LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora

Bu koleksiyon için kalıcı URI

http://hdl.handle.net/11527/19402

Gözat

Yazar "Turanlı Tahir, Eda" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora'a göz atma
  • Öge
    Development of novel BCL-2 inhibitors for glial tumors by using in vitro and in vivo systems
    (Graduate School, 2022-05-31) Çalış, Şeyma ; Turanlı Tahir, Eda ; Avşar, Timuçin ; 521162117 ; Molecular Biology – Genetics and Biotechnology
    Glioblastoma Multiforme (GBM) is the most malign form of glial tumors, which accounts for the majority of brain tumor cases worldwide. There have been different approaches to treat GBM effectively, and with the advancements made for the last decade molecular pathology, target driven therapy, and personalized medicine gained attraction. One of such promising targets for GBM is Bcl-2 induced intrinsic apoptosis pathway. Anti-apoptotic members of Bcl-2 induced intrinsic apoptosis pathway have an important role in the regulation of GBM cell death. In this thesis study, we screened seven potential Bcl-2 inhibitor compounds and evaluated their effects on proliferation of GBM cells as well as their inhibitory capacity of Bcl-2 protein. Of those, I further analyzed three of them namely 58, 243, and ind-199. 58 and ind-199 compounds did not show any significant anti-proliferation effect on GBM cells. Eventually, we decided to elucidate the mechanism of action of 243 compound, a thiazolidine derivative BH3 mimetic, which was the most promising one according to the in vitro proliferation experiments. I performed colony formation assay to assess proliferation of YKG1 GBM cells, additionally to the proliferation assay with A172 GBM cells. While 243 inihibited cell growth significantly compared to control group, Bcl-2 inhibitor ABT-199 did not inhibit cell proliferation. Moreover, I tested 243 on YKG1 tumorspheres to determine its effectivity on tumor initiating cancer stem cells (CSC). Both ABT-199 and 243 had inhibitory effect on CSC proliferation, however 243 was significantly more effective than ABT-199 when compared to control group. Since 243 is a Bcl-2 inhibitor, I analyzed key players of Bcl-2 family and intrinsic apoptosis pathway. I have analyzed gene expression levels of BCL2, BCLXL, BAX, CASP3, CASP7, and CASP9. Furthermore, I also analyzed genes related with cell death which are CASP8 and TP53. Time dependent quantitative RT-PCR results suggested that, GBM cells that are treated with Bcl-2 inhibitors ABT-263 and 243 acts differently in case of gene expressions related to apoptosis. Next, we wanted to show apoptotic cell death with Annexin V-PI assay. Interestingly, we did not detect significantly elevated apoptosis in A172 cells when they are treated with either ABT-199 or 243. Similarly, cell cycle analysis showed that 243 did not have any effect on cell cycle, altough ABT-199 induced G1 phase arrest. Moreover, I determined expression levels of apoptosis related proteins PARP, Caspase-3, and Caspase-9. I used staurosporine treatment as a positive control to induce apoptosis. None of the treatment groups apart from staurosporine increased cleaved-PARP expression. Similarly, I checked if there is a difference in expression of Pro-caspase-3 and Pro-caspase-9, and observed that only stauroporine treated group expressed lower levels of Pro-caspases, indicating that cleaved forms of both Caspase-3 and 9 were produced upon staurosporine treatment only. At this point, we hypothesized that both ABT-199 and 243 could only induce limited apoptotic cell death because BCL2 expression was relatively low in A172 cell line. Expectedly, when I compared gene expression levels among different cell lines, I observed that BCL2 expression was very low in A172 cells, and it was abundant in SH-SY5Y neuroblastoma cells. Therefore, I decided to analyze apoptosis of SH-SY5Y cells after a treatment with ABT-199 and 243. Within only 48 hours of treatment with both inhibitors, I observed apoptotic cell death of SH-SY5Y cells. Hence, we had a new hypothesis that when BCL2 expression is low, upon Bcl-2 inhibitor treatment, cells may die through autophagy since Bcl-2 forms a complex with autophagy related protein Beclin 1. I showed that 243 treatment significantly upregulated autophagy related genes such as BECN1, ATG5, and MAP1LC3B, whereas ABT-199 induced autophagy on limited level. Moreover, autophagy indicative LC3B-II expression was significantly upregulated on a protein level with the 243 treatment, when compared to control as well as ABT-199 treatment. Additionally, I determined protein expression level of p53, which has a role in the interplay between apoptosis, cell cycle, and autophagy. I observed that p53 protein expression was increased upon both ABT-199 and 243 treatment, when compared to control group. Expectedly, when we performed in silico computational analysis, Beclin 1:Bcl-2 interaction and binding of 243 to their BH3 binding domains, we observed that 243 binds to Bcl-2 through important interactions. Since 243 and Beclin 1 binds to Bcl-2 from the same domain, when cells are treated with 243, Beclin 1 cannot bind to Bcl-2 and therefore it is released to initiate autophagy. In addition, we demonstrated that 243 significantly reduced in vivo tumor growth and prolonged survival in orthotropic brain tumor models, compared to vehicle group as well as ABT-263 treated animals. Furthermore, I assessed the anti-proliferative effects of 243 on primary glial cell lines as well. 243 exerted anti-proliferative effect on all patient derived glioma cell lines that have different grades and histopathology, except OLG3 cell line which is a grade 2 oligodendroglioma. According to quantitative RT- PCR results of OLG3, OLG7, and GBM9 cell lines I observed that OLG3 has a lower expression level of BCL2. These results suggest that patients with high BCL2 expression might benefit from 243 treatment. Taken together, our results indicate that 243 disrupts Beclin 1:Bcl-2 complex, hence activates autophagic cell death, and may serve as a potential therapeutic for the treatment of GBM.
  • Öge
    Investigation of familial multiple sclerosis genetics
    (Graduate School, 2022-05-16) Everest, Elif ; Turanlı Tahir, Eda ; 521152104 ; Molecular Biology-Genetics and Biotechnology
    Multiple sclerosis (MS) is a chronic, neuroinflammatory, neurodegenerative disease of the central nervous system. Several lines of evidence have shown that the primary pathophysiological mechanism of MS is the infiltration of autoreactive lymphocytes through the blood-brain barrier, attacking central nervous system components such as myelin and resulting in oligodendrocyte death. This process has been thought to be responsible for axonal pathology and neuronal loss, which result in progressive neuronal dysfunction in some patients. Over the recent years, the roles of astrocytes, microglia, and pericytes have also been increasingly shown in MS pathology. To date, several studies have revealed disease-related cellular pathways that emphasize the different pathological components of the disease; however, underlying mechanisms in MS development and progression are yet to be elucidated. Consistently with its heterogeneous clinical presentation and complex pathophysiology, MS also has a complex inheritance pattern and develops in genetically susceptible individuals under environmental influences. Many studies have been carried out using different approaches and methods to identify genomic regions and variants that cause genetic predisposition to MS, identifying hundreds of common variants as well as candidate rare variants that increase the risk of MS. Today, MS associations of 233 common variants, as well as hundreds of suggestive associations, have been identified. However, all significant common variants, together with the suggestive effects, can cumulatively explain approximately half of MS heritability. Meta-analyses have shown that rare variants can further explain up to 5% MS heritability, still leaving a large proportion of MS genetics unknown. In this thesis study, it was aimed to reveal novel information on MS genetics and pathogenesis. Multiplex MS families with more than two affected family members were collected to identify possible novel genes that contribute to the high MS aggregation in these families. Seven multiplex MS families with the highest number of affected individuals and parental consanguinities were selected, and SNP genotyping (710K or 2.5M, Illumina) was performed (N=41). Candidate MS-associated genomic regions were identified through linkage analysis and homozygosity mapping. Exome sequencing (N=56) revealed that there were no fully penetrant, homozygous, rare, exonic variants segregating within the families. However, two variants were found to be segregated with the disease with an autosomal dominant inheritance pattern in the LRRC6 gene (rs139131485) in family FMS01 and RNF217 (rs73580047) gene in family FMS05, which may increase the risk of MS in corresponding families. Additionally, many incompletely penetrant, rare and low-frequency variants were identified. Subsequently, a weighted sum score analysis including previously identified common MS-associated risk variants and polygenic risk score (PRS) analysis were conducted in MS families (24 affected, 17 unaffected), 23 sporadic MS cases, 63 individuals in 19 non-MS control families, and 1272 independent, ancestry-matched controls to determine whether an increased burden of known MS-associated common variants explain the increased MS risk in these families. Logistic regression analyses showed that familial MS cases had higher sum scores (OR=2.16, P=0.002; OR=2.4, P=0.014) and PRS (OR=1.84, P=0.0077; OR=2.27, P=0.049) compared with the population controls and control families, respectively. Moreover, affected individuals in the MS families had higher weighted sum score and PRS values compared with the unaffected family members; however, the differences were not significant after Bonferroni correction. When individual families were observed, it was seen that the higher sum score and PRS trends in MS cases were evident in only three of the families, and in others, there were no apparent differences in the sum score and PRS values between the affected and unaffected family members or the unaffected individuals had higher sum score and PRS values compared with their relatives with MS, further supporting the polygenic inheritance of MS. Sporadic MS cases had significantly higher PRS compared with both affected and unaffected individuals in MS families, control families, and population controls (P=0.02, P=0.0055, P=0.003, and P=0.0008, respectively), supporting the presence of higher rare risk variation loading in the familial cases. There was no significant difference in the sum scores of familial and sporadic MS cases, possibly due to the high degree of convergence between common and rare risk variation in significant loci for MS. As part of this thesis study, we also performed an integrated bioinformatic analysis using genomic and proteomic data of an unrelated MS group. For this, first, SNP genotyping (300K, Illumina) was performed for 11 unrelated MS cases selected from our MS family cohort whose cerebrospinal fluid samples had been previously included in our proteomic study, in which 2D-gel electrophoresis, mass spectrophotometry, and pathway analyses had been conducted, revealing 151 differentially expressed proteins between MS cases with different clinical MS phenotypes and non-MS controls. To integrate the genomic and proteomic datasets of this patient group to reveal the most relevant disease pathways, pathway enrichment analyses of MS-associated SNPs and differentially-expressed proteins were conducted using the functional enrichment tool, PANOGA. Nine shared pathways were detected between the genomic and proteomic datasets after merging and clustering the enriched pathways. Among those, complement and coagulation cascade was the most significantly associated pathway (hsa04610, P=6.96×10−30). Other pathways involved in neurological or immunological mechanisms included adherens junctions (hsa04520, P=6.64 × 10−25), pathogenic Escherichia coli infection (hsa05130, P=9.03×10−14), and prion diseases (hsa05020, P=5.13×10−13). We conclude that despite the overall increased genetic burden in familial MS cases, weighted sum score and PRS distributions among affected and unaffected family members within individual families revealed that known susceptibility alleles can explain disease development in some high-risk multiplex families, while in others, additional genetic factors remain to be identified through more detailed genomic analyses such as genome sequencing. Additionally, integrating multiple omics datasets of the same patients helps reduce false negative and positive results of genome-wide SNP associations and highlights the most prominent cellular players among the complex pathophysiological mechanisms in MS.
  • Öge
    Investigation of novel genes and functional roles in MEFV negative FMF patients through next-generation sequencing
    (Lisansüstü Eğitim Enstitüsü, 2023-06-20) Önen Özkılıç, Merve ; Turanlı Tahir, Eda ; 521142106 ; Molecular Biology-Genetics and Biotechnology
    The p.Arg228Cys variant and the known pathogenic variant p.A230T are both located on the F-BAR domain, which is important for PSTPIP1 protein to form the functional trimeric complex with pyrin. The computational analyses of the xxi PSTPIP1 structure suggest that the p.Arg228Cys variant may cause a potential destabilization and change in the weak interaction network, leading to a stronger preference for certain interaction partners such as pyrin. Studies have shown that the p.Arg228Cys variant may lead to increased interaction with pyrin and reduced interaction with LYP phosphatase, which is normally required for the regulation of immune responses. The differences in symptoms between patients with the p.A230T or p.E250Q pathgoenic variants causing PAPA syndrome and patients with the p.Arg228Cys variant causing milder autoinflammatory symptoms in the patients in this study may be due to the diverse interaction strength of PSTPIP1 with different phosphatase proteins and pyrin protein. Within the scope of this thesis, it is found that p.Arg228Cys variant appears to increase the binding of PSTPIP1 to pyrin in PBMC samples of the patients. This was indicated by the higher levels of pyrin observed in the IP:PSTPIP1 lanes of our patient samples when compared to healthy controls. The interaction between PSTPIP1 and pyrin was increased in the patients as for P-III-2 1.43 fold and for P-II-1 patient 1.69 fold than HC1, and as for P-III-1 11.7 fold and for P-I-1 14.7 fold than HC2. This increased interaction may lead to hyperphosphorylation of PSTPIP1 and triggering the activation of pyrin inflammasome. Thus, the inflammation model in cultured PBMCs showed increased protein expression levels of pyrin, PSTPIP1, caspase-1, and IL1ß in cell lysates of patients with the p.Arg228Cys variant compared to the patient with a different heterozygote variant (p.A372V) and healthy control in the inflammation-induced condition (LPS+ATP) and secreted caspase1 and IL1ß levels were also found higher in supernatants of patients with p.Arg228Cys variant according to others in the inflammation-induced condition (LPS+ATP). Thus, it is important that other inflammation-related genes involvement should be considered in patients presenting with FMF phenotype negative for MEFV exon 10 recessive mutations. The targeted sequencing approach is useful for detecting rare pathogenic variations in patients with autoinflammatory phenotypes. Further functional analyses of the identified pathogenic variants could be helpful for better understanding the underlying molecular mechanism of FMF development.