Hilay Köse

"Effects of genetic variants and cellular protein degradation mechanisms in the context of neurodegenerativediseases" thesis project

• Investigated the role of cellular protein degradation pathways in the pathogenesis of Sporadic Alzheimer’s disease (AD), with a specific focus on genetic variants and their functional impacts.
• Focused on five major degradation pathways: Ubiquitin-Proteasome System (UPS), Autophagy, Mitophagy, Endoplasmic Reticulum-Associated Degradation (ERAD), and Lysosome-mediated pathways.
• Analyzed Whole Exome Sequencing (WES) data
• Extracted and curated gene lists related to each protein degradation pathway from the KEGG database, and mapped WES-identified variants onto these genes.
• Filtered and annotated non-synonymous exonic variants, evaluating their pathogenic potential using tools including REVEL, SIFT, PolyPhen-2, PROVEAN, PANTHER, Mutation Evaluator, SNAP2, Mutation Taster, Mutation Assessor, Missense3D, and HOPE.
• Selected top candidate variants based on computational predictions and pathway relevance for validation via Sanger sequencing.
• Designed gene-specific primers while using NCBI Primer Blast and Primer3 and conducted Sanger sequencing to validate the healthy and control cases
• Established a functional and molecular link between identified gene variants and disruption in protein degradation pathways in Alzheimer's disease.
• Gained knowledge bioinformatic interpretation of WES data, molecuular and wet-lab validation techniques, pathway curation and variant prioritization
• Contributed to scientific writing and thesis development through critical interpretation of pathway-disease associations and variant functionality.

"Functional analysis of a variant of uncertain significance (VUS) in apurinic/apyrimidinic endonuclease1 (APE1)" project

• Variant selection and interpretation: Identified and prioritized a missense VUS in APE1 based on in silico pathogenicity predictions and biological relevance to base excision repair, focusing on its location within the nuclease domain and physicochemical change (e.g., non-polar to polar amino acid substitution).
• Construct generation: Designed mutation-specific primers and performed site-directed mutagenesis on a myc-DDK-tagged APE1 expression vector to incorporate the variant. Carried out DpnI digestion, bacterial transformation, colony selection, plasmid isolation, and Sanger sequencing to verify the presence of the intended mutation.
• Protein expression: Maintained mammalian cell cultures under sterile conditions, performed transient transfections of both wild-type and variant APE1 constructs, and applied selective conditions as necessary to enrich expression.
• Protein purification and quantification: Prepared cell lysates using optimized lysis buffer, clarified samples, and purified tagged APE1 via affinity capture. Quantified total and purified protein amounts using Bradford assay to ensure equal input for downstream comparisons.
• Expression analysis: Separation of proteins by SDS-PAGE and conducted Western Blotting to evaluate expression levels, protein stability, and potential processing differences between wild-type and mutant APE1. Detected proteins using specific primary antibodies and chemiluminescent imaging.
• Functional assay: Performed endonuclease activity assays with HEX-labeled DNA substrates to directly measure APE1’s DNA incision capability. Reactions were stopped, denatured, and products resolved on high-percentage denaturing polyacrylamide gels. Phosphorimaging was used for sensitive detection and quantification of cleavage efficiency.
• Data integration: Compared biochemical activity of the variant to wild-type, contextualized results within known APE1 biology and Alzheimer’s disease relevance, and synthesized findings with prior literature to infer potential pathogenic consequences of the variant on DNA repair efficacy.

Built on prior variant interpretation and functional characterization work, by extending analysis to circadian rhythm genes in sporadic Alzheimer’s disease patients and the project of "Identification and functional characterization of genetic variants of circadian clock genes associated with sporadic Alzheimer's disease"

• Designed and performed PCR amplification of prioritized circadian clock gene regions from patient-derived samples; assessed DNA quality and concentration with Nanodrop before downstream use.
• Validated candidate variants in circadian genes via Sanger sequencing to confirm genotypes in clinical material and usage of FinchTV to confirm the result of Sanger sequencing
• Conducted targeted gene selection and pathway research to situate circadian gene variants within neurodegenerative and timing-regulation networks, evaluating potential mechanistic links to Alzheimer’s disease phenotypes.
• Integrated circadian-specific findings with the existing multi-tool variant annotation framework to refine prioritization and hypothesis generation without duplicating core bioinformatics or functional assay descriptions.

"Functional characterization of variants of uncertain significance in base excision repair pathway in Sporadic Alzheimer's disease" project:

• Processed Whole-Exome Sequencing (WES) data (case vs. control) to identify coding variants in Base Excision Repair (BER) genes.
• Applied allele frequency filters (e.g., excluding variants with a frequency p-value of less than 0.05 in population databases such as gnomAD and others) to isolate rare, potentially impactful non-synonymous single nucleotide variants (SNVs).
• Conducted systematic variant interpretation using ACMG guideline-informed logic by collecting evidence across categories: population frequency, computational prediction, literature support, and functional relevance.
• Variant Annotation and Interpretation Workflow
• Clinical evidence aggregation: Consulted ClinVar for existing clinical assertions and integrated clinical significance data to support variant classification.
• In silico effect prediction: Applied multiple sequence- and structure-based predictors to evaluate the potential functional impact of coding variants, including ,REVEL, SIFT, PolyPhen-2, PROVEAN, PANTHER, Mutation Evaluator, SNAP2, Mutation Taster, Mutation Assessor, Missense3D, and HOPE.
• Structural and stability assessment: Used DynaMut, mCSM, I-Mutant, I-Stable, and Missense3D to estimate effects of amino acid substitutions on protein stability and conformational dynamics.
• 3D structural insight and modeling: Retrieved experimentally resolved and predicted structures from PDB and AlphaFold to visualize variant positions, assess spatial context, and infer potential disruption of functional domains.
• Evolutionary conservation analysis: Employed ConSurf to evaluate the conservation of affected residues across species, supporting the inference of functional importance.
• Evidence aggregation & guideline application: Varsome and Franklin by Genoox as integrative platforms to consolidate supporting/conflicting evidence, apply ACMG criteria, and generate standardized variant interpretation reports.
• Population frequency & variant catalogues: Queried gnomAD and dbSNP to determine allele frequencies and distinguish rare potentially pathogenic variants from common benign polymorphisms.
• Protein-level functional annotation: Used UniProt for domain architecture, post-translational modification sites, known functional motifs, and cross-referenced expression/localization data.
• Tissue expression context: Incorporated protein and RNA expression patterns from Protein Atlas to understand physiological relevance of the gene in disease-affected tissues.
• Genomic annotation & context: Utilized Ensembl for transcript isoform structure, coding consequences, and gene models, and referenced the UCSC Genome Browser for genomic localization, conservation tracks, and regulatory features.
• Prioritized candidate variants based on combined computational scores and biological plausibility for downstream functional testing.
• Designed sequence-specific primers for introducing point variants using tools such as NCBI Primer Blast, Primer3 and IDT OligoAnalyzer, ensuring appropriate melting temperatures and minimal secondary structure.
• Executed site-directed mutagenesis to generate mutant expression constructs:
• Performed high-fidelity PCR amplification with mutation-bearing primers.
• Treated PCR products with Dpn-I to digest the parental template.
• Transformed into chemically competent bacteria via the heat-shock method.
• Selected colonies on antibiotic-containing media, and prepared glycerol stocks for long-term storage.
• Isolation plasmid DNA using mini-prep/midi-prep kits, and verified the incorporation of intended mutations via Sanger sequencing.
• Handled mammalian cell culture under sterile conditions: thawing, seeding, passaging, counting, and cryopreservation of HEK293T (or comparable) cell lines.
• Maintained an aseptic environment using biosafety cabinets and controlled incubator parameters (temperature, CO₂, humidity).
• Performed chemical-based transient transfections of wild-type and variant expression vectors into mammalian cells to express target BER proteins, applying optimized reagent protocols for efficient uptake.
• Applied selective pressure (e.g., antibiotic supplementation) where needed to enrich for transfected populations.
• Prepared lysis buffers (e.g., RIPA) with protease/phosphatase inhibitors and executed cell lysis on ice to preserve protein integrity.
• Clarified lysates by centrifugation and quantified total protein using Bradford assay with appropriate standards.
• Performed affinity purification of tagged proteins (e.g., Myc/DDK) using antibody-conjugated agarose beads according to kit protocols, followed by elution of purified protein.
• Conducted protein separation via SDS-PAGE (and alternative gel systems as needed), ensuring appropriate sample preparation and loading.
• Executed Western blotting workflows
• Transferred proteins to membranes (semi-dry or wet):
• Blocked non-specific binding,
• Incubated with primary antibodies specific to target proteins and appropriate HRP-conjugated secondary antibodies,
• Detected signal using chemiluminescent imaging (e.g., BioRad ChemiDoc).
• Interpretation of band intensity and size to assess expression levels and potential effects of variants on protein stability or processing.
• Designed and carried out in vitro nuclease (endonuclease) assays using fluorescently labeled DNA substrates to measure BER protein activity.
• Preparation of reaction buffers with defined composition, incubated proteins with DNA substrates under controlled conditions, and terminated reactions with denaturing stop solution.
• Resolved cleavage products on high-percentage denaturing polyacrylamide gels and performed phosphorimaging to visualize and quantify enzymatic activity differences between wild-type and variant proteins.
• Isolatation of genomic and plasmid DNA using standard extraction protocols.
• Performed classical PCR amplifications to validate constructs or amplify regions of interest; verified product size and specificity by agarose gel electrophoresis.
• Prepared and handled LB media (agar and broth), cultured bacterial strains for cloning purposes, and maintained long-term stocks.
• Systematically recorded experimental procedures, observations, and quantitative results.
• Integrated computational predictions, experimental readouts, and literature evidence to construct mechanistic interpretations of how BER variants might influence Alzheimer’s disease pathology.
• Prepared figures, summaries, and presentations using Microsoft Word, Excel, and PowerPoint to communicate findings to the research group.

• Learned and applied ACMG (The American College of Medical Genetics and Genomics) guidelines for variant classification in hereditary cancer genes.
• Utilized online genomics resources, including UniProt, OMIM, ClinVar, and VarSome, to gather and integrate evidence for variants of uncertain significance (VUS).
• Performed functional characterization and reclassification of variants of uncertain significance (VUS) in hereditary breast/other cancers.
• Conducted a literature review on breast cancer-associated variants and mechanisms of DNA damage and repair, with a focus on ATM and BRCA1.
• PBMC isolation from blood samples of breast cancer patients carrying specific mutations.
• Executed Comet Assays and analyzed DNA damage using the Comet Score tool to quantify genomic instability.
• Carried out protein quantification (Bradford assay), gel electrophoresis, and Western Blotting to assess the expression and modification of DNA repair proteins.
• Utilized light microscopy for cellular, assay validation, and imaging.
• Integrated molecular and functional data to support variant interpretation in the context of hereditary cancer risk.
• Performed systematic variant curation by collecting population frequency data, functional annotations, disease associations, and clinical assertions to inform pathogenicity assessments.

Investigated the effects of plant extracts on gut microbiota composition and their potential therapeutic impact on disease-related symptoms.

• Researched associations between specific diseases and gut bacterial taxa, assessing bacterial growth, inhibition, and suppression in response to selected phytochemicals.
• Observation DNA isolation from blood and gaita samples using commercial extraction kits, and prepared extracted DNA for downstream sequencing applications, ensuring quality and integrity.
• Collected, curated, and integrated multi-source data to inform hypothesis generation for novel treatment strategies aimed at symptom reduction and quality-of-life improvement.
• Assisted in developing a data accessibility platform to streamline retrieval and visualization of microbiota and clinical data.
• Observed and used R and Python for preliminary data analysis, visualization, and statistical exploration.
• Utilized Microsoft Office suite for data organization (Excel), reporting (Word), and presentations (PowerPoint)

Exome sequencing analysis: Worked on exome data from a family with multiple members affected by Multiple Mclerosis (MS). Filtered out common variants

• Annotation across databases: Looked up each candidate gene in sources like OMIM, CADD, UCSC Genome Browser, MGI, PDB, and KEGG to understand their function and relevance to disease.
• Gene prioritization: Chose four genes (CNDP1, KLHDC4, GSE1, PSD) that best matched the disease context based on their pathways, known links to disease, and how they might fit the family pattern. Mapped them on the family pedigree to see if their inheritance made sense.
• Use of GEMINI: Used the GEMINI framework to explore genetic variants together with their annotations, helping form ideas about which variants might matter.
• Putting evidence together: Combined published research, pathway information, and family inheritance patterns to argue that these rare variants could be involved in familial MS, suggesting them for follow-up validation.
• Thinking about complexity: Reflected on how rare variants behave differently in families versus sporadic cases and considered how combining linkage data with exome sequencing could improve discovery in complex hereditary diseases.

• Researched cancer-associated genes and molecular mechanisms in naked mole rat and blind mole rat, focusing on their cancer resistance phenotypes.
• Queried and integrated data from OMIM, UniProt, GeneCards, KEGG Pathways, Protein Atlas, and PDB to characterize gene/protein function and pathway involvement.
• Performed protein structure visualization and modeling using UCSF Chimera to analyze structural features of key cancer-related proteins.
• Mapped and compared conserved domains, post-translational modification sites, and interaction partners relevant to tumor suppression and oncogenesis.
• Synthesized multi-omics data to generate hypotheses about species-specific cancer resistance mechanisms.
• Gained hands-on experience with bioinformatics databases and structural bioinformatics tools; improved literature curation and mechanistic interpretation skills.

• Participated in the formulation and optimization of nanoparticles for drug delivery applications using hydrogel matrices.
• Assisted in selecting appropriate hydrogel systems to achieve controlled and sustained drug release.
• Gained hands-on experience in laboratory safety rules and standard operating procedures (SOPs), including sterile work practices.
• Practiced accurate micropipette use, solution preparation, and concentration calculations.
• Observed the use of a 3D bioprinter in tissue engineering research, including scaffold design and bio-ink preparation.
• Learned the operation principles of laboratory equipment such as centrifuges, incubators, and spectrophotometers.
• Conducted basic characterization experiments for nanoparticle size, distribution, and drug loading.
• Collaborated in reviewing scientific literature and documenting research findings.