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Analysis of enhancer activity changes in late-stage prostate cancer
Prostate cancer is one of the most common cancer types in Turkish men. At nearly all stages of the disease, the androgen receptor (AR) is the main driver of initiation and growth. When activated, the AR binds to enhancer cis-regulatory elements and induces gene expression through chromatin loops that connect enhancers with target promoters. Given its critical function, targeting AR activity is the standard of care to treat metastatic or recurrent prostate cancer. While treatment is initially effective, resistance inevitably occurs. Yet even in these resistant tumors, AR remains the critical driver of proliferation in the vast majority of patients. There is increasing evidence that alterations to enhancer regulatory elements, either through cistrome reprogramming or somatic mutations, can act as acquired drivers of resistance. While both chromatin looping and AR's transcription factor function play an important role in enhancer activity, the extent of their contribution is not fully understood. In this work, we hypothesize that resistance in late-stage prostate cancer is driven by alterations in enhancer activity. Enhancer activity can be influenced by both changes in transcription factor activity and alterations in enhancer-promoter interactions. To investigate the changes in transcription factor activity, we functionally characterized the enhancer activity of a constitutively active AR splice variant using a massively parallel reporter assay. Unexpectedly we found that ARv7, a variant linked with resistance in clinical disease, repressed enhancer activity of AR. Next, we integrated the chromatin looping data from models of distinct stages of prostate cancer and identified significant changes in enhancer- promoter interactions in later stages of the disease. We detected new enhancer-promoter interactions in the late-stage model that are absent in the early-stage counterpart. This suggests that new chromatin interactions can change enhancer activity and may potentially contribute to resistance in late-stage prostate cancer. These results highlight the complex interplay between AR and chromatin organization, shedding light on novel mechanisms driving late-stage prostate cancer.
Nutrigenetik ve mikrobiota testlerinin bireye özgü klinik kullanımı için test panellerinin oluşturulması
İnsan genom projesinin 2003 yılında tamamlanmasının ardından insanlar arasındaki genetik çeşitliliğin insan sağlığı üzerindeki etkisi daha iyi anlaşıldı. 4P (Predictive (Tahmin Edici), Preventive (Önleyici), Personalized (Kişiselleştirilmiş), Participatory (Katılımcı) tıp olarak da adlandırılan hassas tıp, kişisel sağlık risklerinin belirlenmesinde, kronik hastalıkların önlenmesinde, teşhis edilmesinde ve tedavisinde kullanılmakta ve bireysel sağlığı etkileyen fenotipik, genotipik ve çevresel faktörlerin belirlenmesini amaçlamaktadır. Beslenmede hastalıkların önlenmesinde, tedavisinde veya desteklenmesinde önemli unsurlardan biridir. Hassas sağlık sistemleri anlayışında kişiye özel yaklaşımın kullanıldığı ve kişiye özel beslenmenin önemi artık daha da artıyor. Kalıtsal genetik varyantların nutrasötiklerin alımı ve metabolizması üzerindeki etkilerini araştırmayı ve hastaların nutrigenetik temelli epigenetik takviye tercihlerinin etkisini vurgulamayı amaçlamaktayız Kişilerin beslenmesinde nutrigenetik temelli beslenmenin önemi anlaşılmakla beraber. Nutrigenetik ve nutrigenomik araştırmaları multidisipliner klinik uygulamaya dönüştürmek en zorlu olanıdır. Artık fenotip ve genotip ile ilgili verilerin entegre edilmesi gerektiği açıktır. Fenotip ve bireylerin genetiğine uygun beslenme, yaşam tarzı ve takviyelerin kullanılması klinik başarıyı artıracaktır. Burada önemli olan, eğer epigenomik bir yaklaşım benimsemek istiyorsak, nutrigenetiği, mikrobiyotayı ve kişiselleştirilmiş risk analizlerini test sonuçlarından çıkarmamız gerektiğidir. Nutrigenomik alanını daha derinlemesine araştırdıkça, bir bireyin genetik yapısını anlamanın, potansiyel riskleri en aza indirirken diyet seçimleri ve takviyelerinin potansiyel faydalarını optimize etmek için hayati önem taşıdığı giderek daha açık hale geliyor. Anahtar kelimeler: Kişiselleştirilmiş beslenme, Nutrigenetik, Nutrigenomik, Epigenetik, Nutrigenomik, Mikrobiyota, Halk Sağlığı
Transcriptional profiling in Arabidopsis thaliana SLIM1 mutant exposed to boron toxicity
The effects of B (B) toxicity on plants have been investigated for a long time, but the interaction between sulfur metabolism and B has not been sufficiently clarified in this process. Sulfur metabolism plays an important role, especially in the production of compounds such as cysteine, methionine and glutathione (GSH), and is considered a critical component in the stress response of plants. In this study, the effects of sulfur metabolism in response to B toxicity and the role of the SLIM1 transcription factor in this process were investigated. SLIM1 is an important transcription factor that controls the synthesis of sulfur-containing amino acids by regulating sulfur uptake and metabolism. The deficiency of this factor in slim1 mutants leads to disruptions in the functioning of sulfur metabolism. In this study, the effects of sulfur metabolism were investigated in slim1 mutant Arabidopsis thaliana plants exposed to B toxicity.To better understand the functions of sulfur (S) uptake and metabolism in plants at the molecular level, it is necessary to investigate these processes under the effects of B toxicity. For this purpose, in this study, wild-type and slim1 mutant plants of Arabidopsis thaliana exposed to 1 mM and 2 mM B and RNA-Seq analysis were performed. As a result of the analyses, significant changes were observed in genes related to sulfur metabolism and transport in the slim1 control group. While a significant increase in ribosome biogenesis, RNA processing and binding activities was detected in slim1 mutants under B toxicity, glucosinolate biosynthesis processes were suppressed in all slim1 mutants. In addition, it was revealed that ribosome biogenesis and RNA processing processes, together with antioxidant mechanisms, play critical roles in the regulation of defense responses against B toxicity.This study provides important contributions to the understanding of the mechanisms of resistance to B toxicity and reveals that targeting SLIM1 and related mechanisms provides valuable clues for the development of plant varieties resistant to B stress conditions.
Çinko oksit nanopartiküllerinin yeşil sentezi, karakterizasyonu, sitotoksisitesi ve antimikrobiyal aktivitesi
Bu çalışmada, Sideritis akmanii bitkisinden elde edilen ekstrakt kullanılarak yeşil sentez tekniği ile çinko oksit nanopartikül (ZnONP)'ler sentezlendi. Elde edilen ZnONP'lerin Ultraviyole-Görünür Spektrofotometrisi (UV-VIS), X Işını Difraksiyonu (XRD) spektrofotometresi, Taramalı Elektron Mikroskopisi (SEM) ve Fourier Dönüşümlü Kızıl Ötesi Spektroskopisi (FT-IR) teknikleri ile karakterizasyonu gerçekleştirildi. Yeşil sentez tekniğiyle sentezlenen ZnONP çözeltisinin maksimum absorbans değeri 365 nm olarak kaydedildi. Nanopartiküllerin XRD analizi sonucu elde edilen Bragg tepe noktalarının JCPDS modeli 36-1451 ile uyumlu olduğu ve nanopartiküllerin altıgen yapıda olduğu tespit edildi. XRD ve SEM analizi sonucunda 2θ açısındaki en yüksek pik 36.262, FWHM değeri 0.437 ve ZnONP partikül boyutu 21.26 nm ve küresel şekilde olduğu tespit edilmiştir. Sideritis akmanii ekstraktının ve sentezlenen ZnONP'lerin A549 hücre hattı üzerindeki sitotoksik etkileri konsantrasyon ve zamana bağlı olarak değerlendirildi. ZnONP için LD50 dozu NP50 (5 mg/mL) olarak belirlendi. ZnONP'lerin, Sideritis akmanii özütüne kıyasla çok düşük konsantrasyonlarda bile daha yüksek sitotoksik aktivite gösterdiği tespit edildi. ZnONP'lerin ve bitki ekstraktının A549 hücre hattı üzerindeki genotoksik etkileri 24 ve 48 saatlik periyotlar boyunca karşılaştırıldı. A549 hücre hattı üzerinde ZnONP'lerin farklı dozlarda farklı genotoksik etkilere sebep olduğu gösterildi. Çalışmada uygulanan bitki ekstraktının üç konsantrasyonunun da 24 ve 48 saatlik uygulamada genotoksik hasarı azaltıcı yönde etki oluşturduğu görüldü. Sentezlenmiş ZnONP 25'in hem 24 saat hem de 48 saat uygulamasında kontrol grubuna kıyasla genotoksik hasarı gerilettiği tespit edildi. ZnONP'lerin Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Salmonella enteritidis (ATCC 13076), Klebsiella pneumoniae (ATCC 700603) ve Candida albicans (ATCC 90028) mikroorganizmaları üzerindeki antimikrobiyal aktivitesi disk difüzyon yöntemi kullanılarak analiz edildi. Disk difüzyon testi sonuçları, ZnONP'lerin 8-13.67 mm arasında değişen inhibisyon zonu çapları ile 5 mikroorganizma suşu üzerinde etkili olduğunu ortaya koymuştur. 24, 48 ve 72 saatlik ZnONP doz uygulamalarının en etkili olduğu suşun Salmonella enteritidis olduğu tespit edildi. En yüksek inhibisyon çapı ise 72 saatlik ve 250 mg/mL ZnONP doz uygulaması yapılan Salmonella enteritidis suşunda 13.67 mm olarak kaydedildi. Elde edilen sonuçlar, Sideritis akmanii ekstresi ve sentezlenen ZnONP'lerinin çeşitli kanser tiplerine, bakteriyel ve fungal enfeksiyonlara karşı alternatif tedavi yöntemlerinin geliştirilmesinde faydalı olabileceğini göstermektedir.
Molecular identification and antimicrobial resistance profiles of Escherichia coli isolated in urine cultures from Shamiya State Hospital (Iraq)
In this study, Escherichia coli strains isolated from urine samples of patients admitted to Shamiya State Hospital (Iraq) were identified by conventional and automatic microbiological methods. Escherichia coli by percentage 29%, Staphylococcus aureus by percentage 21%, Klebsiella pneumoniae by percentage 18%, Proteus mirabilis by percentage 12%, Enterococcus faecalis by percentage 16% and Pseudomonas aeruginosa by percentage 4%. In our study, we used 11 Antibiotics (CFM, AM, AK, CN, CTX, ATM, CIP, LEV, IPM, TET, MRP) to show resistant, intermediate and susceptibility of Escherichia coli. The presence of Tet-A, Qnr-B, Ctx-M, TEM resistance genes in the identified E. coli strains was investigated by PCR. The CTX gene is a resistant gene compared to other genes in the total of bacterial isolates. The TET-A and TEM genes are a weakly resistant genes compared to the CTX gene. The QNRB gene is the weakest resistant gene compared to the CTX gene among the total isolates of bacteria. Demographic characteristics of individuals from whom E. coli strains were isolated were determined. The results obtained contributed to the development of clinical strategies for the prevention and management of E. coli infections causing UPEC in the Shamiya region.
In vitro analyzes of synergistic anticancer effects of sorafenib and lipoic acid combination on hepatocellular carcinoma cells
Hepatocellular carcinoma (HCC), the most common type of liver cancer, represents the third leading cause of cancer death worldwide. Its complex pathogenesis and molecular heterogeneity complicate the treatment of liver cancer cases. Therefore, the need for the development of targeted therapies is increasing. It is anticipated that the use of Alpha Lipoic Acid (ALA) together with various antineoplastic drugs will be promising for cancer control and treatment due to its ability to interact with cells at molecular resolution and its protective effects. This study aimed to determine the synergistic, anti-proliferative and anti-cancer effects of different combinations of ALA on HepG2 cell line by cell viability test (MTT) and expression levels of some genes (Tnf-α, Bax, Caspase-3 and Bcl2) and to evaluate the protein levels of the same genes by ELISA method, as well as to detect possible DNA damage by Comet test. According to the findings obtained from MTT test, the group that was administered only Sorafenib showed approximately 42% cell viability after 24 hours of application, indicating that the compound alone showed significant cytotoxic effect. Similarly, the group that was administered only 5 µM ALA showed approximately 56% cell viability, and it was observed that ALA alone reduced cell viability. As a result of the application of ALA + Sorafenib combination, 85% cell viability was detected. In the 48 hour application, cell viability was approximately 45% in the Sorafenib group, and 47% in the 5 µM ALA group. In the ALA + Sorafenib combination group, cell viability was 48%. In the comet test results, DNA damage was determined as 20.33±2.52 for 24 hours at the Sorafenib 20 µM dose and 25.33±4.16 for 48 hours. The lowest DNA damage in the data was determined to be the ALA dose with 4.67±2.08 at 24 hours. An increase in DNA damage was observed in 48 hour ALA application, and this damage was determined as 15.67±1.15. DNA damage caused by Sorafenib decreased in both 24 and 48 hour applications compared to the combination application. The effects of Sorafenib and ALA combinations for 24 and 48 hours in HepG2 liver cancer cell line were evaluated by examining the expression levels of Bax, Bcl-2, Caspase-3 and Tnf-α genes involved in apoptotic and inflammatory processes. According to the qRT-PCR data obtained, significant changes were observed in gene expressions in both periods. Again, the levels of key gene products related to apoptosis and inflammation, such as Bax, Bcl-2, Caspase-3 and TNF-α, were determined in both 24 hour and 48 hour applications, and it was found that gene expression levels and protein products were generally consistent. The findings from all these studies suggest that combination therapy produces more pronounced effects compared to single agents in most cases. While increases in the expression of pro-apoptotic genes, especially Bax and Caspase-3, reveal the effects of the applied agents on cell death mechanisms, changes in anti-apoptotic genes, such as Bcl-2, are critical to understanding how this process is regulated. Keywords: Alpha-Lipoic Acid; Hepatocellular Carcinoma; HepG2 Cell Line; Sorafenib; Cytotoxicity, Genotoxicity, BAX, BCL-2, CASPASE-3, TNF-α
Eylais setosa (Acari, hydrachnidia) türünde kadmiyum (cd2+) uygulamaları ve antioksidan enzim aktivitelerinin belirlenmesi
Bu tez kapsamında, Eylais setosa (Acari, Hydrachnidia) türü su kenesinde kadmiyumun antioksidan sistem üzerine etkisi araştırıldı. Çalışmanın ana materyali olan su kenesi türü Çivril-Işıklı Gölü'ndeki sazlıklardan ve sığ sulardan arazi çalışmaları sonucunda temin edildi. Kadmiyum nitratın farklı konsantrasyonlarına [10-5 M Cd(NO3)2, 10-4 M Cd(NO3)2 ve 10-3 M Cd(NO3)2] bir hafta süre ile maruz bırakılan Eylais setosa türünde ICP-MS analizi ve enzim aktivite tayinleri (CAT, SOD, GSH-Px, GSH-ST ve GSH-Rd) gerçekleştirildi. Elde edilen veriler doğrultusunda ağır metal absorplama oranları ve enzim aktivitelerindeki değişimler değerlendirildi. ICP-MS analizi sonucunda Eylais setosa türünün kadmiyum ağır metalini artan konsantrasyonlarla doğru orantılı bir şekilde bünyesinde biriktirdiği tespit edildi. Kadmiyuma maruz kalmış grupların enzim aktivitelerinde ise kontrole göre istatistiksel açıdan önemli oranda düşüşlerin olduğu (p<0.05) belirlendi. Çalışmada elde edilen veriler bir bütün olarak değerlendirildiğinde enzim aktiviteleri ile sucul kirlilik arasındaki ilişkinin kullanışlı biyobelirteçler olduğu açıkça görülmektedir.
Pık3ca mutant meme kanserlerinde alpelisib (Byl-719)-fulvestrant aracılı büyüme engellemesine direnç oluşturabilecek sinyal ağı etkinliklerinin belirlenmesi
Luminal A breast cancer, characterised by hormone receptor positivity and human epidermal growth factor receptor 2 negativity, represents approximately 70% of all reported cases of breast cancer. Mutations in components of the PI3K pathway, particularly p110 and PTEN, are commonly observed subsequent to p53 mutations in these cancers. Consequently, PI3K inhibitors are employed to treat breast tumours characterised by PI3KCA mutations. Nevertheless, inhibiting the PI3K pathway may give rise to the activation of other signalling pathways, hence inducing the development of drug resistance. The objective of this study is to examine the potential activation of pathways associated with the FDA-approved combined use of Alpelisib (BYL-719) and Fulvestrant in the treatment of PIK3CA mutant breast cancer. In this study, MCF7 and T47D cell lines developed acquired resistance to the Alpelisib/Fulvestrant combination. The resistant cells were then subjected to comparative transcriptome analysis to identify putative resistance networks. Our results implicate various components of the MAPK pathway as a potential resistance factor in both cellular models. We also corroborated these results via immunoblot analysis of the respective signalling cascades. Therefore, we assessed the potency of the combination of Pemigatinib, an FGFR inhibitor and alpelisib and have shown that this combination is successful in inhibiting the growth of the resistant cell lines. We also supported our findings by performing 3D growth assays with this combination. The findings of our study will contribute to the advancement of therapeutic strategies for luminal A breast cancer, particularly in the context of Alpelisib/Fulvestrant drug resistance.
Astımı olan pedı̇atrı̇k covıd-19 hastalarında ekstraselüler vesı̇küllerı̇n rolü
SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected millions of people, and asthma was initially considered a risk factor for COVID-19. Although numerous studies on asthma and COVID-19 have been conducted, the role of plasma-derived extracellular vesicles (EVs) in COVID-19 patients with asthma remains unknown. In this study, we assessed the influence of EVs from healthy controls, severe and mild COVID-19 pediatric patients with or without asthma during acute and convalescent periods on healthy naïve CD4+T cells and monocytes. While plasma cytokines and anti-SARS-CoV-2 antibodies were similar between the groups with and without asthma, immune responses varied depending on the severity of COVID-19. In the severe acute group, whereas all cytokines increased, IFNγ, CD4+T cell counts, and monocyte numbers decreased. Stimulating healthy cells with EVs from severe acute patients led to increased PDL1 expression, Th2 and Treg cell proportions, decreased IFNγ secretion, Th1, and Th17 cell ratios. Patient EVs also reduced proinflammatory cytokine secretion from monocytes. Severe acute patient EVs caused a decline in healthy CD4+T cell and monocyte populations. Overall, our results indicate immunological responses and EV-related outcomes depending on the severity of COVID-19 rather than the presence of asthma, immunosuppression seen in severe acute COVID-19 and potential contribution of EVs to this immunosuppressive pattern in severe cases.
Farkli adjuvantlar ve li̇pozom kompleksasyonuyla geliştirilmiş SARS-CoV-2 VLP aşinin farelerdeki̇ bağişiklik gücünün değerlendi̇ri̇lmesi̇
SARS-CoV-2, emerging in December 2019 in Wuhan, China, led to a swift global pandemic declaration in March 2020, prompting widespread vaccine development. Most vaccines target specific regions, mainly the spike protein. In our lab, we have employed an innovative virus- like particle (VLP) vaccine approach encompassing all four structural proteins of the virus: spike, nucleocapsid, membrane, and envelope. Our study utilizes sterically stabilized cationic liposomes (SSCL) to encapsulate VLPs expressing the Delta variant spike protein, along with various adjuvants: CpG ODN, poly(I:C), and 2'3'-cGAMP. We characterized liposomes using tunable resistive pulse sensing for size and concentration. In C57BL/6 mice, we administered primary and two booster injections on Day 0, Day 15, and Day 73, respectively, collecting blood samples at intervals (Day 14, Day 28, Day 42, Day 72, and Day 90). To assess vaccine impact on mouse humoral immunity, we conducted ELISAs for total IgG, IgG1, and IgG2c antibodies against recombinant Spike and the receptor-binding domain (RBD). IgG titers increased until Day 42, remained stable or slightly decreased on Day 72, and significantly rose on Day 90. We calculated IgG2c/IgG1 ratios, reflecting Th1 immune responses, revealing enhanced cellular immunity potential in groups with adjuvants compared to the VLP-only group. This study underscores the effectiveness of our VLP vaccine strategy in stimulating robust immune responses and opens avenues for further research and development.
Adenozin almaçlarının hedeflenerek aşı etkinliğinin arttırılması
Vaccination is the major protection method against many diseases caused by pathogens through creating acquired immunity. Vaccines can be classified in two major groups, which are subunit vaccines and attenuated vaccines. Attenuated vaccines can create effective immunity however; they also can induce many different side effects such as fever and allergic reactions. On the contrary, with subunit vaccines side effects are decreased but the efficacy of the vaccines is also decreased and there is a need for repetitive vaccinations to provide long lasting immunity. That is why, there is a need for developing more efficient vaccines and particularly vaccine adjuvants. Adenosine receptors, as part of purinergic signaling, have a regulatory role in immune system. Adenosine and 4 different adenosine receptors have an immunosuppressive role in major immune cells to create acquired immunity such as DCs, macrophages and lymphocytes. That is why, we hypothesize that, the efficacy of vaccines can be decreased by endogenous adenosine and the usage of antagonists in adjuvant formulations can increase this efficacy by inhibiting the suppressive effects caused by endogenous adenosine. To be able to test this hypothesis, we first determine the specific adenosine receptor and antagonists taking a role in this immunosuppressive effect. For this purpose, we use primary dendritic cells and macrophages. We see that A2A and A2B receptors create most effective immunosuppression and SCH 58261 (A2A antagonist) and PSB 603 (A2B antagonist) are the main antagonists taking a role in the inhibition of this suppression. We then evaluated these two molecules in a vaccine formulation comprising MPL-A and AddaVax. As a result, these antagonists do not significantly change the general initial immune responses significantly however they create more antigen specific response. On the other hand, after antigen re-stimulation, mice taking these antagonists shows more antigen specific response and they also create higher antibody titers. With this study, adenosine receptor antagonists used in adjuvant formulations for the first time and it was shown that, with more study, they can be important in increasing vaccine efficacy created by immunostimulatory adjuvants. Keywords: Adenosine, Adenosine Receptors, Vaccines, Adjuvants,
Ökaryotik canlılarda korunmuş olan Loc115098 geninin işlevsel tanımlandırılması
Loc115098 is a newly identified gene in our laboratory. The gene was firstly discoveredin an unrelated study in which the cis-acting regulatory elements of a neighboring geneNIS (Sodium-Iodide Symporter) were investigated. It is composed of 4 exons. Accordingto the computational predictions, it has rather interesting features such as having acomposition of mostly charged amino acids, carrying a nuclear localization signal andbeing well conserved throughout the eukaryotic kingdom. Our aim in this study was tounderstand the function of this new gene. Firstly we confirmed that it is expressed inseveral different tissues, later we conducted a subcellular localization study whose resultsled us to hypothesize that the function of loc115098 could be related to cell cycleregulation. According to our hypothesis we decided to utilize a lower eukaryote carryinga human homologue of loc115098 which we selected to be Aspergillus nidulans. It was avery useful model organism for our study not only because it carries a very similarhomologue of loc115098 but also it is an easily handled organism which is widely usedfor genetic studies. Firstly we compared the expression profile of loc115098 in severalcell cycle mutants of A. nidulans as well as a wild type strain, the results indicated thatthe expression of loc115098 decreases in the restrictive temperature of cell cycle mutantstrains. We also conducted subcellular localization studies in A. nidulans and the resultsindicated that it was mostly cytoplasmic and sometimes perinuclear. Our other aim was toutilize A. nidulans as a model for our knock-out experiments. We designed a knock-outsystem by the help of a sophisticated method called Double-Joint PCR. Our first attemptssupported that the knock-out of loc115098 was likely to cause lethality in the organismand therefore we improved our system to conditionally knock out the gene. Ourpreliminary results indicated that the knock out of loc115098 causes cell death and wesuspect that it could be due to a damage in one of the crucial metabolic events for cellviability such as mitosis. Our attempts to functionally characterize the function ofloc115098 are currently going on.
Protein hücre içi yerleşim ve gen ifadesi verileri için analizler ve örün arayüzleri
In order to beneï¬t maximally from large scale molecular biology data gener-ated by recent developments, it is important to proceed in an organized mannerby developing databases, interfaces, data visualization and data interpretationtools. Protein subcellular localization and microarray gene expression are twoof such ï¬elds that require immense computational eï¬ort before being used asa roadmap for the experimental biologist. Protein subcellular localization is im-portant for elucidating protein function. We developed an automatically updatedsearchable and downloadable system called model organisms proteome subcellu-lar localization database (MEP2SL) that hosts predicted localizations and knownexperimental localizations for nine eukaryotes. MEP2SL localizations highly cor-related with high throughput localization experiments in yeast and were shownto have superior accuracies when compared with four other localization predic-tion tools based on two diï¬erent datasets. Hence, MEP2SL system may serve asa reference source for protein subcellular localization information with its inter-face that provides various search and download options together with links andutilities for further annotations. Microarray gene expression technology enablesmonitoring of whole genome simultaneously. We developed an online installablesearchable open source system called diï¬erentially expressed genes (DEG) thatincludes analysis and retrieval interfaces for Aï¬ymetrix HG-U133 Plus 2.0 ar-rays. DEG provides permanent data storage capabilities with its integration intoa database and being an installable online tool and is valuable for groups whoare not willing to submit their data on public servers.Keywords: protein subcellular localization prediction, microarray gene expression,eukaryotic model organisms, web interface and database, proteome.iv
Jüvenil idiyopatik artrit hastalığında bozuk x-kromosomu etkinsizleştirilmesi
Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease withfemale predominance and an incidence between 7-21/100,000. There are several explanationsfor the reason of disease development, such as environmental factors that triggerautoimmunity and genetic basis. The genetic basis of JIA is not well defined. It rarelymanifests familial recurrence. But the monozygotic twin data suggest that there is aconsiderable genetic basis, which is likely to involve multiple epigenetic events. It wasproposed that a disturbance in mosaicism of females may cause autoimmune diseasedevelopment. Recently, in our lab, an association between extremely skewed X-chromosomeinactivation (XCI) patterns and female predisposition to autoimmunity was identified. SinceJIA is thought to have an autoimmune etiology, we hypothesized that skewed XCI might playa role in the disease development. To determine XCI status, androgen receptor locus wasanalyzed by methylation sensitive Hpa II digestion followed by PCR by using of 72 femalepatients diagnosed with JIA and 183 female controls, which comprised of newborns (n=91)and children with no history of an autoimmune condition (n=92). A male control (46, XY)was used for complete digestion in the analysis of XCI pattern. We expect to see anassociation between extremely skewed XCI and female predisposition to JIA.
İnsan hepatoselüler karsinom hücre hatlarının tek nükleotid polimorfizm yongaları kullanılarak karyotiplendirilmesi
Hepatocellular carcinoma (HCC) etiology is genetically heterogeneous; multiple different mechanisms have been shown to promote hepatocarcinogenesis. However, chromosomal aberrations (CAs) and signaling pathways that they alter are still poorly understood. Changes in chromosome number (aneuploidies) or structural chromosomal aberrations, such as; amplifications, deletions, loss of heterozygosity and recessive mutations are important mechanisms for tumor evolution. Recently developed single nucleotide polymorphism (SNP) microarrays provide high-throughput quantitative and qualitative screening of genomic DNA with higher resolution compared to conventional methods such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). In cancer research, SNP arrays ease the screening of structural changes as well as aneuploidies with exact physical position. In the framework of this study, we aimed to detect DNA copy number alterations in a panel of 14 HCC cell lines. We screened all the autosomal chromosomes and the X-chromosome and found previously undescribed novel regions that harbor homozygous and hemizygous deletions at 13q12 and Xq21; amplifications at 8p23, 8q13, 8q24, 9p22-21, 12p1, 14q12, 15q21, 16q23, 17p12-p11, 17q11, 22q11 and Xp22. In our knowledge, our results are the first comprehensive high-throughput screen of commonly used HCC cell lines.
Doğala yakın öldürücü T hücre alt gruplarının karakterizasyonu
iNKT cells are a unique subset of T cells. Unlike T cells, they recognize glycolipids via MHC1 like molecule, CD1d. After activation, they can produce both Th1 and Th2 cytokines. iNKT cells have several subsets. These subsets are distinguished by the transcription factors they express and the cytokines they produce. NKT1, NKT2, and NKT17 cell express the transcription factor PLZF, TBET, and RORγt at different levels. NKT2 cells are known as the main producer of iNKT cell-derived Th2 cytokines. However, in this study, splenic NKT2 cells produced less IL-4 than NKT1 cells after activation with either αGalCer, or PMA/Ionomycin. Therefore, we hypothesize that peripheral NKT2 cells are hypo-responsive. Additional work on cell signalling (NUR77) confirmed this hypothesis. Knowledge of the main source of iNKT cell-derived Th2-cytokines is essential to understand the role iNKT cells can play in various diseases and to improve iNKT cell-related diagnosis and therapy.
3 boyutlu hepatik organoidlerde lizozomal asit lipaz eksikliği modelinin geliştirilmesi
In the last decade, organoids have become the most powerful cell culture tool to study human biology in health and disease. Organoids are three-dimensional structures grown in an extracellular matrix that resemble organ structure and show organ-specific functions. Recently established protocols for liver-specific organoids enables the understanding of disease pathology and improvement of patient-specific therapies. Our aim in this work is to utilize organoid technology to model Lysosomal acid lipase deficiency (LALD). Lysosomal acid lipase (LAL) is an enzyme, mainly found in the liver, that breaks down cholesterol esters into free cholesterol. In the absence of the LAL enzyme, cholesterol esters begin to accumulate in liver cells and a spectrum of symptoms can be observed. The mild form with 3%-5% enzyme activity is called cholesterol ester storage disease (CESD) and it can go undiagnosed for years but the severe form is Wolman's disease with less than 1% LAL enzyme activity. Unless treated, WD patients have a life span of maximum a year. LALD models are limited to animal models. These models are completely inadequate when a personalized approach is desired to be developed. Until recently, there were no cell lines that model LALD, however, a recent study has established Wolman Disease organoids from patient-derived iPSCs and thus, paved the way for the understanding of molecular mechanism and treatment options for the disease. In this thesis, first, we have established iPSC clones stably expressing an inducible shRNA vector for the LIPA gene, then we have created an iPSC derived 2D hepatocyte and 3D endodermal hepatic organoid (eHEPO) model for the LALD. LAL deficient 2D hepatocytes have shown to have decreased levels of LAL enzyme activity in comparison with healthy counterparts. On the contrary, our eHEPO model has not been successful to show LAL enzyme activity impairment after a 40% reduction in gene expression. Thus, we conclude that a more dramatic decrease in the gene expression level is needed to mimic the disease phenotype. This suggests that strict regulation of the enzyme activity is present in the 3D organoid model and further investigation of molecular changes and regulation pathways is needed to understand the disease mechanism.
P53 mutant kolon kanseri hücrelerinde 1,8-cineolenin antikanser etkilerinin araştırılması
Kolon kanseri (KK), dünya genelinde en yaygın kanser türlerinden biri olup, hem erkekler hem de kadınlar arasında önemli bir sağlık sorununu teşkil etmektedir. Erken teşhis ve tedavi yöntemlerindeki ilerlemelere rağmen, ileri evre kolon kanseri vakalarının tedavisinde günümüzde de zorluklar yaşanmaktadır. Bu zorlukların başında, kanser hücrelerinin kemoterapiye ve diğer tedavi yöntemlerine karşı geliştirdiği ilaç direnci gelmektedir. Kolon kanseri hücrelerinde gözlenen P53 mutasyonları hücresel ilaç direncinin gelişmesine aracılık eder ve buda kanser hücrelerinin apoptozdan kaçmasına ve artan metastaz yeteneğine neden olur. Bu olumsuzlukların aşılmasında kombine tedavi seçeneklerine ek olarak kanser ilaçlarının etkinliğini arttırıcı ve ilaç direncini azaltıcı doğal bileşiklerin tedavide kullanılması gün geçtikçe artan bir yaklaşımdır. 1,8-cineole, doğada çeşitli bitkilerde bulunan monoterpen sınıfı uçucu yağ bileşenidir. Bu bileşiğin antienflamatuar, antioksidan ve antikanser özellikler ortaya koyduğu rapor edilmektedir. Buna ek olarak yapılan çalışmalar kanser hücrelerinde 1,8-cineole'nin ilaç direncini azaltarak kemoterapik etkinliği arttırabileceği bildirilmektedir. Bu çalışmada P53 mutasyonlu kolon kanseri hücrelerine karşı 1,8-cineole'nin sitotoksik, genotoksik ve ilaç direnci genleri üzerine etkilerini ortaya koymayı amaçladık. Çalışmada HT-29 insan kolon kanseri hücreleri üzerine standart kemoterapotik ilaç 5-Fluorourasil (5-FU) ve 1,8-cineole'nin ayrı ayrı ve kombine uygulamaları sonrası hücre canlılık düzeyleri MTT analizleriyle belirlendi. Sonrasında bileşiklerin DNA hasarına etkileri tek hücre jel elektroforezi yöntemiyle analiz edildi. Çalışmada ayrıca 1,8-cineole'nin kanser hücrelerinde ilaç direncine neden olan ABCB1, ABCG2 ve ABCC2 genlerinin mRNA ifade düzeylerine etkileri de araştırıldı. Sonuçları uygun istatistiksel yöntemlerle analiz edildi. Sonuçlarımız, 1,8-cineole'nin tek başına ve 5-FU ile kombinasyon halinde HT-29 kolon kanseri hücre canlılığını anlamlı düzeyde azalttığını gösterdi (p<0.05). 5-FU uygulanan gruplarda DNA hasar düzeyi kısmen artış gösterdi (p<0.05). Ancak 1,8 cineole uygulaması sonrasında hücrelerde genotoksik etki gözlenmedi. 1,8-cineole 5-FU uygulaması sadece 5-FU uygulanan gruba kıyasla ABCC2 gen ifadesinde azalmaya neden oldu. Elde edilen sonuçlar 1,8-cineole'nin 5-FU ile sinerjik etki gösterebileceğini işaret etmektedir.
NRG1 genindeki iki SNP'li bir haplotip ile şizofreni hastalığının genetik ilintisinin incelenmesi
The earliest gene reported to be linked with schizophrenia is NRG1 which has a number of susceptibility alleles at many SNP (single nucleotide polymorphism) loci. In this study, the genetic association of schizophrenia with two SNPs located in the NRG1 gene -rs4560751 and rs3802160- has been investigated. The study has been planned in case-control format. The case group was consisted of 96 schizophrenia patients and the control group was 100 healthy controls living in Malatya-Turkiye. Two SNPs mentioned above have been genotyped in these groups and the distributions of their alleles and genotypes were compared between two groups. The statistical significances of differences were tested with Pearson's Chi-Squared method. The statistical tests showed there were no significant difference between the case and control groups for the distributions of the alleles or genotypes of two NRG1 SNPs. As conclusion, our study revealed that the SNPs rs4560751 and rs3802160 in the NRG1 gene were not associated to schizophrenia in our sample.
CRY4BA ve CRY11AA ile etkileşen Asya kaplan sivrisineği proteinlerinin insektisit mekanizması
The Asian tiger mosquito (Aedes albopictus) is a mosquito species native to Southeast Asia that has now spread across Türkiye. Given its vector potential and remarkable ability to colonize various environmental conditions, Ae. albopictus is considered one of the most significant invasive mosquito species globally. Under experimental conditions, this mosquito species has demonstrated the potential to transmit four dengue serotypes, yellow fever, chikungunya, Ross River virus, and at least 22 other arboviruses. Consequently, preventing the spread of invasive mosquitoes necessitates the use of environmentally friendly pesticides that are species-specific and have minimal impact on non-target organisms. In this study, we aimed to investigate the larvicidal effects of Cry4Ba and Cry11Aa toxins, which are among the most effective biopesticides for targeting Aedes species. Furthermore, we sought to identify the receptors for Cry4Ba and Cry11Aa toxins to elucidate their mechanisms of action in Ae. albopictus. Despite the critical role of toxin-receptor interactions in Cry toxin efficacy, the receptors that interact with Cry toxins in Ae. albopictus have not yet been characterized. Our findings revealed that two alkaline phosphatases, a vacuolar-type ATPase subunit B, and a maltase-like receptor serve as functional receptors for Cry4Ba and Cry11Aa in Ae. albopictus larvae. Additionally, we demonstrated that the susceptibility of mosquito larvae to Cry toxins is influenced by the downregulation of these receptor gene expressions using RNA interference. These results provide new insights into the mechanisms of Cry toxin action and may contribute to developing more effective mosquito management strategies.
Hücre yüzeyi proteinlerinin analizi için yeni bir metod geliştirilmesi
Plasma membrane is an evolutionary conserved cellular compartment for all cells. It directly or indirectly participates in vital pathways in the cell such as nutrition uptake, signal transduction, cell to cell communication, migration, adhesion, and cell division. Therefore, to elucidate molecular mechanisms of the cell or regulation of these pathways, we need to understand how plasma membrane components interact with inner part of the cell and their specific roles in these pathways. One unbiased way to address these questions is to take proteomics approach. Yet, unique structure of the plasma membrane and low abundancy of the cell membrane proteins make it challenging to study plasma membrane proteins with traditional proteomics approach. Therefore, there is a need for more effective approaches for identification of plasma membrane proteins. In the first part of my thesis, I developed a novel approach for plasma membrane protein enrichment. In this method, I used centrifugation and BioID enrichment methods subsequently. To that extent, I modified biotin ligase (BirA) enzyme to target and biotinylate cell membrane proteins. Then, by using subsequent physical and affinity-based enrichment methods, plasma membrane protein levels increased in the sample. Proteins then identified by using mass spectrometry. In the second part of my thesis, I focused on two members of Chloride Intracellular Channel (CLIC) proteins, CLIC1 and CLIC4. In a recent quantitative proteomics study from our laboratory showed that CLIC1 and CLIC4 enriched at the plasma membrane during cell division Currently, CLICs are not associated with any cell cycle dependent function. In the second part of my thesis, I focused on the characterization of the CLIC1 and CLIC4 proteins in cell cycle dependent manner. To that extent, I generated CRISPR-Cas9 mediated knockout cell lines and investigated their effects on cytokinesis fidelity. CLIC1 and CLIC4 knockout cells increased cytokinesis abnormalities indicating their role at the plasma membrane during cell division.
CCDC57 mikrosefali proteini CEP63 ile birlikte sentriol çoğalmasını ve mitotik progresyonu düzenler
Centrosomes play critical roles in diverse cellular processes ranging from cell division to cellular signaling. At the core of the centrosomes are two centrioles, which duplicate only once per cell cycle and this duplication cycle is tightly regulated. Accordingly, their deregulation causes diseases such as cancer and developmental disorders like primary microcephaly. Complete understanding of the mechanisms that regulate centrosome biogenesis and function is required to elucidate the disease mechanisms. In this study, we identified a new centrosome protein CCDC57, which also localizes and binds to centriolar satellites and microtubules. Proximity-labeling and interaction studies showed that CCDC57 forms a complex with Cep63, centriolar satellites and microtubules. Importantly, characterization of CCDC57 deletion mutants revealed that its N-terminal 1-502 amino acid region mediated its centrosomal localization and interactions, whereas the C-terminal 606-916 amino acid region mediated its localization and binding to microtubules. Loss of CCDC57 causes defects in canonical centriole duplication and centriole amplification, and results in a failure to localize microcephaly-associated proteins Cep63 and Cep152 to the centriole during initiation of centriole duplication. Additionally, CCDC57 depletion resulted in prolonged mitosis, increased apoptosis and a higher ratio of mitotic defects such as misaligned chromosomes and multipolar spindles. Together, our results identify CCDC57 as dual regulator of the Cep63 centriole duplication module and microtubule-mediated mitotic processes and provide new mechanistic insight into defects underlying cancer and primary microcephaly.
Hidrofobik protein DewA'nın Pichia pastoris ile rekombinant üretimi ve biyoteknolojik uygulamaları
Hidrofobinler, adeze olarak yüzey karakteristiğini değiştirebilme özelliklerinden dolayı birçok biyoteknolojik uygulamada büyük potansiyele sahiptir. Son yıllarda, hidrofobinlerin biyoteknolojik uygulamalarında belirgin bir artış olmakla birlikte, endüstriyel boyutlarda üretimi verim problemlerinden dolayı hala başarılmış değildir. Bu nedenle hidrofobinlerin rekombinant üretimi üzerine daha fazla çalışmaya ihtiyaç vardır. Bu tez çalışmasında literatürde yüksek temas açısına sahip olduğu belirlenen Aspergillus nidulans'a ait sınıf I hidrofobin DewA proteininin rekombinant olarak üretimi amaçlanmıştır. Bu amaçla ilk olarak, DewA geninin (UNIPROT: 52750) salgı sinyal dizisi ve dur kodonu çıkarılmıştır. Daha sonra elde edilen diziyi kesmeyen restriksiyon enzimleri biyoinformatik programı kullanılarak tespit edilmiş ve diziye EcoRI ve XbaI enzim kesim bölgeleri eklenmiştir. Ardından kodon optimizasyonu gerçekleştirilerek, dizi sentezlettirilmiştir. Sonuçta DewA proteini pPICZα-A vektörüne aktarılarak AOX1 promotörü altında Pichia pastoris X-33 suşu kullanılarak rekombinant olarak üretilmiştir. En yüksek verim 96. saatte 77 mg/L olarak %1'lik metanol konsantrasyonunda elde edilmiştir. Elde edilen rekombinant proteinin moleküler ağırlığı yaklaşık 15 kDa olarak bulunmuştur. DewA proteinin cam yüzeye kaplanması sonucunda, yüzeyin karakteristiğini değiştirerek hidrofobik hale getirdiği; teflon yüzeylerde ise yine yüzey karakteristiğini değiştirerek, hidrofilik hale getirdiği kanıtlanmıştır. Ardından bu yüzeylere sıcak SDS ve UV uygulaması yapılarak proteinin yüzey stabilitesi değerlendirilmiştir. Sonuçta yüzeye kaplanmış olan DewA proteininin hem cam hem de teflon yüzeylerde sıcak SDS uygulamasına dirençli olduğu; UV uygulamasında ise, cam yüzeylerde UV maruziyeti ile protein degrede olurken, teflon yüzeylerde yapısını koruduğu anlaşılmıştır. Tez çalışması ile A. nidulans'a ait DewA proteini, pPICZα-A vektörüne klonlanarak, P. pastoris X-33 suşunda AOX promotörü kontrolünde ilk kez rekombinant olarak üretilmiştir.
Turning the spotlight on a long-overlooked pathway: molecular dynamics investigation of ras family members with RalGDS
The Ras superfamily of GTPases is capable of switching on and off their downstream effector proteins. Activated effector proteins modulate a significantly complicated and wide range of cellular processes. Ras sub-family proteins are the most extensively studied branch of the superfamily as 30% of all human tumors have activating mutations in one of the RAS genes. Ras isoforms share high sequence and structural similarity yet adopt different behaviors upon the same oncogenic mutations. Another fact is particular Ras mutations preferentially activate different effector pathways for the same isoform. Considering these challenges, targeting a mutant Ras protein requires a prior investigation in an isoform-specific and effector-specific manner. Until a few years ago, downstream Raf and PI3K pathways have attracted more attention as they are directly responsible for tumor initiation and proliferation in Ras-driven cancers. Recent studies have shown that the RalGDS-Ral pathway is more decisive than Raf and PI3K pathways for the progression of Ras-driven colon and pancreatic cancers. In this study, a long-overlooked Ras-RalGDS interaction was investigated by the molecular dynamics method. Simulated Ras isoforms were selected based on the mutation frequencies and distinct functions in RalGDS (RBD) pathway. Gibbs free energy results showed that Rap1-RBD was the strongest while M-Ras -RBD was the weakest interaction among the selected systems. Upon G12V and G12D mutations, both K-Ras and H-Ras proteins showed an increased affinity toward RBD compared to their wild-type controls. It noted that Ras uses the same acidic interface residues while binding to complementary basic residues of RalGDS and Raf. E31 and D33 residues on Ras isoforms interact with the α-helix containing parts of the RBD, whereas E37 and D38 residues participate in intermolecular β-sheet interaction. GLU37Ras(β2)-ARG7RBD(β1) and ASP33Ras(SI)-LYS39RBD (h1) hydrogen bonds were conserved on both the Ras and RalGDS side among all simulation systems and suggested as a scaffold for Ras-RalGDS interaction. GLU37K-Ras-TYR18RBD bond was only observed in K-Ras mutant systems but not in K-Ras control and proposed as a suitable candidate for further drug-design studies. LYS31Rap1 residue was proposed to be responsible for the tightest interaction between Rap1 and RalGDS. ASP33Ras(SI)- LYS19RBD(β2) interaction which is only present in the M-Ras system and considering the locations of the residue pair this bond likely to distort the shape of the β2 of the RBD that cause destabilization in the M-Ras-RBD interaction and responsible for the weak binding affinity. Trajectory clustering analysis showed that H-RasCONTROL adopted state 1, and KRasCONTROL adopted state 2 conformations while interacting with RBD. Upon G12D mutation, H-Ras was switched between state 1 and 2 while H-RasG12V has remained in state 1. The distinct switch I profile of H-RasG12D and H-RasG12V in complex with RBD were suggested to arise from that reason. Another remarkable finding was increased cooperation between switch I and II in K-RasG12D, G12V, and H-RasG12V. On the contrary, the switch I region of H-RasG12D has exhibited an increased correlation with the allosteric lobe exclusively with helix 3. In mutant isoforms, the stability of switch I appeared to be achieved via diverse cooperation mechanisms yet can promote the overall strength of the interaction. In this study, only the G-domains of Ras proteins were used when modeling the interaction with RBD. Most of the results were consistent with the literature hence proved that even G-domain was enough to make some deductions about differential behaviors of H-Ras and K-Ras in complex with RBD. Here, represented interaction dynamics might be a good reference point for the studies which aim to develop mutant specific targeting against the tumors harboring Ral over-activity.
Genome-wide mapping of nucleotide excision repair in arabidopsis root and shoot & the fate of LYM1/2 proteins during nodulation in Medicago truncatula
Plants, unlike many other organisms, are not mobile, which makes them vulnerable to a variety of hazardous factors such as ultraviolet (UV) light. UV light is known to cause DNA damage, which in turn leads to genetic instability. Organisms have found ways to cope with DNA damage by different molecular mechanisms to preserve genetic information flow properly. Nucleotide excision repair (NER) is a conserved mechanism widely utilized by many organisms, including plants. In this study, The Excision Repair Sequencing (XR-seq) method has been applied to shoot, root, and seedling tissues in Arabidopsis thaliana to map genome-wide DNA damage types: CPD and (6-4) PP. Arabidopsis thaliana has been shown to have transcription-coupled repair (TCR) in addition to global genome repair (GG-NER). Interestingly, root tissue, despite not being exposed to UV light, has demonstrated a significant amount of nucleotide excision repair, whose levels are similar to tissues exposed to UV. Root tissue might exhibit such repair due to the chemicals in soil, which might give rise to the formation of various DNA damage products repaired by the NER mechanism. Together with this finding, the repair dynamics could be further investigated in the model organism Arabidopsis to understand better essential players of DNA damage, gene activity, and chromatin accessibility throughout the plant genome. Medicago truncatula is a model legume that undergoes a mutualistic relationship with rhizobia under nitrogen-limited conditions to obtain ammonium (NH3). In this interaction, rhizobia produce ammonium from atmospheric nitrogen (N2) and provide it to the plant and in return, use the carbon source supplied by the plants. However, the question and mechanism of how the bacteria are allowed to maintain their cellular processes inside nodule organs within the root tissue of plants remain to be addressed in great detail. Lysin motif-containing receptor (LysM) proteins, LYM1 and LYM2, reside on plants' plasma membranes and are responsible for triggering innate immunity in response to patterns of bacterial peptidoglycan structures. Furthermore, upon arrival of rhizobia, the GPI anchors of LYM1 and LYM2 proteins are known to be cleaved that releases these proteins from symbiosome membrane. Thus, these proteins are inactivated to initiate an immune response so the intracellular rhizobia could survive, and the symbiotic relationship can be attained. This study includes cloning of LYM1 and LYM2 genes into plant expression vectors and genotyping lym1 and lym2 mutants. The questions of what the other interacting partners of LYM proteins are and what happens to LYM proteins in the absence of DNF2 protein need to be resolved in further studies.