Kök Hücre Bilimleri
https://hdl.handle.net/11655/9127
Kök Hücre Bilimleri2024-03-28T11:17:42ZOptimization and Risk Assessment of RAB27A-/- Gene Therapy Alternatives Using Current Molecular Techniques and Technologies
https://hdl.handle.net/11655/34716
Optimization and Risk Assessment of RAB27A-/- Gene Therapy Alternatives Using Current Molecular Techniques and Technologies
EROL DUYAR, Özgür Doğuş
EROL DUYAR, Ö.D. Optimization and Risk Assessment of RAB27A-/- Gene Therapy Alternatives using Current Molecular Techniques and Technologies. Hacettepe University Graduate School of Health Sciences Stem Cell Doctor of Philosophy Thesis, Ankara, 2024. Griscelli Syndrome Type 2 (GS-2) is caused by a mutation in RAB27A which plays a role in exocytosis and membrane trafficking, resulting in immune deficiency. Here, we investigated 1) the efficacy of CRISPR/Cas9 gene editing of RAB27A using GS-2 MSCs and iPSC lines; 2) the efficacy of LV vectors to transduce MSCs and express RAB27A under the control of the SFFV, PGK and UCOE promoters, and 3) potential risks related to overexpression of RAB27A in immune deficient mice. We designed CRISPR/Cas9 constructs that target mutations in RAB27A and tested the gene correction efficacy on GS-2 MSC and iPSC lines. We generated a new GS-2 iPSC line and optimized cell culture/cryopreservation. Although editing of RAB27A using CRISPR/Cas9 is possible, survival of the stem cells was low. Transduction of GS-2 MSCs with LV vectors showed the highest RAB27A expression with SFFV, followed by PGK and UCOE. RAB27A+ MSCs and HSCs were transplanted into immune deficient mice but did not cause tumor formation. However, RAB27A overexpression may affect stem cell function. In conclusion, we compared the efficiency of gene editing using CRISPR/Cas9 with gene addition using LV vectors for the development of gene therapy for GS-2. Although gene editing results in acceptable levels of repair, the technology results in low cell viability. LV gene transfer was easy and robust, but high expression of RAB27A affected stem cell function. Thus, both methods can be developed into gene therapy for GS-2, but optimization of the procedures, increasing cell viability and fine-tuning of expression levels may be necessary before these therapies are ready for clinical use.
Keywords: Griscelli Syndrome Type 2, CRISPR/Cas9, lentivirus, stem cells.
(*) This study was supported by grants from the Scientific and Technological Research Council of Turkish Government TÜBİTAK no: 219S675 and Hacettepe University Scientific Research Projects Coordination Unit grant no: TUK-2019-17760 and THD-2022-19940.
TÜBÜTAK 219S615
THD-2022-19940
2024-02-09T00:00:00ZOksidatif Stres Altında Mezenkimal Kök Hücrelerde Src-YAP Aktivitesi ve Apoptoz ile İlişkisi
https://hdl.handle.net/11655/34557
Oksidatif Stres Altında Mezenkimal Kök Hücrelerde Src-YAP Aktivitesi ve Apoptoz ile İlişkisi
Bozdemir, Özlem
One of the reasons for the post-transplantation loss of mesenchymal stem cells (MSCs), which are used in the treatment of many diseases with their immunomodulatory and regenerative properties, is oxidative stress-induced cell death. Therefore, investigating and elucidating the cellular signalling responses occurring in MSCs under oxidative stress will contribute to the development of future treatments. In the thesis study, it was aimed to examine the changes in Src-YAP activity in MSCs under oxidative stress caused by H2O2 and to correlate this with apoptosis through Bax expression and pYAPY357-p73 interaction. For this purpose, the H2O2 concentration was determined as 300 μM by WST-1 analysis to induce oxidative stress in MSCs isolated from lipo-aspirate and characterized. It was determined by flow cytometry that intracellular ROS levels increased after H2O2 applications for 15, 30, 60, and 120 minutes. In Western blot analyses, it was determined that Src phosphorylation increased, followed by an increase in YAP and phosphorylations. In western blot and immunofluorescence analyses, it was determined that the nucleus-cytoplasm distribution of YAP and its phosphorylations changed after H2O2 application. It was determined that the Bax/Bcl-2 ratio increased after H2O2 application. In the immunoprecipitation analysis, the pYAPY357 protein band was detected in the samples precipitated with p73 in the nuclear extract. In line with the stated purpose, this thesis is the first study to detect the Src-YAP relationship in MSCs under oxidative stress, and it is unique in that it shows that pYAPY357 increases and is localized in the nucleus. The interaction of pYAPY357 and p73 in MSCs was also shown for the first time in this study.
Bu tez çalışması, Hacettepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi (Proje No: TYL-2022-19813) ve TÜBİTAK 1002 (Proje No: 122Z314) kapsamında desteklenmiştir.
2024-01-01T00:00:00ZMikrorna-21 Baskılanmış Meme Kanseri Kök Hücrelerinin İnsan Kemik İliğinden İzole Edilen Telositler ve Telosit Kaynaklı Mitokondriler ile Ko-Kültürü
https://hdl.handle.net/11655/34152
Mikrorna-21 Baskılanmış Meme Kanseri Kök Hücrelerinin İnsan Kemik İliğinden İzole Edilen Telositler ve Telosit Kaynaklı Mitokondriler ile Ko-Kültürü
Babadağ, Sena
The aim of this thesis study was to investigate the contribution of telocytes and telocyte-derived mitochondria, which have also been identified in breast tumours, to the tumour development of breast cancer stem cells via miR-21-5p. Within the scope of this thesis study, firstly, CD34 and CD117 positive telocytes were isolated from human bone marrow mononuclear cells and characterised by flow cytometry, immunofluorescent staining and TEM analysis methods and mitochondria were isolated from telocyte cells and characterised by immunofluorescent staining. In the next step, CD24- and CD44+ cancer stem cells were isolated from MDA-MB-231 breast cancer cell line and characterised by flow cytometry. Then, miR-21-5p expressions of cancer stem cells were suppressed by transfection of anti-miR-21-5p inhibitor. Changes in vimentin and ALDH1A1 protein levels were analysed by Western blot to investigate the role of bone marrow-derived telocytes and mitochondria in miR-21-5p suppressed cancer stem cell lines. SNAI1, LZTFL1, OCT3/4, ABCC11, CHD1 (E-Cadherin) and CHD2 (N-Cadherin) gene expression changes were analysed by RT-qPCR. Isolated telocytes expressed CD44/CD117/α-SMA/CD31/CD45 and CD34 surface markers and were also positive for vimentin and PDGF-β. Mitochondria isolated from telocytes were followed by immunofluorescent staining. Isolation and characterisation of cancer stem cells resulted in miR-21-5p inhibition and a 132-fold decrease in miR-21-5p gene expression was confirmed by RT-qPCR. After co-culture of telocytes and mitochondria with miR-21-5p inhibited cancer stem cells, miR-21-5p, SNAI1, ABCC11, N-Cadherin, vimentin and ALDH1A1 expressions decreased; E-Cadherin and LZTFL gene expressions increased. In this thesis, it has been demonstrated for the first time that telocytes and telocyte-derived mitochondria can reduce EMT transition via miR-21-5p in the progression of breast cancer and have the potential to reduce cancer metastasis by analyses at the gene/protein level.
2023-01-01T00:00:00ZApc Mutant Fare Modelinde Azaltılmış Bdnf Ekspresyonunun İntestinal Tümör Gelişimi Üzerine Etkilerinin Araştırılması
https://hdl.handle.net/11655/33890
Apc Mutant Fare Modelinde Azaltılmış Bdnf Ekspresyonunun İntestinal Tümör Gelişimi Üzerine Etkilerinin Araştırılması
Gök, Ayşenur
Gök, A., Effects of Reduced Bdnf Expression on Intestinal Tumor Development in Apc Mutant Mouse Model, Hacettepe University Graduate School of Health Sciences Department of Stem Cell Sciences Doctor of Philosophy Thesis, Ankara, 2023. A mutation in adenomatous polyposis coli (Apc) tumor suppressor gene, which negatively regulates Wnt signaling, is the most common mutation in colon cancers. Brain-derived neurotrophic factor (Bdnf), one of the genes regulated by the Wnt signaling pathway, is expressed at high levels in many tumor tissues such as colorectal cancers. In this thesis study; It was aimed to investigate the effects of decreased Bdnf expression on intestinal tumor development in Apc mutant mice. In this context; we followed up Apc+/-, Bdnf+/-, Apc+/-Bdnf+/- and wild-type mouse experimental and control groups for 36 weeks and investigated the effects of Bdnf mutation on Apc mutant mice phenotypically and molecularly. When the experiments are finished; compared to Apc+/- and wild-type mice, while half of the group had normal body weight, a significant weight gain was observed in the other half in Bdnf+/- female and male mice. A similar trend of weight gain occurred in Apc+/- Bdnf+/- mice. Apc+/- Bdnf+/- mice did not show any disease phenotypes such as anemia, weight loss, discolored fur, or altered posture during the first 6 months compared to Apc+/- mice. Survival times of Apc+/- Bdnf+/- mice increased by approximately 2 months compared to Apc+/- mice. While it slightly decreased the number of polyps in the small intestine of Apc+/- Bdnf+/- mice, it significantly increased the number of polyps in the colon of Apc+/- Bdnf+/- mice. Bdnf, Trk B, beta catenin and Lgr5 gene expressions and mature and immature Bdnf and beta catenin protein levels were decreased in tumors of Apc+/- Bdnf+/- mice compared to Apc+/- mice, so it was thought that stem cell dynamics might also change. As a result of this study; we think that BDNF may be an important candidate molecule for the treatment of intestinal tumors starting with Apc mutation.
2023-04-01T00:00:00Z