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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.