer through a software application (Molero Martin, Laird, Hwang & Salis 2013). We collected normative data from healthy older adults (N=22-24) using digits, real words (one- and two-syllables) and non-words (one- and two- syllables). Their performance was scored following two systems. The Highest Span system was the highest span length (e.g. 2-8) at which a participant correctly responded to over 7 out of 10 trials at the highest sequence length. Test re-test reliability was also tested in a subgroup of participants. The test will be available as free of charge for clinicians and researchers to use

Racionalizace obrábění součásti s využitím CNC obráběcích strojů v podmínkách OKD, BASTRO, a.s.

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta strojní. Katedra (346) obrábění a montáž

The effect of adding organic polymers on the handling properties, strengths and bioactivity of a Ca-Sr-Zn-Si glass polyalkenoate cement

This work demonstrates the addition of a number of naturally occurring proteins/polymers to a Zinc based Glass Polyalkenoate Cements (GPCs). Chitin (Chi.) Collagen (Col.), Cysteine (Cys.) and Keratin (Ker.) were added with the intention of improving the bioactivity of this cement. Initial testing involved characterization of the glass with X-ray Diffraction (XRD) and Differential Thermal Analysis (DTA) before and after sterilization with γ-irradiation. No significant changes occurred as a result of sterilization. Handling properties of the modified cements were not significantly different from those of the control, BT 101 (Working Tw - 36s, and Setting time Ts - 70s) except for Chi. (30s, p≤0.016) and Cys. (105s, p≤0.0001) respectfully. Comparison of the mechanical properties of BT 101 (compression - σc and biaxial flexural - σf) to the modified cements revealed a significant decrease in σc with Chi. and Col., after 1, 7 and 30 days. However, there were little changes occurring in σf. Cement structural testing was investigated and found that the addition of these polymers greatly reduced the cements surface area, however the only significant change to occur in the solubility testing was Ker. (p≤0.009). Simulated Body Fluid (SBF) testing resulted in increased calcium phosphate (CaP) deposition of Chi. and Col. compared to BT 101. Cell culture studies determined only Col. significantly increased (p≤0.0001) in comparison to the control cement