Biotehnoloogia magistritööd - Master's theses

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    The novel trans-complementation system for chikungunya virus
    (Tartu Ülikool, 2024) Makhotina, Anna; Merits, Andres, juhendaja; Naumenko, Krystyna, juhendaja; Wang, Sainan, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Alphaviruses (Togaviridae) are mosquito transmitted positive-sense RNA viruses that cause severe and sometimes even lethal diseases in their vertebrate hosts. Numerous outbreaks have been brought on by alphaviruses worldwide in the last few decades. Chikungunya virus (CHIKV) stands out as a major global health concern due to recurrent outbreaks and debilitating chronic arthralgia caused by CHIKV infection. The arthralgia might be due to the persistent replication of CHIKV. CHIKV non-structural protein 4 (nsP4) is the RNA-dependent RNA polymerase (RdRp), which enables the replication of the viral genome and therefore plays a critical role in virus infection. In this work, we established the inducible stable cell lines for CHIKV nsP4 expression. NsP4 expressed in these cells can functionally trans-complement CHIKV mutant genomes harboring lethal mutations in nsP4 or lacking the nsP4 region entirely. This system enables the production of CHIKV virions that are infectious for nsP4 expressing cell lines but limited to early stages of infection (attachment, internalization, and replicase protein expression) in other cells. Moreover, our system was found to be suitable for the analysis of compounds inhibiting CHIKV replication; for example, RdRp inhibitor 4’-Fluorouridine significantly inhibited viral replication in nsP4 expressing cells. Additionally, our study revealed that CHIKV nsP4 can form functional replicases with P123 polyproteins of heterologous alphaviruses using trans-replication assay. This finding expands the versatility of the CHIKV nsP4 stable cell lines. We extended these findings and constructed inducible cell lines that express both nsP1 and nsP4 components of replicase and demonstrated that these cells can complement for lethal defects introduced into both nsP1 and nsP4 of the CHIKV genome. Taken together, CHIKV nsP4 inducible stable cell lines are valuable tools for alphavirus research, can be used to produce virions that are infections only in specific cell lines and therefore be handled without biosafety risk, such tools open new possibilities for studies of alphavirus replication as well as for screening and analysis of inhibitors of alphavirus replication.
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    T-cell receptor repertoire analysis in CVID patients post Comirnaty vaccination
    (Tartu Ülikool, 2024) Aghayari, Avishan; Kisand, Kai, juhendaja; Elsakova, Alexandra, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Common variable immunodeficiency (CVID) is one the most diagnosed primary immunodeficiencies (PID) with a complex etiology, characterized by low levels of serum immunoglobulins mainly due to B-cell deficiencies with potential T-cell dysregulations and abnormalities. This immune system dysfunction puts CVID patients at a higher risk for developing severe infections, including COVID-19. Despite its critical importance, their immune response to SARS-CoV-2 infection and vaccination remains unclear. Studying the T-cell-receptor (TCR) repertoire of these patients sheds light on the dynamics of their cellular immune response, knowing that T-cells recognize antigens through their TCR. In this study, we utilized high throughput sequencing to investigate and profile the TCR repertoire of PID patients, particularly those with CVID following the second dose of the Pfizer-BioNTech Comirnaty-BNT162b2 vaccine. We report on the diversity and clonality of TCR repertoires between diseased and healthy individuals post-vaccination. Investigating gene segment usage, we found a bias in the frequency of TCR V beta gene segment usage between patients and controls. Lastly, we examined the clonal expansion of T-cells following Spike-antigen (S Ag) stimulation to provide insights into the T-cell response post-vaccination.
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    Exploring the microbial communities of ferromanganese concretions and pockmarks in the Gulf of Finland
    (Tartu Ülikool, 2024) Lukaša, Līva Kristiāna; Laas, Peeter, juhendaja; Kisand, Veljo, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Ferromanganese concretions are aggregates of precipitated minerals, of which the mechanism of formation is not clear. They are found in aquatic environments and may be formed by abiotic and/or biotic processes. Pockmarks are depressions in the seafloor that can be caused by groundwater breaking through it during a process called submarine groundwater discharge. This work was conducted to assess the bacterial community composition of ferromanganese concretions and pockmark sediments using molecular biology methods and comparing them to seawater and other sediment samples from the Gulfs of Finland and Riga, and Väinameri. The concretion microbiome was significantly different from all the other sample types, as was the sediment microbiome from one pockmark site. Both microbiomes were distinguished by a high abundance of bacteria from the phylum Patescibacteria, which may be involved in the formation of concretions. Pockmark sediments from a different site resembled sediments not affected by groundwater, signifying the inactivity of this pockmark. Different layers of sediment cores were homogenous in their microbial community composition, pointing to the occurrence of sediment resuspension.
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    Analysis of Chikungunya Virus Capsid Interactome
    (Tartu Ülikool, 2024) Bratslavskaia, Elizaveta; Varjak, Margus, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    The ongoing global spread of alphaviruses presents a significant challenge, underscoring the urgent need for the development of novel antiviral treatments. Understanding the intricate host virus interactions that underlie virus infection is crucial for uncovering potential pro- or antiviral targets for such treatment strategies. In this study, we investigated the interactome of the Chikungunya virus (CHIKV) Capsid protein (CP), a key player in viral replication, assembly, and cell-virus interactions. Here, the quantitative label-free proteomics analysis was used to study CHIKV CP interactors in two different types of cells, in the human host and mosquito vector. A number of host and vector factors were identified, among them were many homologous proteins to be focused on in further downstream analysis. The effect of selected interactors on CHIKV pathogenesis was evaluated in mosquito and human cells. Notably, silencing of PGAM5, SRRT, and VIRMA proteins significantly reduced the level of viral replication, demonstrating a shared pro-viral effect in both, human and mosquito, cell types tested. These results underscore the conservation of CP function across different organisms and highlight the potential significance of these cellular factors in the context of CHIKV infection. Overall, this study provides valuable insights into the molecular mechanisms underlying CHIKV pathogenesis and identifies potential targets for the development of novel antiviral treatments.
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    Insights into gas fermentation optimisation for enhanced acetate production
    (Tartu Ülikool, 2024) Mishchuk, Anatolii; Acuna Lopez, Pedro, juhendaja; Quataert, Koen, juhendaja; Valgepea, Kaspar, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Conversion of CO2 utilising gas fermenting acetogens is a feasible and environmentally beneficial solution to the global problem of greenhouse gas emissions. Homoacetogens are an especially intriguing type of microorganisms since they can yield acetate as their primary metabolic product via the Wood–Ljungdahl pathway. A product inhibition mechanism is observed when acetate accumulates in high concentrations, adversely impacting bacterial growth and acetate production. The tolerance of these microorganisms towards varying concentrations of acetate and under identical conditions has not previously been investigated. However, it has been defined as crucial for selecting the most robust acetogen for its use at industrial scale. The designed experimental setup for acetate tolerance studies, including positive and negative controls, was intended to answer this scientific question. Following the pre-screening test, it was observed that the carbon source affected the microbial ability to tolerate acetate; bacteria performed substantially better when a more energy-rich carbon source, glucose, was supplemented. The latter finding was accounted for when designing the main screening setup for acetate tolerance in four well-known homoacetogenic strains, where Moorella thermoacetica and Thermoanaerobacter kivui performed the best. Then, T. kivui was utilised to perform pH-controlled fermentations in pressurised gas bioreactors. Furthermore, the outcomes of this master's thesis support the hypothesis that gram-positive anaerobes undergo variation in their Gram staining due to oxygen exposure. This thesis provides valuable information for the future selection and screening of homoacetogenic strains and the effect of pH-controlled cultivation in a pressurised bioreactor.
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    Development Of Glucose-independent Yarrowia Lipolytica Processes
    (Tartu Ülikool, 2024) Kucukozden, Yigit; Bottoms, Scott, juhendaja; Dahlin, Jonathan, juhendaja; Mans, Robert, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    As the global population continues to expand, there is a rising demand for animal meat production. However, the consumption and production of animal-based protein have detrimental effects on human health and the environment. Furthermore, challenges such as zoonotic diseases, climate change, and land scarcity underscore the need for exploring alternative meat sources. The study aims to investigate the feasibility of using Yarrowia lipolytica in mycoprotein research, focusing on enhancing its growth rate on environmentally friendly carbon sources that mitigate industrial CO2 emissions. This involves employing molecular and genetic approaches to optimize natural metabolic pathways and utilizing adaptive laboratory methods for fermentation. This study, a part of the Yummowia project, advances scientific understanding in synthetic biology and food science and holds promise in shaping the future landscape of sustainable and appealing meat alternatives. The collaborative and multidisciplinary nature sets the stage for continued innovation, with the potential to positively influence the broader food industry.
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    Systematic interpretation of large-scale GWAS analyses of 5,035 phenotypes
    (Tartu Ülikool, 2024) Alekseienko, Anastasiia; Võsa, Urmo, juhendaja; Abner, Erik, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    This thesis presents the systematic interpretation of 5,035 genome-wide association studies (GWAS) conducted within the Estonian Biobank, aiming to elucidate the genetic determinants influencing a diverse array of phenotypic traits. Through a review of existing literature and the application of advanced bioinformatic tools, the work done in this thesis outlined the results of main post-GWAS methods, such as the identification of novel variants, SNP heritability estimation, fine-mapping of causal variants, and prioritization of genes associated with complex traits. Around 26% of the variants identified as lead ones in this work are novel and had not been implicated by GWASs before. Fine mapping prioritized single genetic variants for 10.3% of investigated loci, providing hypotheses for further functional studies, and the gene prioritization approach identified the 2,402 lead variants to be related to 804 genes, several of those biologically interpretable. Heritability was reliably inferred for 25% of studied phenotypes, the most heritable traits being “Other specified hypothyroidism” (ICD-10 code E03.8), “Obesity due to excess calories” (E66.0), “Obesity” (E66), “Myopia” (H52.1), and “Hypertension” (I10). These findings are a good starting point for a more in-depth interpretation of loci associated with complex diseases in the Estonian Biobank.
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    Optimization of plasmid curing from genetically engineered Clostridium autoethanogenum
    (Tartu Ülikool, 2024) Udemezue, Victoria Chinonyerem; Valgepea, Kaspar, juhendaja; Shaikh, Kurshedaktar Majibullah, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    The accumulation of greenhouse gases (GHGs) released by harmful human activities involving the combustion of fossil-fuels is a driver of climate change that threatens biosustainability on Earth. Microbial gas fermentation provides an attractive option to capture CO2 while also enabling biomanufacturing of chemicals, fuels, and proteins. Acetogens are the preferred biocatalysts for gas fermentation as they can use CO2 as their sole carbon source (with H2 as energy source). However, genetic engineering of acetogens to better understand their metabolism and develop cell factories is challenged by slow growth, very low transformation efficiencies, and inefficient plasmid curing. In this thesis, we developed a CRISPR/Cas9-based curing plasmid (C-plasmid) tool for optimized plasmid curing from the model-acetogen Clostridium autoethanogenum. Firstly, the C-plasmid was constructed to express Cas9 and a gRNA targeting the ColE1 origin of replication in both the C-plasmid and an editing plasmid (E-plasmid). Next, the C-plasmid and the non-template gRNA plasmid (N-plasmid) were electroporated into C. autoethanogenum harboring an E-plasmid used for gene deletion and culture were plated on agar. Plate counting and PCR screening showed no presence of plasmids in colonies transformed with either C-plasmid or N-plasmid. This implies that cells were cured of plasmids by the act of electroporation and transformation of a C-plasmid might not be needed. In any case, this thesis seems to have identified a significantly more efficient plasmid curing method for C. autoethanogenum. Further tests are needed to confirm these observations and its applicability to other genetically-engineered C. autoethanogenum strains. The methodology has potential to contribute towards improving genetic engineering workflows for acetogens.
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    Carbon dioxide sensing in Arabidopsis thaliana mediated by liquid-to-liquid phase separation of Protein Phosphatase Type 2C homologs
    (Tartu Ülikool, 2024) Heincz, Stefan; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Carbon dioxide sensing is crucial for plants to maintain a balance between exchange of gas and water vapor which directly affects tolerance of environmental stresses and ensures plant survival. Understanding CO2 sensing in plants will therefore help to be more prepared for future challenges in food crop production caused by increased environmental stresses due to climate change. An up to this point nearly unstudied process in plants, the phase separation (PS) of proteins, has recently been shown in vitro to be the main mechanism of CO2 sensing of a protein phosphatase type 2C (AP2C3) from Arabidopsis thaliana. PS describes the process of protein condensate droplet formation due to local enrichment of protein caused by interactive motifs usually located in an intrinsically disordered region (IDR) within the polypeptide backbone. In this study, we attempted to reproduce published results and screen for further IDR/PS mediated CO2 sensitivity of PP2Cs by cloning, expression, and purification of AP2C1, AP2C4 and PP2C74, along with AP2C3, for in vitro imaging experiments. We successfully established a reliable laser scanning confocal microscopy imaging setup for monitoring the phase separation of purified proteins fused to fluorescent tags. However, CO2-condensation of PP2C proteins could not be detected. Furthermore, we aimed to study PS and verify intracellular localisation of selected PP2C homologs in planta via a transient overexpression assay in Nicotiana benthamiana. Additionally, a connection of AP2C3 with the guard cell CO2 signalling pathway by a bimolecular fluorescence complementation assay with Mitogen-activated Protein Kinase 12 (MPK12) was investigated. These experiments verified the intracellular localisation of selected homologs, the presence of biomolecular condensates for AP2C3/4 in planta and indicated interaction between AP2C3 and MPK12.
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    Emotion Recognition using EEG signal data from EMO2018 Dataset
    (Tartu Ülikool, 2024) Rebriks, Aleksandrs; Avots, Egils, juhendaja; Juuse, Liina, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Emotion Recognition (ER) is developing area within the artificial intelligence field that is focused on comprehending and further interpreting of human emotions through various modalities. Despite that, these approaches are often not ubiquitous as they are affected by external factors. With recent physiology research connecting development of emotions to the central nervous system, usage of brain signals became a highly practical option for emotion recognition. One of the most promising methods of emotion recognition using brain signals for emotion recognition involves using Electroencephalography (EEG). Despite being more complex than classical machine learning or deep learning approaches, EEG-based emotion recognition is potentially more accurate and robust, with applications in mental health monitoring, researches in applied physiology or human-computer interactions. This thesis studies existing approaches of EEG-based emotion recognition methods for private EMO2018 dataset. We adopted methods of Fast Fourier Transform with additional processing for key features extraction and tested different Deep Learning models. Our results show performances of utilized Deep learning models with best accuracy of 88.6% from Hybrid Neural Network approach.
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    Screening for genes that underlie organs’ 3D structure formation using fruit fly as a model organism
    (Tartu Ülikool, 2024) Elias Elias; Shimmi, Osamu, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Organ formation involves dynamic cell shape changes, adaptations and responses to signaling molecules. Despite numerous studies on the involvement of various cellular structures and signaling pathways in organogenesis, current knowledge regarding the genetic control over dynamic tissue morphogenesis remains limited. The pupal wing of Drosophila melanogaster has been shown to include cellular structures and pathways common to the organogenesis of other animals. This project utilized Drosophila wings to study specific candidate genes associated with organogenesis and their contributions to final wing development. The screening of genes of interest has been carried out by employing tissue- and stage-specific RNAi-mediated gene knockdown methods. The results show that seven candidate genes, which have been characterized as regulating cellular homeostasis and structures, play important roles in wing morphogenesis and vein formation. Furthermore, most RNAi-mediated wing phenotypes resemble the loss-of-function phenotypes of conserved signaling pathways such as Notch, Wg/Wnt and BMP. Therefore, these observations suggest that candidate genes may regulate conserved signaling pathway during Drosophila wing development.
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    Methane Flux Dynamics and Driving Mechanisms in Constructed Wetland
    (Tartu Ülikool, 2024) Efakwu, Gideon; Okiti, Isaac, juhendaja; Kasak, Kuno, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Methane (CH4) is a greenhouse gas primarily produced through natural processes and human activities. Constructed wetlands (CWs), while effective for treating diffused agricultural runoff, can also emit CH4 due to the anaerobic decomposition of organic matter. This study investigates CH4 emissions and their driving mechanism in Vända CWs (designed to treat agricultural runoff) in Uhti village, southeast Estonia. The CWs have two sections (wetlands one and two). CH4 emissions were measured from October 2022 to December 2023. The result reveals that both wetlands emit CH4 emissions. However, the second wetland has higher emission with a median of 619.2 μg m⁻² hr⁻¹ (5306.1 μg m⁻² hr⁻¹ ± 936.5 μg m⁻² hr⁻¹ mean ± standard error SE) and a peak emission of about 35,000 μg m⁻² hr⁻¹ compare to the first wetland which shows a median of 218.9 μg m⁻² hr⁻¹ (2424.4 μg m⁻² hr⁻¹ ± 743.9 μg m⁻² hr⁻¹) and a peak of above 15,000 μg m⁻² hr⁻¹. Also, both wetlands exhibited seasonal variations, with summer months displaying the overall CH4 emission with a median of 2666.8 μg m⁻² hr⁻¹ (8393.3μg m⁻² hr⁻¹ ± 3351.6 μg m⁻² hr⁻¹) in the first wetland and a median of 11150.8 μg m⁻² hr⁻¹ (19430.7 μg m⁻² hr⁻¹ ± 3563.4 μg m⁻² hr⁻¹) in the second wetland. Winter months showed the lowest overall CH4 median emission of 4.9 μg m⁻² hr⁻¹ (103.5 μg m⁻² hr⁻¹ ±36.1 μg m⁻² hr⁻¹) in the first wetland and a median of 4.9 μg m⁻² hr⁻¹ (114.1 μg m⁻² hr⁻¹ ±47.4 μg m⁻² hr⁻¹) in the second wetland. Spearman's rank correlation analysis was used to assess the strength of the relationships between CH4 emission and various measured parameters. The analysis revealed that several measured parameters, including dissolved oxygen (O2), pH, oxidation-reduction potential (ORP), and conductivity, exhibited statistically significant correlations (<0.001) with CH4 emission when compared to other parameters. Based on these findings, we recommend regular harvesting of wetland biomass and installation of aeration techniques as mitigation strategies for reducing CH4 emissions from Vända-constructed wetlands.
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    Enhanced Speech Emotion Recognition Using Averaged Valence Arousal Dominance Mapping and Deep Neural Networks
    (Tartu Ülikool, 2024) Rizhinashvili, Davit; Anbarjafari, Gholamreza, juhendaja; Sham, Abdallah Hussein, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    This thesis delves into advancements in speech emotion recognition (SER) by establish ing a novel approach for emotion mapping and prediction using the Valence-Arousal Dominance (VAD) model. Central to this research is the creation of reliable emotion to-VAD mappings, achieved by averaging outcomes from multiple pre-trained networks applied to the RAVDESS dataset. This approach adeptly resolves prior inconsistencies in emotion-to-VAD mappings and establishes a dependable framework for SER. The study also introduces a refined SER model, integrating the pre-trained Wav2Vec 2.0 with Long Short-Term Memory (LSTM) networks and linear layers, culminating in an output layer representing valence, arousal, and dominance. Notably, this model exhibits commendable accuracy across various datasets, such as RAVDESS, EMO-DB, CREMA-D, and TESS, thereby showcasing its robustness and adaptability, an improvement over earlier models susceptible to dataset-specific overfitting. The research further unveils a comprehensive speech analysis application, adept at denoising, segmenting, and profiling emotions in speech segments. This application features interactive emotion tracking and sentiment reports, illustrating its practicality in diverse applications. The study recognizes ongoing challenges in SER, especially in managing the subjective nature of emotion perception and integrating multimodal data. Although the research marks a progression in SER technology, it underscores the need for continuous research and careful consideration of ethical aspects in deploying such technologies. This thesis contributes to the SER domain by introducing a dependable method for emotion to VAD mapping, a robust model for emotion recognition, and a user-friendly application for practical implementations.
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    The N-terminus of Melanoma-associated antigen A10 interacts with peripheral nucleoporins
    (Tartu Ülikool, 2024) Nesterovich, Alexey; Kurg, Reet, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Showing common features of tumorigenesis, Melanoma-associated antigen A10 (MAGEA10) possesses the potential for cancer therapy. MAGEA10 is a highly immunogenic MAGEA family member that acts as a hub protein and is associated with global hypomethylation. The N-terminus of MAGEA10 is involved in its expression, nuclear transport, and electrophoretic mobility shift. This thesis aims to characterise further the N-terminus of MAGEA10 and find its potential interaction partners.
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    Isolation of extracellular vesicles induced by Chlamydia pneumoniae infection
    (Tartu Ülikool, 2024) Pelypenko, Vadym; Kurg, Reet, juhendaja; Hanski, Leena, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Chlamydia pneumoniae is an obligate intracellular parasite distinguished by the increased resistance based on the unique life cycle including the switches between several morphological forms of high pathogenicity, resistance or metabolic activity. Despite the high antibody prevalence and diverse transmission ways, these days there is no optimal method of diagnostics of chronic form of chlamydial pneumonia. Here we introduce the study aimed to establish and optimize the protocol for isolating the extracellular vesicles derived from infected A549 lung epithelial cells and THP-1 monocytes, as well as for initial evaluation of vesicles amount produced by treated and non-treated cells via the total protein content and the level of CD63 exosome marker.
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    Investigating Ribosomal Catalysis: Optimizing Assays with Thermostable Bacterial Ribosomes for Defining Minimal Components Necessary for Peptidyl Transferase Activity
    (Tartu Ülikool, 2024) Novoshinskaia, Mariia; Forster, Anthony, juhendaja; Bao, Letian, juhendaja; Remme, Jaanus, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Ribosomes are cellular molecular machinery, facilitating the peptidyl transferase activity – a crucial reaction for the elongation of the peptidyl chain during translation. The process of how exactly ribosome catalyzes peptidyl transferase activity remains a subject of study. Researchers are determined to establish the minimal ribosomal components essential for ribosomal activity. Studies with E. coli 70S ribosome showed that only large ribosomal subunit 50S is essential for performing the peptidyl transferase activity. Later, it was discovered that only one of 50S rRNAs, 23S rRNA, is essential, as it contains the peptidyl transferase center. Later studies with more thermophilic bacteria species paved the way to create a minimal synthetic ribosome reconstructed from the in vitro synthesized ribosomal rRNAs. This project aims to create a minimal Thermus thermophilus ribosome from in vitro transcribed (IVT) rRNAs and naturally extracted 50S total protein components and assay its peptidyl transferase activity with fragment reaction to test the functionality of the reconstructed large ribosomal subunit. For this purpose, a new assay with f[35S]Met-tRNAfMet was assembled. The results of this work show that using [35S] radiolabel leads to more sensitive peptidyl transferase activity detection. On the other hand, the detected activity of the reconstructed Thermus thermophilus 50S subunit is still low, which significantly differs from studies with closely related bacteria species. That opens a question of whether post-translational modifications matter for the activity of Thermus thermophilus ribosomes.
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    A Study of Anti-adenoviral activity of antimicrobial peptides
    (Tartu Ülikool, 2024) Nabeel, Muhammad; Rausalu, Kai, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Adenoviruses, non-enveloped DNA viruses, pose a significant health concern. This study explores the antiviral potential of four recombinant proteins with attached cationic antimicrobial peptides (H1mod1R, H2mod2R, HELP and HIn) against the human adenovirus (AdV-Gluc) in vitro. The proteins presented some antiviral activity, but this inhibitory effect was independent of the concentrations of the proteins within the tested range. The results are not aligned with the previous research on the cationic antimicrobial peptides with other viruses. Currently, these proteins are not ideal for use as therapeutic against adenovirus, but the good cytotoxicity profile suggests that further research is needed to optimize the efficacy and to explore their mechanism of action for beneficial future applications.
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    The Role of CISD2 in Autophagy of Endoplasmic Reticulum
    (Tartu Ülikool, 2024) Pieris, Kurukulasuriya Anne Nicola Rashali; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Wolfram syndrome (WS) is an uncommon neurological condition distinguished by the presence of diabetes insipidus, diabetes mellitus, optic atrophy, and hearing loss. The condition is attributed to genetic alterations in either the WFS1 gene (WS1) or the CISD2 gene (WS2). CISD2 genes for a 15-kDa transmembrane protein that is found in the endoplasmic reticulum (ER). It is involved in several cellular functions such as maintaining calcium and iron levels, as well as regulating autophagy. This work examines the function of CISD2 in autophagy, specifically focusing on ER-phagy, which is a type of selective autophagy that targets the endoplasmic reticulum. We utilized HeLa cell models with CISD2 deletion (CISD2-KO) and wild-type (CISD2-WT) conditions to evaluate the levels of ER-phagy using confocal microscopy and Western blot analysis. The findings suggest that the lack of CISD2 results in heightened ER-phagy, whereas its presence or excessive expression diminishes ER-phagy, hence illustrating the protective function of CISD2. Furthermore, it was demonstrated that interaction partners such as CISD1 and Bcl-2 had an impact on ER-phagy in cells lacking CISD2, indicating prospective targets for therapeutic intervention in disorders related to CISD2 deficiency. This study offers valuable information about the functional role of CISD2 in ER-phagy and its possible implications in WS2 and other related diseases.
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    Unsupported Solution Blow Spinning: Dependence on Polymer, Solvent, and Blowing Parameters
    (Tartu Ülikool, 2024) Bambals, Daniels; Must, Indrek, juhendaja; Vihmar, Marie, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Fabrication of unsupported micro/nanofiber structures using Solution Blow Spinning (SBS) is a versatile method for creating fine fibers with potential applications in various fields such as biomedicine, filtration and soft robotics. While many methods exist for investigating non-woven tensile properties, the self-tensioning behavior of fibers post-spinning is less explored. This study addresses this gap by focusing on how internal tensions within the fibers influence their structure. The primary focus is on developing and refining methodologies to optimize fiber formation for applications requiring unsupported, freestanding structures during spinning. This work varied the solution composition, the type of polymer and solvent used, and incorporating additives like ionic liquids to enhance fiber formation properties. A methodology was developed for investigating tensioning of fibers post-spinning. Polyethylene oxide revealed limitations due to inadequate post-tensioning properties, the fibers were not able to endure their own tensioning force and ruptured. Poly(vinylidene fluoride-co-hexafluoropropylene) showed potential yet also faced challenges in maintaining structural integrity under tension. Incorporating 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ionic liquid into the poly(vinylidene fluoride-co-hexafluoropropylene) solution enhanced post-tensioning and reduced material fractures. This modification led to fibers capable of sustaining higher strains making them suitable for robust structural applications such as biomedical scaffolds. The study establishes a framework for the scalable production of unsupported fiber structures. The findings suggest that with further optimization, SBS could be used to produce different unsupported fiber-based constructs directly on anchors/supports instead of creating the structures after achieving supported fibrous mats.
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    The analysis of novel Far1 motif important for Cdk1 inhibition
    (Tartu Ülikool, 2024) Mukhadze, Juli; Faustova, Ilona, juhendaja; Maljavin, Artemi, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    The cell cycle progression in yeast is regulated by cyclin-dependent kinases (CDKs), which phosphorylate different substrate proteins to ensure proper order and timing of cell cycle events. In the presence of pheromones, cell division can be interrupted in the G1 phase via activation of the mating mitogen-activated protein kinase (MAPK) pathway. In this pathway, MAP kinase Fus3 phosphorylates Far1 protein. Consequently, phosphorylated Far1 can bind to the CDK complex and suppress its activity. Although T306 phosphorylation of Far1 was shown to be crucial for CDK inhibition, the exact mechanism and main determinants of this process in the Far1 protein sequence remain unclear. In this study, we analysed the previously undescribed amino acid region of Far1 and identified key residues with the strongest impact on CDK activity, which likely points to their importance for protein-protein interactions. Our results suggest that the analysed Far1 region may potentially serve as a CDK docking site. However, further analyses are required to reveal the exact mechanism of inhibition.