BIASES AND BLIND-SPOTS IN GENOME-WIDE CRISPR-CAS9 KNOCKOUT SCREENS

Adaptation of the bacterial CRISPR-Cas9 system to mammalian cells revolutionized the field of functional genomics, enabling genome-scale genetic perturbations to study essential genes, whose loss of function results in a severe fitness defect. There are two types of essential genes in a cell. Core essential genes are absolutely required for growth and proliferation in every cell type. On the other hand, context-dependent essential genes become essential in an environmental or genetic context. The concept of context-dependent gene essentiality is particularly important in cancer, since killing cancer cells selectively without harming surrounding healthy tissue remains a major challenge. The toxicity of traditional cancer treatment protocols to the normal cells stresses the need for new strategies that can identify and address the weaknesses specific to cancer cells. Studies showed that CRISPR monogenic knockout screens can identify specific processes that cells rely on for growth and proliferation, which is a crucial step in identifying candidate cancer-specific therapeutic targets. While it is widely accepted that CRISPR screening is both more specific and more sensitive than previously established methods, the limitations of this technology have not been systematically investigated. In this dissertation, through several lines of integrated analysis of CRISPR screen data in cancer cell lines from the Cancer Dependency Map initiative, I will describe several computational approaches to demonstrate that CRISPR screens are not saturating. In fact, a typical screen has a ~20% false-negative rate, saturating coverage requires multiple repeats and false negatives are more prevalent among moderately expressed genes. I will then introduce a solution to the false negative problem and describe another method that provides a cleaner analysis of the data, rescuing the false negatives observed in these screens. Moreover, I will show that half of all constitutively expressed genes are never observed as essential in any CRISPR screen. Notably, these never-essentials are highly enriched for paralogs, suggesting that functional redundancy masks the detection of a substantial number of genes. Finally, I will describe our efforts to investigate functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening technology and discuss the paralog synthetic lethal interactions that we have identified, which have escaped detection in monogenic CRISPR-Cas9 knockout screens. Collectively, these observations reveal significant biases and blind-spots in the analysis of CRISPR-based functional genomics approaches and offer new opportunities for the discovery of novel candidate drug targets

RNA2DNAlign: nucleotide resolution allele asymmetries through quantitative assessment of RNA and DNA paired sequencing data.

We introduce RNA2DNAlign, a computational framework for quantitative assessment of allele counts across paired RNA and DNA sequencing datasets. RNA2DNAlign is based on quantitation of the relative abundance of variant and reference read counts, followed by binomial tests for genotype and allelic status at SNV positions between compatible sequences. RNA2DNAlign detects positions with differential allele distribution, suggesting asymmetries due to regulatory/structural events. Based on the type of asymmetry, RNA2DNAlign outlines positions likely to be implicated in RNA editing, allele-specific expression or loss, somatic mutagenesis or loss-of-heterozygosity (the first three also in a tumor-specific setting). We applied RNA2DNAlign on 360 matching normal and tumor exomes and transcriptomes from 90 breast cancer patients from TCGA. Under high-confidence settings, RNA2DNAlign identified 2038 distinct SNV sites associated with one of the aforementioned asymetries, the majority of which have not been linked to functionality before. The performance assessment shows very high specificity and sensitivity, due to the corroboration of signals across multiple matching datasets. RNA2DNAlign is freely available from http://github.com/HorvathLab/NGS as a self-contained binary package for 64-bit Linux systems

Retrospective analysis of BCL-2 and laminin expression in cases of rectosigmoid Hirschsprung disease with histopathological diagnosis and its relationship with Hirschsprung-associated enterocolitis

Hirschsprung hastalığı (HH) etyolojisi netlik kazanmamış, histopatolojik tanısı güç bir hastalıktır. Hastalıkla ilişkili Hirschsprung enterokoliti (HEK) etyolojisi de belirsizliğini korumaktadır. İntrauterin dönemde nöral krest hücrelerinin enterik sinir sistemini oluşturmak üzere, kraniokaudal yöndeki göçünde görevli ekstrasellüler matriks proteinlerinden Laminin ve anti-apoptotik, nöroprotektif BCL-2 ekspresyonunun, post-operatif HEK geçiren ve geçirmeyen hastalardaki durumu karşılaştırmalı olarak incelenmiştir. Merkezimizde opere olmuş, histopatolojik tanılı 20 rektosigmoid Hirschsprung hastasına (post-operatif HEK görülen 10, görülmeyen 10 hasta) ve kontrol grubu olmak üzere, HH dışı nedenlerle opere edilmiş 10 hastaya ait, patoloji arşivinde bulunan bağırsak dokusu retrospektif olarak incelenmişir. Dokular immunohistokimyasal yöntemlerle protein ekspresyonu, moleküler genetik incelemelerle mRNA ve DNA düzeyinde değerlendirilmiş, BCL-2 ve Lamininin, HH ve HEK ilişkisi araştırılmış, yöntemlerin sonuçları karşılaştırılmıştır. BCL-2 immunohistokimyasında HH’de aganglionik dokulardaki ekspresyonun, ganglionik dokulara göre anlamlı şekilde azaldığı görülmüştür. BCL-2 mRNA ekspresyonu ise hem aganglionik hem ganglionik dokularda, kontrol grubuna kıyasla anlamlı ölçüde düşük belirlenmiştir. Laminin immunohistokimyasında aganglionik dokular anlamlı düzeyde pozitif boyanırken, ganglionik dokular negatif-hafif boyanmıştır. Laminini mRNA düzeyinde değerlendirmede kullanılan LAMA1 molekülünün ekspresyonu, aganglionik dokularda daha yüksek olma eğilimi gösterse de her dokuda farklı değerler elde edilmiştir. BCL-2 ve Lamininin HEK ile ilişkisine yönelik incelemelerde immunohistokimyasal ve moleküler anlamlılık belirlenememiştir. BCL-2 geninde mutasyon araştırmak amacıyla yapılan DNA dizi analizinde, bir olguda HH için literatürde tanımlanmamış bir değişim tespit edilmiştir. Bu hastanın aganglionik bağırsak dokusundaki BCL-2 mRNA ekspresyonu da ganglionik dokusuna göre belirgin olarak azalmıştır. Bunların sonucunda BCL-2’nin, HH ve HEK etyopatogenezinde üzerine düşülmesi gereken bir molekül olduğu kanaatindeyiz. Elde edilen sonuçlar, geniş hasta serileriyle yapılacak çalışmalarla anlamlılık gösterebilecek potansiyeldedir.Hirschsprung's disease (HD) is a condition whose etiology has not been clarified and its histopathological diagnosis is tough. The etiology of the Hirschsprung associated enterocolitis (HAEC) also remains unclear. The expression of Laminin which is extracellular matrix protein involved in the craniocaudal migration of neural crest cells in the intrauterine period to form the enteric nervous system and BCL-2 which is anti-apoptotic, neuroprotective were compared in patients with and without post operative HAEC. Retrospective analysis was performed on the intestinal tissue from 20 patients with the histopathological diagnosis of Rectosigmoid Hirschsprung who underwent surgery in our center (10 patients with post-operative HAEC, 10 patients without) and 10 patients as the control group who underwent surgery for an unrelated condition. Immunohistochemical techniques were used to evaluate the protein expression in tissues and molecular genetic studies were used to evaluate in the terms of mRNA and DNA analyses. Investigations were done into the connections between BCL-2, Laminin, HD and HAEC. The outcomes of the various procedures were compared. When compared to ganglionic tissues, BCL-2 immunohistochemistry revealed that HD patients' aganglionic tissues had significantly lower levels of BCL-2 expression. In HD patients' aganglionic and ganglionic tissues, BCL-2 mRNA expression was shown to be considerably lower than in the control group. Aganglionic tissues stained strongly positively with Laminin immunohistochemistry, whereas ganglionic tissues stained lightly or negatively. Although aganglionic tissues tended to have increased levels of LAMA1 molecule expression, which is used to evaluate Laminin at the mRNA level, various values were found in each tissue. Immunohistochemical and molecular significance could not be determined in the investigations regarding the relationship between BCL-2 and Laminin and HAEC. During DNA sequencing analysis for BCL-2 gene mutation investigations, a mutagenic change for HD that has not yet been identified in the literature was detected. Aganglionic tissue from this patient expressed BCL-2 mRNA substantially less than ganglionic tissue appears to have done. Hence, we conclude that BCL-2 is a molecule that should be considered while figuring out the etiopathogenesis of HD and HAEC. Results from research studies with larger patient series could discover the outcomes to be meaningful

Meslek hastalığı olarak brusella saptanan ve kas-iskelet sistemi bulguları olan aynı köyde yaşayan iki olgu

Brucellosis is a systemic and infectious disease which spreads all around the world but mostly Mediterranean and Middle Asian countries. Brucellosis begins with non-specific symptoms such as fewer, night sweating, anorexia, weight loss, weakness, severe headache and polialtralgia. Appearance of symptoms is connected with duration of disease. One of the systems which brucellosis holds onto is the musculoskeletal system. Peripheral arthritis, sacroiliitis, spondylitis, tenosynovitis, bursitis and osteomyelitis can be seen on musculoskeletal system. In two case, brucellosis spondylitis, olecranon bursitis and arthritis which is detected as industrial illness on two men living in the same village, having arthralgia, backache, elbow pain and swelling is reported. © 2018, Duzce University Medical School. All rights reserved

2018 Fen Bilimleri Dersi Öğretim Programına Yönelik Öğretmen Görüşleri

Bu çalışmanın amacı 2018 yılında yenilenen fen bilimleri dersi öğretim programına yönelik öğretmen görüşlerini belirlemektir. Araştırma yöntemi olarak olgubilim (fenomenoloji) deseni kullanılmıştır. Araştırmanın örneklemini Kilis, Adana, Şanlıurfa, Osmaniye ve Gaziantep illerinde görev yapan, deneyimleri 1 ile 35 yıl arasında değişen 18 fen bilgisi öğretmeni oluşturmaktadır. Örneklem ölçüt örnekleme yöntemi kullanılarak belirlenmiştir. Veri toplama aracı olarak iki fen eğitimi alanı uzmanı tarafından uzaman görüşü alınarak araştırmacılar tarafından geliştirilmiş olan yarı yapılandırılmış görüş formu kullanılmıştır. Elde edilen veriler içerik analizi yöntemi ile çözümlenmiştir. Çalışmanın güvenirliği Miles-Huberman formülü ile incelenmiştir. Çalışmanın sonucunda fen bilgisi öğretmenlerinin öğretim programının kazanım ve içerik boyutları için olumlu görüş bildirirken, öğretmenlerin yarısının programın öğrenme-öğretme süreci boyutu ile ilgili olumlu, ölçme-değerlendirme boyutu ile ilgili olumsuz görüş bildirdiği tespit edilmiştir. Ayrıca çalışmada öğretim programının güçlü olduğu yönlerine ilişkin görüşlerin daha fazla olduğu, çağın ve toplumun ihtiyaçlarına daha çok kısmen cevap verebildiği, genel olarak öğretim programında güncelliğinin yitirmiş konu ve uygulamaların bulunmadığı%253B öğretim programının işlevselliği ve uygulanabilirliğine yönelik olumlu görüşlerin daha fazla olduğu%253B öğretim programına yönelik memnuniyet düzeyinin orta düzeyde olduğu%253B öğretim programının evrensel ahlak değerleri, milli ve kültürel değerler ile bilimsel etik ilkelerinin benimsenmesini sağlamaquot%253B amacı ile fen, mühendislik ve girişimcilik uygulamalarına yönelik olumlu görüşlerin fazla olduğu sonuçları elde edilmiştir. Elde edilen bulgular ışığında, 2018 fen bilimleri dersi öğretim programına ilişkin önerilerde bulunulmuştur

Function generation with two loop mechanisms using decomposition and correction method

Method of decomposition has been successfully applied to function generation with multi-loop mechanisms. For a two-loop mechanism, a function y = f(x) can be decomposed into two as w = g(x) and y = h(w) = h(g(x)) = f(x). This study makes use of the method of decomposition for two-loop mechanisms, where the errors from each loop are forced to match each other. In the first loop, which includes the input of the mechanism, the decomposed function (g) is generated and the resulting structural error is determined. Then, for the second loop, the desired output of the function (f) is considered as an input and the structural error of the decomposed function (g) is determined. By matching the obtained structural errors, the final error in the output of the mechanism is reduced. Three different correction methods are proposed. The first method has three precision points per loop, while the second method has four. In the third method, the extrema of the errors from both loops are matched. The methods are applied to a Watt II type planar six-bar linkage for demonstration. Several numerical examples are worked out and the results are compared with the results in the literature

Conceptual design of a 2-DoF planar high-speed industrial parallel manipulator

This study focuses on conceptual design alternatives for a planar high-speed/high-precision manipulator in terms of mechanism structure, control strate-gy, and drive system selection. These concepts are investigated specifically for planar 5-bar based parallel linkages. An over-constrained 6-bar linkage with par-allelogram loops and its simply constrained version are selected for detailed de-sign. In addition, a model-based control strategy including a stiffness model is discussed for future studies. Alternative drive systems are evaluated. Finally a prototype is presented.TÜBİTAK "Proje no: 116M272