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Farklı çinko ve azot uygulamaları altında yetişen buğday ve mısırda çinkonun alımı, taşınması ve tanede birikimi
Chronic zinc (Zn) deficiency is a major health issue affecting over two billion people, caused by heavy reliance on staple crops (i.e. wheat, rice and maize) which are inherently low in Zn. This project was devoted to reveal the individual and combined effects of genetic and agronomic Zn biofortification in wheat and maize. The first part focused on understanding the mechanisms involved in differences in uptake and translocation of foliar-applied Zn among wheat and maize species. It was shown that wheat has a greater capacity of leaf uptake and translocation of foliar-applied Zn compared to maize. The second part investigated the effect of nitrogen (N) supply on uptake and accumulation of Zn in maize and wheat. Improving N supply significantly enhanced the shoot accumulation as well as leaf uptake of Zn from foliar Zn sprays in wheat and maize. The third part studied the effectiveness of Zn fertilizers in the form of soil, foliar and soil + foliar for improving growth, grain yield and nutrients uptake by genetically biofortified HarvestPlus wheat genotypes. It was demonstrated that the genetically biofortified genotypes have higher capacity to uptake, utilize and translocate Zn from soil and/or foliar applications as compared to conventional cultivars. These results conclude that the most sustainable way of tackling human Zn deficiency would be to improve grain Zn concentration of cereal crops by unifying genetic and agronomic biofortification strategies.
Küçük hücreli dışı akciğer kanserinde p53 tarafından regule edilen hk1b metabolic tedavi için yeni bir hedeftir
Deregulation of glycolysis is common in non-small cell lung cancer (NSCLC). p53 controls the cellular metabolism pathway by activating hexokinases (HKs) to increase glycolysis. HK enzymes catalyze the phosphoryl-group-transfer in glucose metabolism. Unlike HK2, HK1 has several transcript variants. However, the functional differential roles of HK1 isoforms in glucose metabolism and the malignant tumor progression are still elusive. Here, we show that primary NSCLC patient tumor cells metabolically differ from the normal lung epithelium in that they display predominant expression of one of the HK1 isoforms, hexokinase1b (HK1b). Interestingly, we show that p53 positively regulates HK1b expression using CRISPR-Cas9 system. We utilized CRISPR-Cas9 system to selectively target specific HK1b isoform in NSCLC and show that silencing HK1b in NSCLC cells inhibits tumorigenesis through diminishing glycolysis and proliferation. Finally, HK1b deletion sensitizes NCLC cells to cisplatin treatment and the combination therapy synergistically increases both the p53- mediated apoptotic cell death by cisplatin and autophagic cell death by increased formation of LC3-II associated autophagic vesicles and myelinoid bodies in an AMPK-independent manner. Our findings reveal that targeting HK1b isoform alone or in combination with cisplatin may represent a novel strategy for NSCLC patients.
P53 ve MDM4 etkileşimini bozan nanobodilerin keşfi
The p53 protein is considered as the guardian of the genome thanks to its important tumor suppressor roles such as cell-cycle arrest, apoptosis and senescence. Because these roles are extremely vital, the p53 pathway is strictly regulated. During unstressed conditions, p53 protein levels are kept in control by both ubiquitination of the p53 protein and inhibition of its transcriptional activity through the MDM2 and MDM4 proteins, respectively. Although MDM2 is the main modulator of p53 activity, there is a collaboration between MDM2 and MDM4 proteins to enable the control of p53. Thus, MDM4 is as important as MDM2 in this mechanism. In most human cancers, there is either a mutation in the Tp53 gene or an overexpression of its negative regulators. Thus, targeting the p53-MDM2-MDM4 interplay is one of the main aims of cancer therapeutics. Also, in some cancers, where there is overexpression of negative regulators, the use of inhibitors for only MDM2 is not enough to activate the p53 protein. For this reason, exploring inhibitors for MDM4 are vital for therapy. In this study, we aimed to optimize the purification of in silico designed nanobodies targeting the MDM4-p53 interaction and test their affinity and effectiveness by surface plasmon resonance (SPR) and a fluorescent two-hybrid (F2H) assay.
Buğdayın yapısal genomik içeriklerinin ortaya çıkarılması
Production rate of wheat, an important food source worldwide, is significantly limited by both biotic and abiotic stress factors. Development of stress resistant cultivars are highly dependent on the understanding of the molecular mechanisms and structural elements in wheat and/or wheat interacting species. The huge and complex genome of bread wheat (BBAADD genome) has stood as a vital obstruction for understanding the molecular mechanisms until the recent availability of wheat reference genome. In this study, we provided improved and/or novel methodologies to reveal structural elements in plants. These methodologies include miRNA identification, manual curation of lncRNAs, identification of lncRNAs using wheat specific prediction models and a comparative analysis of WES data analysis tools. Using these techniques, we here focused on the uncovering of structural genomic contents of wheat. With an improved identification methodologies and manual annotation of lncRNAs, we revealed several miRNAs and lncRNAs in Triticum turgidum species and Wheat stem sawfly (WSS), a major pest of wheat. We provided a comprehensive transcriptome analysis of tetraploid wheat varieties and revealed drought responsive transcripts. Additionally, we presented the first clues of miRNA mobility between WSS larva and hexaploid wheat. Thereby, besides enrichment of the genetic information available for wheat species, this study provides important elements driving both abiotic and biotic stress responses in wheat. In this study, we also applied machine learning approaches for the fast and accurate prediction of lncRNAs in wheat species. With annotated genomes of hexaploid and tetraploid wheats, we provided better accuracy scores (99.81%) over the most popular tools available. Finally, we conducted a comparative analysis of the tools used for variant discovery. Among eight aligners and three callers, we chose the best combination for the variant calling in wheat. Later, we performed variant calling in 48 lines of elite wheat cultivars using the best tool sets. Overall, this study focused on the improvements on the identification of miRNAs, lncRNAs and structural variations in wheat.
Hepatoselüler karsinoma gelişim ve metastazında mikrornaların rolü
Autophagy is a cellular stress response mechanism whose activation leads to the degradation of proteins as well as damaged organelles for maintaining cellular homeostasis. Autophagic capacity or alteration of its activity was found to relate with several pathologies, including cancer and metabolic diseases. Besides, microRNAs (miRNAs) have implicated in several fundamental biological processes such as cellular transformation, carcinogenesis, and even autophagy. Deregulation of both autophagy and miRNA pathways have found to contribute to the pathogenesis and progression of hepatocellular carcinoma (HCC), which is one of the mortal types of cancer. Deregulation of autophagy-regulating miRNAs addressed almost all stages in HCC tumorigenesis from manifestation of low-grade dysplastic nodules to advanced disease. Thus, manipulation of autophagy by miRNAs might provide novel targeted therapy options for HCC.
Sarılı-sarmal bölge bulunduran 124 (CCDC124) proteı̇nı̇nı̇n doğal bağışıklık sı̇stemı̇nı̇n antı̇vı̇ral yanıtındakı̇ rolü
The innate immune system acts as the first line of defense in a non-specific manner against infectious diseases as well as malignant transformation. Natural Killer (NK) cells are members of innate immune system which are particularly responsible for killing virus-infected cells and tumor cells. Distinct properties of NK cells are remarkable in terms of cancer immunotherapy. Among several approaches, genetic modification of NK cells to enhance their immune function is widely studied with promising results but in vitro gene delivery into NK cells is highly challenging. HIV-1 based lentiviral vector systems for stable gene transfer have been used in most of the studies that aim genetic modification of NK cells. However, viral resistance of NK cells causes low efficiency and reduced stability, but enhancement of gene delivery efficiency is possible to achieve with small-molecule kinase inhibitors, such as BX795. Stress granule assembly is known to be associated with antiviral responses. This study aims to study the effect of CCDC124 gene which may be associated with stress granule formation and antiviral response during lentiviral gene transfer to NK cells. To investigate the mechanism, CRISPR/Cas9 system was used to knock out CCDC124 and other genes that may be involved in the intracellular response against lentiviral vectors in HCT116, NK-92 and YTS cell lines. We compared the responses of different cell lines to lentiviral transduction and observed significant change in transduction efficiencies. Additionally, stress granule formation in CCDC124 knockout NK-92 cells is examined. Our findings present novel insights into the resistance of NK cells to lentiviral gene delivery and provide useful tools to improve genetic modification of NK cells.
Fındıkta külleme hastalığının tanımlanması ve genetik olarak iyileştirilmesi
Corylus avellana L. (hazelnut) is an economically important nut crop worldwide due to its nutritional and nutraceutical properties. Powdery mildew is a fungal infection caused by obligate biotrophic fungal plant pathogens distributed nearly worldwide and infects a wide range of plants. Being a highly contagious pathogen that effects the crop quality, a fast and correct genetic diagnosis efforts are highly demanded. In the content on this thesis work initial effort is given to identify the types and characteristics of two pathogens of powdery mildews that mostly infects hazelnut leaves in Turkey. As a result of unique microscopy analysis and genetical ancestor tracing they have been identified as Erysiphe corylacearum and Phylactinia guttata where first is more abundant and destructive and latter is less frequently observed. In order to be able to diagnose the type of pathogen on-field, a novel fast and cost-effective methodology based on the use of newly designed E. corylacearum and P. guttata specific primers is developed. Results suggest that the proposed approach reduces the pathogen diagnosis time 10 to 1 when compared with diagnosis with the use of conventional ITS primers. Focusing specifically on E. corylacearum specific primers limit of detection is identified quantitatively with calculation of pathogen copy number. Loop Mediated Isothermal Amplification (LAMP) method is employed with E. corylacearum specific 28S-LAMP primers that enables the on-field identification of pathogen. Lastly, the genome sequencing of Corylus avellana cv. Tombul have been correctly sequenced that enabled the identification of resistant gene family Mildew Locus O (MLO) that paves way to powdery mildew resistant hazelnut crops.
BTB domaın içeren transkripsiyon faktörlerinin etkileşim matriksi
The BTB domain is a protein-protein interaction unit found in eukaryotes. It forms a distinct multimeric structure with a large interaction surface. The exposed residues of each monomer are highly variable and can allow dimerization, oligomerization, and interactions with several other proteins such as NCOR and SMRT corepressors. BTB-containing transcription factors are diverse and control various physiological processes ranging from immune system development to cell cycle regulation. To understand the structural basis of these functions, we assessed the interaction networks of these proteins. In this study, we developed specific and systematic assays to screen the interactions between various BTB domain-containing transcription factors and their interaction partners in vitro and in vivo, using surface plasmon resonance (SPR) and fluorescent two-hybrid (F2H) assays. We constructed a homo- and hetero-dimerization matrix of several BTB domains of interest.
Hepatoselüler karsinomada kanser-bağlantılı fibroblastlar ile kanser hücrelerinin karşılıklı etkileşiminin analizi
Most anticancer treatment approaches focus on malignant cells. However, tumors do not only comprise of malignant cells, but also contain many other cell types such as fibroblasts, mesenchymal cells, epithelial cells and non-cellular components. Hence, the progression of a tumor also depends on its crosstalk with neighboring cells called tumor stroma. Stroma can be activated by various stimuli and affects tumor progression, metastasis and drug resistance. Among stromal cells, fibroblast have attracted a significant amount of attention in the last decade, due to their unique functions. Naive fibroblasts are quiescent, but they are activated during physiological events, such as wound healing. On cancer, in response to interactions with tumor cells, fibroblasts (and some other cell types) undergo transdifferentiation and turn into cancer-associated fibroblasts (CAFs). CAFs are distinguished from other types of activated fibroblasts. CAFs were shown to support tumor growth, invasion, metastasis and even cancer resistance to treatment. In this study we isolated primary fibroblasts of normal liver and CAFs from hepatocellular carcinoma (HCC) tissues, and analyzed their effects on cancer behaviour. Co-culture experiments showed that, HCC-derived CAFs, but not normal liver fibroblasts, supported the growth of HepG2 and Huh7 tumor cell lines. Moreover, tumor promoting effects of CAFs did not require direct cell contact, indicating involvement of secreted factors. We identified a factor playing a key role in the observed cancer cell-CAF crosstalk. Secretion level of the factor was higher in conditioned media from CAFs that were co-cultured with cancer cells compared to those co-cultured with tissue fibroblasts. We demonstrated that the factor was predominantly secreted from CAFs, and cancer cells showed much lower expression levels. Importantly, proliferative effects of CAFs on cancer cells was reversed when a factor-specific neutralizating antibody was added to co-cultures. Hence, we defined a key regulator of CAF-induced tumor growth in HCC. The identified factor might be a potential target for new anticancer therapy approaches.
Nükleik asit tespit platformları için yöntem geliştirilmesi ve iyileştirilmesi
The nucleic acid tests abbreviated as NAT, is a technique requires amplification and detection to provide guidance on the diagnosis of genetic materials. Although the genetic material of every living consists of DNA or RNA, there are variations in genome sequences. This genetic variation makes NAT an ideal technique for identifying, genetically modified organisms (GMOs), infectious diseases, cancer, genetic disorders, and mitochondrial disorders, helping to improve diagnostic technologies. Nucleic acid amplification requires a laboratory environment with special equipment and technical expertise. Loop Mediated Isothermal Amplification (LAMP) is technically simpler than Polymerase Chain Reaction (PCR). LAMP has ideal properties for nucleic acid detection applications. LAMP assays are robust and has ability of pyrophosphate production in the presence of target, which enables detection with naked eye. Polymerase inhibitors in samples do not affect the amplification process. Most importantly, LAMP makes the reaction suitable for simple target-response diagnostic systems with simplified sample preparation. In this thesis, LAMP was primarily developed and optimized according to highlight the strong diagnostic aspects of detection platforms, and their effects on healthcare and its benefits to society. The systems we worked on enlarges the target DNA using LAMP method. In less than 30 minutes, it reacts with pH-dependent dyes (such as hydroxynaphtol blue (HNB)) and enables colorimetric DNA detection with naked-eye. Detection of DNA fragments were performed parallelly in thermal cycler and our platforms. Results show LAMP is an advantageous method because it is highly sensitive, cheap, user-friendly, and safe; in addition, does not usually require DNA extraction (in colony-LAMP). The LAMP reaction is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually.
Cd70 genindeki primer immün yetersizlik hastalığına sebep olan yeni bir mutasyonun fonksiyonel analizi
Immunity is the broad definition that embodies all protective mechanisms employed by the body against pathogens. T cell activation is central to a functional immune response, as T cells are fully activated by antigen-specific-interactions, co-stimulation, and instructive cytokines, according to the three-signal hypothesis. Primary immunodeficiencies (PIDs) are the heterogenous group of congential immune system defects that result in either partial or complete loss of immune responses against pathogens. Individuals with the PIDs are highly prone to recurrent infections. Epstein- Bar virus (EBV) is a ubiquitous oncogenic virus that is mostly asymptomatic, yet it can cause lymphoproliferative disorders (LPDs) in individuals with genetic defects. In this thesis, we identified a novel point mutation in the CD70 gene that leads to EBV- associated PID. The CD27/CD70 signalling pathway was previously shown to be responsible for the expansion and maintanence of EBV-specific CD8+ T cells, and humoral immunity. To analyze further, we generated stable cell lines through HIV-1 based lentiviral vector production, and transduction to transfer the wild-type and mutant CD70 proteins to K-562 and Namalwa cell lines. We performed cell surface and intracellular staining experiments to investigate wild-type and mutant CD70 gene products with flow cytometry. We also aimed to construct an in vitro functional assay employing CD27-Fc fusion protein production to evaluate the functionality of the identified CD70 mutations. Overall, we report a novel mutation in the CD70 gene that causes CD70 deficiency that can potentially contribute to the diagnosis of the suspected PID cases.
UV kaynaklı DNA hasarının kesip çıkarmalı onarımı ile DNAreplikasyonunun genom çaplı etkileşimi
Replication can cause unrepaired DNA damages to turn into mutations that might lead to cancer. Nucleotide excision repair is the leading repair mechanism that prevents melanoma cancers by removing UV-induced bulky adducts. However, the role of replication on nucleotide excision repair, in general, is yet to be clarified. Recently developed methods Damage-seq and XR-seq map damage formation and nucleotide excision repair events respectively, in various conditions. Here, we applied Damage-seq and XR-seq methods to UV-irradiated HeLa cells synchronized at two stages of the cell cycle: early S phase, and late S phase. We analyzed the damage and repair events along with replication origins and replication domains of HeLa cells. We found out that in both early and late S phase cells, early replication domains are more efficiently repaired relative to late replication domains. The results also revealed that repair efficiency favors the leading strand around replication origins. Moreover, we observed that the repair efficiency of the strands around origins is inversely correlated with the number of melanoma mutations.
PATZ1 BTB alanının karakterizasyonu
The BTB domain is a conserved, multi-functional protein interaction domain engaged in the coordination of various large protein complexes, including ion channels, degradation signaling complexes and DNA transcription factors. The ZBTB protein family of transcription regulators is characterized by an N-terminal BTB domain followed by a variable number of Zinc Fingers and it is involved in cellular proliferation, immune cell lineage differentiation, and cancer development. Among the 49 human ZBTB proteins, PATZ1 stands out with a unique extra 31-amino acids long sequence in the center of the BTB domain. The central loop of PATZ1 is an intrinsically disordered region (IDR) conserved in mammals but absent in fish and has high binding potential. PATZ1 is a ubiquitous transcription factor that plays a role in numerous cancer types and interferes with the p53 tumor suppressor. In this thesis work, the determination of the unique structural features of the PATZ1 BTB domain was primarily accomplished with the solution of its crystal structure as a homodimer in both mouse and zebrafish (PDB IDs: 6GUV and 6GUW). Although similar in the 3D fold and the dimerization interface to the BCL6 BTB domain, the new crystal structures suggest that the PATZ1 co-repressors binding site contains a divergent sequence. The molecular dynamics of the new protein structures compared to other previously known structures allowed us to classify ZBTBs family members. Finally, the formation of stable BTB heterodimers with selected ZBTB proteins was computationally demonstrated. Keywords: BTB domain, PATZ1 transcription factor, Protein Crystallization, Dimerization, Molecular Dynamics
Glukoserebrosidaz (GBA) genindeki kodlanan tek nükleotid polimorfizmlerinin (SNP) in siliko yöntemler ile analiz, ve hucrenin biyolojik fonksiyonları üzerindeki etkisi
Gaucher disease (GD, ORPHA355) is a rare, autosomal recessive genetic disorder. It is caused by an insufficiency of the lysosomal enzyme, glucocerebrosidase (GCase), due to the severity of GBA1 gene's mutations. Such conditions lead to GD through massive accumulation of GCase substrate, glucosylceramide, in the lysosomes. Accordingly, this research analyzed the most frequent Gaucher Disease-linked single nucleotide polymorphisms (SNPs) on the GBA1 gene by applying various bioinformatics algorithms. We have classified and characterized the L296V mutation in the GD-linked deleterious SNPs spectrum. We also showed an increase in in-vitro enzymatic activity after lysosomal reacidification and evaluated the mutant GCases' reactions to constant pH experiments. Our results showed that L296V, N370S, L444P, and D409H variants are harmful mutations with different levels of hydrolysis disturbance, tertiary structure unbalance, activation interruption, and transportation of the GCase protein as well as enzymatic efficiency reduction. Yet, we have seen lysosomes' reacidification by over-expressing hydrogen pumps (V-ATPases), which reverted the decreased stability and increased enzymatic activity. These findings may suggest a research background for the therapeutic applications of Gaucher Disease through lysosomal re-acidification, and can be extended for future studies.
Kesip çıkarmalı DNA onarım etkinliğinin epigenetik belirleyicileri
Passing through the atmosphere, UV components of sunlight reach the earth's surface. Long exposures of cells to UV-A and UV-B result in cellular dysfunctionalities by causing DNA damage. Nucleotide excision repair (NER) is a mechanism that identifies and removes bulky DNA adducts such as UV-induced dipyrimidines. NER consists of two sub-pathways with respect to its damage recognition step: global (G-NER) and transcription-coupled repair (TC-NER). TC-NER takes place on the transcribed strand of the genes, whereas G-NER is globally active throughout the genome. It has been reported some chromatin states affect the efficiency of NER which consists of G-NER and TC-NER, in combination. TC-NER is associated with transcription and related genomic features. However, epigenetic factors affecting the G-NER efficiency has been underexplored. Here, we processed the genome-wide datasets derived from DNA damage and repair maps as well as histone modification maps of the three cell lines. With the genomic DNA damage, repair, and histone modification datasets, we built machine learning models to reveal epigenetic factors that can be predictive of NER and particularly G-NER efficacy. Our models resulted in high accuracy prediction of DNA repair potential of the genomic regions. We suggest that cells' epigenetic architecture is likely the key determinant of global DNA repair bias, therefore, mutagenesis in cancer.
Biyotik ve abiyotik stres koşulları altında fındık genom ve transkriptom analizleri
European hazel (Corylus avellana L.) is a crop tree of well-known health benefits and great economic importance, but only limited number of studies exist about it at the molecular level. The aim of this thesis to demonstrate the mechanisms of genes under biotic and abiotic stresses, and analysis of the cv 'Tombul' genome, one of the most important Turkish varieties. Firstly, the emerging powdery mildew pathogen, Erysiphe corylacearum, and the degree of genetic diversity within the growing epidemic were analyzed with DNA barcode analysis and molecular markers. No genetic variation was observed within these samples, suggesting that the current outbreak originated from a single recent transmission event. In order to utilize available hazelnut genetic resources for crop improvement, annotation for cv Tombul was carried through a fully assembled genome sequence produced with a hybrid sequencing strategy. The genome includes 27,270 high-confidence protein-coding genes, over 20,000 of which were functionally annotated based on homology to known plant proteins. Furthermore, the complete chloroplast genome was assembled and analyzed with multiple annotation tools, having a typical quadripartite structure. This can provide comprehensive genetic insight into the evolution of genus Corylus. Lastly, transcriptome analysis was conducted to obtain new insight on the genes and gene networks involved in chilling stress. A total of 2,440 differentially expressed transcripts were generated, and of these, 1,368 were upregulated under chilling stress. It may facilitate further study on freezing tolerance mechanisms which could be useful for breeding approaches in tree species.