Heterochromatin protein 1 recruitment and chromatin conformational dynamics at the single-molecule level

Chromatin is the template on which DNA-associated transactions take place in eukaryotic organisms. Nucleosomes consisting of the four histones H2A, H2B, H3 and H4 each organize ~150bp of DNA and constitute a first layer of chromatin. The three-dimensional organization of chromatin as well as histone post-translational modifications (PTMs) regulate recruitment of chromatin-associated effector proteins (effectors). Heterochromatin protein 1 (HP1) is an effector associated with silenced genome regions. HP1 recognizes histone H3 trimethylated at lysine 9 (H3 K9me3) and can dimerize. This results in a protein with two binding domains allowing multivalent engagement of target chromatin. HP1 can further promote chromatin condensation and inter-fiber contacts. The effector p53 binding protein (53BP1) is a key regulator in the DNA damage repair pathway. It is known to target a trio of PTMs; H4 dimethylated at K20 (H4 K20me2), H2A(.X) ubiquitylated at K15 (H2A.X K15ub) and H2A.X phosphorylated at S139 (H2AX S139ph). Although details about the function of the individual domains of these proteins have been uncovered, little is known about the binding mechanism of the holoproteins and how this affects chromatin conformation. The aim of this thesis was to develop chromatin engineering and single-molecule fluorescence based methods to; i) Interrogate recruitment kinetics of HP1 to post-translationally modified chromatin. ii) Understand how HP1 binding alters the conformational dynamics of chromatin secondary structure. iii) Prepare histones carrying the PTM signature recognized by 53BP1. We established an assay to monitor binding kinetics of HP1α to modified chromatin using co-localization single-molecule microscopy (CoSM). H3 K9me3 octamers, labeled HP1α, dimerized HP1α and labeled array DNA formed the basis for this. With this, we found that HP1a multivalency induced by dimerization functions as a platform to enhance HP1a binding to target chromatin up to 9-fold by both accelerating association and prolonging retention. This was further corroborated by FRAP measurements using specific mutants in live mouse fibroblasts. Chromatin conformational dynamics were investigated by ensemble and single-molecule FRET (smFRET). Multiple combinations of FRET positions allowed us to obtain multi-perspective information on the conformational changes in chromatin upon compaction. This showed distinct steps in the local folding of chromatin. Histone acetylation of histone H4 prevented the last steps of this folding pathway. HP1-mediated compaction promotes earlier steps of compaction while maintaining conformational dynamics. Finally, towards similar studies with 53BP1, we devised schemes for synthesis of histones containing the target PTMs. Histones with the individual PTMs were prepared by ligation and desulfurization. H2A.X with both an N-terminal ubiquitin and C-terminal phosphorylation was prepared through a convergent route using recombinant SUMO and a split intein from Nostoc Punctiforme as orthogonal recombinant protection groups. Together the work described in this thesis combines advanced protein and chromatin engineering with CoSM and smFRET. This resulted in mechanistic insight into the spatio-temporal regulation of HP1 recruitment, chromatin conformational dynamics, templates for similar investigations with 53BP1, and tools for further investigation of nucleosome function in the context of chromatin

VISUOMOTOR AND AUDIOMOTOR REACTION TIME IN ELITE AND NON-ELITE BADMINTON PLAYERS

The ability to quickly perceive appropriate motor response is essential in the badminton sport under the critical time pressure. This study aimed to evaluate the visual and auditory reaction time, speed, anaerobic power and vertical jump between elite and non-elite badminton athletes. With this purpose, various anthropometric measurements, hexagonal obstacle test, vertical jump test, anaerobic power measurement and auditory and visual reaction time tests were performed to the elite and non-elite athletes. When auditory reaction time, vertical jump and anaerobic power measurements were evaluated, there was no significant difference between the elite and non-elite groups, but it was noticed that there was a significant differences in quickness and visual reaction time in favor of elite athletes. It is also seen that speed and visual reaction time have a positive effect on badminton athletes are able to get to the high performance level in other literature information. For this reason, it has been thought that training programs designed for badminton athletes by considering these physiological parameters and training systems designed to increase the reaction time may be beneficial.  Article visualizations

Secondary prevention of coronary heart disease in elderly population of Turkey: A subgroup analysis of ELDERTURK study

Background: Secondary prevention plays an important role after acute coronary event due to high risk of adverse events in elderly. In present study we aimed to evaluate the lifestyle, management of risk factors and medical treatment for secondary protection in elderly patients with known coronary heart disease (CHD). Methods: ELDERTURK is a non-interventional, multi-centered, observational study, which included total of 5694 elderly patients ( > 65 years) from 50 centers in Turkey. In this study elderly patients from the ELDERTURK population with known CHD were evaluated for cardiovascular risk factors, comor- bidities and medication usage. Results: A total of 2976 (52.3% of study) out of 5694 patients included in the ELDERTURK study were evaluated. All had known CHD with a mean age of 73.4 ± 6.2 years and 60.3% were male. 13.0% of patients were smokers, 42.4% were overweight and 21.1% were obese. Only 23.6% of patients reported to do regular exercise, 73.4% had history of hypertension, 47.4% had dyslipidemia and 33.9% had diabetes mellitus. The rate of patients with systolic blood pressure > 140 mmHg were 31.1% and only 13.9% of patients had a recommended ≤ 70 mg/dL level of low-density lipoprotein cholesterol. Anti- platelet, statin, beta-blocker and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker usage was limited to 27.3%. Conclusions: The ELDERTURK study shows that many patients with CHD have a high prevalence of modifiable risk factors and unhealthy lifestyle. Apart from this, many patients are not receiving thera- peutic intervention and as a consequence most were not achieving the recommended goals.   

Lymphocyte-to-monocyte ratio is a valuable marker to predict prostate cancer in patients with prostate specific antigen between 4 and 10 ng/dl

Objective: To evaluate the diagnostic value of serum inflammation markers derived from complete blood count in diagnosis of prostate cancer (PCa). Methods: We retrospectively analyzed the data of 621 patients who underwent prostate biopsy between March 2013 and April 2018. Age, prostate specific antigen (PSA), free PSA, platelet count, neutrophil count, lymphocyte count, monocyte count, prostate volume (PV) and pathology result of the patients were recorded. Patients were grouped as benign prostatic hyperplasia (BPH), prostatitis and PCa. Patients were also grouped according to PSA values, as PSA 10 ng/dl. Results: The mean lymphocyte-to-monocyte ratio (LMR) value of the patients with PCa was significantly lower in the entire cohort (p = 0.047). In the PSA 4-10 ng/dl range, LMR value wassignificantly lower in patients with PCa than those with BPH or prostatitis (p = 0.012). In this PSA range, free/total PSA ratio and LMR were significant factors to predict PCa. The cut-off values of LMR, free/total PSA were 3.05 and 0.15 respectively. The sensitivities, spesificities, positive predictive values (PPV) and negative predictive values using LMR cut-off, free/total PSA cut-off and their combination were assessed. Specificity and PPV of the combination group were higher (97.2%, 83.3% respectively) compared to free/total PSA cut-off group (91.6%, 76.6%) and LMR cut-off group (67.8%, 43.7%). Conclusions: LMR is a useful tool at detecting PCa especially in patients with PSA value between 4 and 10 ng/dl. The combination of free/total PSA ratio and LMR improves the diagnostic accuracy more than the use of free/total PSA ratio alone

Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability

Characterization of greater middle eastern genetic variation for enhanced disease gene discovery

The Greater Middle East (GME) has been a central hub of human migration and population admixture. The tradition of consanguinity, variably practiced in the Persian Gulf region, North Africa, and Central Asia1-3, has resulted in an elevated burden of recessive disease4. Here we generated a whole-exome GME variome from 1,111 unrelated subjects. We detected substantial diversity and admixture in continental and subregional populations, corresponding to several ancient founder populations with little evidence of bottlenecks. Measured consanguinity rates were an order of magnitude above those in other sampled populations, and the GME population exhibited an increased burden of runs of homozygosity (ROHs) but showed no evidence for reduced burden of deleterious variation due to classically theorized ‘genetic purging’. Applying this database to unsolved recessive conditions in the GME population reduced the number of potential disease-causing variants by four- to sevenfold. These results show variegated genetic architecture in GME populations and support future human genetic discoveries in Mendelian and population genetics

The IDENTIFY study: the investigation and detection of urological neoplasia in patients referred with suspected urinary tract cancer - a multicentre observational study

Objective To evaluate the contemporary prevalence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC] and renal cancer) in patients referred to secondary care with haematuria, adjusted for established patient risk markers and geographical variation. Patients and Methods This was an international multicentre prospective observational study. We included patients aged ≥16 years, referred to secondary care with suspected urinary tract cancer. Patients with a known or previous urological malignancy were excluded. We estimated the prevalence of bladder cancer, UTUC, renal cancer and prostate cancer; stratified by age, type of haematuria, sex, and smoking. We used a multivariable mixed-effects logistic regression to adjust cancer prevalence for age, type of haematuria, sex, smoking, hospitals, and countries. Results Of the 11 059 patients assessed for eligibility, 10 896 were included from 110 hospitals across 26 countries. The overall adjusted cancer prevalence (n = 2257) was 28.2% (95% confidence interval [CI] 22.3–34.1), bladder cancer (n = 1951) 24.7% (95% CI 19.1–30.2), UTUC (n = 128) 1.14% (95% CI 0.77–1.52), renal cancer (n = 107) 1.05% (95% CI 0.80–1.29), and prostate cancer (n = 124) 1.75% (95% CI 1.32–2.18). The odds ratios for patient risk markers in the model for all cancers were: age 1.04 (95% CI 1.03–1.05; P < 0.001), visible haematuria 3.47 (95% CI 2.90–4.15; P < 0.001), male sex 1.30 (95% CI 1.14–1.50; P < 0.001), and smoking 2.70 (95% CI 2.30–3.18; P < 0.001). Conclusions A better understanding of cancer prevalence across an international population is required to inform clinical guidelines. We are the first to report urinary tract cancer prevalence across an international population in patients referred to secondary care, adjusted for patient risk markers and geographical variation. Bladder cancer was the most prevalent disease. Visible haematuria was the strongest predictor for urinary tract cancer

Student Teachers' Perspectives on the Cognitive and Metacognitive Strategy Practices in Instructional Design Course

This is a case study which aims to investigate student teachers' perspectives on the practices related to the use of cognitive and metacognitive strategies integrated in the course content. 90 sophomore student teachers at two different state universities participated in this study. Cognitive strategy practices are conducted in the first participating group while metacognitive strategy practices are conducted in the latter group. Structured interview consisting of open-ended questions is employed with the aim of determining participants' perspectives. Descriptive analysis is used for data analysis. Findings show that their satisfaction levels in terms of the practices are high, and that they believe in the usefulness of the practices in terms of the knowledge and skills in the use of strategy and content area. Also, it is found that they are satisfied with the practices in terms of opportunities for the revision of the previous knowledge and adapting studying. However, it is clear that they are annoyed with the weekly conducted practices

A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage

The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play a key role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair