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Kanser tedavisi için protein mühendisliği yaklaşımları ile geliştirilmiş anti-vegf antikorlarının araştırılması
Monoclonal antibodies (IgGs) are powerful therapeutics for many diseases, including cancer, due to their high specificity and other favorable properties. However, their large size limits their penetration into large masses of biological tissue, such as solid tumors. The single-chain variable fragments (scFvs), on the other hand, have better tissue penetration due to their smaller size. Yet, due to the absence of constant domains, scFvs are not as thermally stable as their parental IgGs or even Fab (fragment antigen-binding) fragments, which poses a challenge for therapeutic applications. Therefore, in this thesis, we aimed to develop a general method for increasing the thermal stability of scFvs. Anti-angiogenic therapy has a critical importance in tumor-mediated angiogenesis, which is a hallmark of solid tumors. Bevacizumab (IgG) was the first therapeutic developed for anti-angiogenic therapy, and it is still used in the clinic for a variety of solid tumor types, mostly in combination with chemotherapy. In this thesis, we developed anti-VEGF scFvs derived from bevacizumab and they demonstrated better anti-angiogenic efficacy compared to bevacizumab on a transgenic zebrafish model. Next, by utilizing rational design approaches including computational methods, and directed evolution techniques including yeast surface display we obtained thermally more stable scFv variants, the highest of which has around 10 ºC higher transition mid-point (T1/2) compared to wild type scFv. This method described in this thesis can be used to improve the thermal stability of any kind of antibody or antibody fragment.
Hesaplamalı yöntemler kullanılarak beyin neoplazmasında prognoz ve teşhis işaretlerinin belirlenmesi
Low-grade gliomas (LGG) are central nervous system Grade I tumors, and as they progress become one of the deadliest brain tumors. Today, there is still great need for timely and accurate diagnosis and prognosis. The aim of this study, was to identify diagnostic and prognostic biomarkers associated with LGG, by employing using diverse computational approaches. For this purpose, differential gene expression analysis between LGG tissue and corresponding normal tissue was performed by using three methodologies. A total of four thousand four hundred and ninety six common differentially expressed genes (DEGs) between LGG and healthy brain tissue were detected. Next, those DEGs were used to conduct Weighted Gene Co-Expression Network Analysis (WGCNA) in order to identify clusters of co-expressed genes. In this way, two modules significantly correlated with clinical traits were detected. The DEGs comprising the modules were further used to construct gene co-expression and protein-protein interaction networks. Based on this analysis, we derived a consensus of eighteen hub genes, namely, CD74, CD86, CDC25A, CYBB, HLA-DMA, ITGB2, KIF11, KIFC1, LAPTM5, LMNB1, MKI67, NCKAP1L, NUSAP1, SLC7A7, TBXAS1, TOP2A, TYROBP, and WDFY4. All detected hub genes were up-regulated in LGG, and were also associated with unfavorable prognosis in LGG patients. These findings could be applicable in the clinical setting for diagnosing and monitoring LGG.
Nörotrofin reseptörü ile ilişkili ölüm bölgesi proteininin Wnt/ β - katenın sinyal iletiminin düzenlenmesindeki rolünün zebrabalığı embriyonik modeli ve CRISPR/Cas9 knockout modeli ile araştırılması
Wnt/β-catenin signaling pathway plays essential roles in many biological processes including cell proliferation, migration, differentiation and apoptosis during development and adulthood. Many regulatory molecules of the pathway were revealed by researchers over the years. Yet, the whole mechanism is a puzzle with missing pieces, waiting to be solved. In this study, we identified neurotrophin receptor-associated death domain (nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative feedback regulator of Wnt/β-catenin signaling in zebrafish embryos. We revealed that nradd overexpression significantly suppresses Wnt8-mediated/β-catenin signaling which controls mesoderm and neuroectoderm patterning during gastrulation. Also, its overexpression suppresses the expression of Wnt-target genes: Lymphoid Enhancer Binding Factor 1(Lef1), Sp5 transcription factor-like (Sp5l), and conductin (Axin2) and can rescue the effect of high Wnt8-mediated/β-catenin signaling activity, loss of forebrain and eyes. In addition, we investigated nradd F0 knockout model generated by CRISPR/Cas9 gene-editing. To observe nradd loss-of-function efficiently in F0 embryos, we injected Cas9 protein with 4 guide RNAs targeting nradd gene to one-celled embryos. Our approach is significantly suppressed nradd expression in F0 crispants. In crispants, Wnt-target genes' expressions are significantly upregulated, supporting the negative regulator role of Nradd in Wnt/β-catenin signaling. Moreover, we also analyzed the apoptosis level in crispants to evaluate the defined apoptotic function of nradd by previous studies. Interestingly, we observed an increased apoptosis level in crispant embryos at 24 hours-post-fertilization (hpf), confirmed by increased expression level of pro-apoptotic genes and increased immunoreactivity of cleaved-caspase-3 antibody. In summary, Nradd acts as a negative feedback modifier of Wnt/β-catenin signaling and a regulator of cell death during embryonic development. As the pathway modifiers serve as attractive drug targets especially in targeted therapies for cancer, further characterization of Nradd at the molecular level will significantly contribute to both elucidations of as yet unknown pathway mechanisms and the discovery of novel drugs targeting the pathway.
Ekzozomal miR-34a Aracılığıyla Mikroglia – Nöron Etkileşimi
Microglia are extra-embryonic yolk sac-derived myeloid cells in the Central Nervous System (CNS) playing roles in regulating brain development, repairing injuries and maintaining neuronal networks. When signaling dangers to CNS by dead cells, microbes, unnecessary synapses and soluble antigens, they remove them and maintain homeostasis. Because of being the primary source of proinflammatory cytokines, microglial cells mediate neuroinflammation and regulate cellular responses that have extensive spectrum. Exosomes are membrane-encapsulated vesicles that are released via various cell types. They have functions in regulating cell-to-cell transmission via carrying their cargo to recipient cells containing microRNAs (miRNAs), mRNAs, proteins and DNA. Microglia-derived exosomes promote neural regeneration by stimulating neurite outgrowth and might have a role in neurological disorders. MiRNAs within exosomes have functional roles when being delivered into the recipient cells and are thought to be the key functional elements. The aim of this thesis is to identify the impacts of microglial cells on neuronal cells via exosomal miR-34a. As a requirement of this purpose, SH-SY5Y neuronal cell line will be treated with different concentrations of hydrogen peroxide (H2O2) and its cytotoxicity will be evaluated via LDH (Lactate Dehydrogenase) assay and PI (Propidium Iodide) visualization. Then, the cytotoxic effect of hydrogen peroxide on neurite outgrowth will be determined by assessing the number of protrusions per cell, and neurite lengths will be analyzed and expressed as mean process length. After that, HMC3 microglial cell line will be transfected with miR-34a mimic and their exosomes will be given to SH-SY5Y cells. The impacts of miR-34a on SH-SY5Y cells in terms of neuroprotection will be determined.
Amsacta moorei entomopoxvirus (AMEV) muhtemel kaspaz orf'si̇ni̇n (AMV063) fonksi̇yonel anali̇zi̇
Amsacta moorei entomopoxvirus (AMEV), tarımsal açıdan önemli zararlıları enfekte ettiği için, AMEV'nin genomunda yapılan genetik mühendisliği çalışmalarıyla hem bu virüslerin biyoteknolojik ajan olarak kullanımının artırılması amaçlanmakta hem de henüz keşfedilmeyen gen fonksiyonları aydınlatılmaktadır. AMEV'nin, LD652 ve CF-70-B2 gibi böcek hücre hatlarını enfekte edilebildiği ve viral yaşam döngüsünü devam ettirmek için apoptozise sebep olduğu gösterilmiştir. Kaspazlar, sistein proteaz ailesinin üyeleridir ve apoptozisin düzenlenmesinde önemli rollere sahiptir. AMEV'ye ait muhtemel kaspaz proteinini kodladığı belirlenen 63. ORF'nin, AMEV protein kinazlarıyla etkileşimi belirlenmiş olmasına rağmen, AMV063 proteininin apoptozis ve kaspaz aktivitesi daha önce araştırılmamıştır. Bu tez kapsamında, AMEV063 ORF'sinin fonksiyonel analizi yapıldı. AMV063 proteini prokaryotik sistemde ifade edildi. Homolog rekombinasyonla AMV063 dizisi silinen nakavt Am∆063/gfp oluşturuldu. Apoptozise duyarlı CF-70-B2 hücreleri, AMV063 proteini ve Am∆063/gfp kombinasyonları ile muamele edilerek apoptozis ve kaspaz-2 aktiviteleri araştırıldı. AMV063 proteini ile muamele edilen CF-70-B2 hücrelerinde, kaspaz-2 ve TUNEL aktivitesi belirlenemedi. Am∆063/gfp virüsü ile muamele edilen CF-70-B2 hücrelerinin kaspaz-2 ve TUNEL aktivitesi, kontrol Am∆sph/gfp virüsü ile muamele edilenlere göre önemli derecede azaldı. Bu sonuçlar, AMV063 dizisinin kaspaz-2 proteinini kodladığını ve doğal sisteminde üretilen AMV063 proteininin, kaspaz-2 ve apoptozis aktivitesine sahip olduğunu açık bir şekilde gösterdi. Anahtar Kelimeler: Böcek virüsleri, Amsacta moorei entomopoxvirus, AMV063, Am∆063/gfp, kaspaz-2, apoptozis