Science and Technology - Bachelor's theses

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    Investigating ternary template-assisted mechanochemical synthesis for Fe/Ni-N-C bifunctional electrocatalyst: fabrication, characterization, and performance evaluation
    (Tartu Ülikool, 2023) Wojsiat Boere Rodrigues da Cunha Souza, Ivan Tito; Kosimov, Akmal, juhendaja; Kongi, Nadežda, juhendaja
    The transition to alternative energy sources to mitigate global climate issues has led to increasing interest in renewable energy generation. However, the intermittent nature of renewables and the significant upfront investments required pose challenges in ensuring a reliable and cost-effective energy supply. Metal-air batteries (MABs) have emerged as a promising efficient and low-cost energy storage solution. However, the kinetics of the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) in the MAB cathode limit their performance. Extensive research has focused on developing bifunctional electrocatalysts to optimize MAB efficiency and stability during charge-recharge cycles. This study investigates a novel strategy for synthesizing a Fe/Ni-N-C bifunctional electrocatalyst using a multifunctional templating approach via neat, liquid-assisted ball- milling grinding. The synthesized catalyst is characterized using various techniques, and its electrochemical performance is evaluated. The findings contribute to developing cost- effective and efficient electrocatalysts for sustainable energy storage.
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    Methanolysis of Benzoyl hydrazines in the presence of Trifluoromethanesulfonic acid
    (Tartu Ülikool, 2023) Ul Haq, Intasar; Mastitski, Anton, juhendaja
    Methanolysis of variously substituted benzoyl hydrazines in the presence of CF3SO3H was explored for the first time. The effects of CF3SO3H acid concentration and substituents attached to benzoyl hydrazine on yield and reaction times were also examined. It was found that doubling the acid concentration results in an approximately four-fold decrease in reaction times. Moreover, reaction times are strongly dependent on the nature of substituents. Presence of electron-donating substitutes increases the reaction rate, while presence of electron-withdrawing groups has the opposite effect. The obtained results clearly demonstrate that acyl hydrazines could be converted to esters in good to excellent yields in the presence of 6-9 eq of CF3SO3H.
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    Stoma-inspired valve
    (Tartu Ülikool, 2023) Makarov, Aleksandr; Must, Indrek, juhendaja
    Soft robotics and microfluidics are interdisciplinary fields with shared methods for developing flexible and functional devices. However, manufacturing durable, efficient, and robust devices remains a challenge. One of the key hurdles is the limited number of valve designs available. Currently employed microvalves are either normally closed with negative control pressure or normally open with positive control pressure. However, there has not been a microvalve design reported which is normally closed and has positive control pressure, while it was present for an extended period in nature in the form of a plant stoma. Plant stoma is a robust normally closed valve with positive control pressure, with robustness arising from the fact that when the plant dries the valve closes. It was used as an inspiration source for the valve in this work, which is normally closed and opens at positive as well as negative control pressure, which are improvements over previously reported designs. The findings suggest the potential of this technology for applications ranging from artificial leaves to bioreactors. By considering nature as an inspiration source, a contribution was made towards more sustainable solutions.
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    Testing antibacterial drugs against nongrowing bacteria
    (Tartu Ülikool, 2023) Bērziņš, Normunds; Kaldalu, Niilo, juhendaja
    All antibiotics currently in use were discovered due to their ability to stop bacterial growth. However, only a few of them can kill nongrowing bacteria. During chronic and recurrent infections, a large fraction of the pathogenic bacteria is nongrowing or grow very slowly. Drugs are needed that kill nongrowing bacteria and it is therefore reasonable to look for potential candidates in already existing drug libraries. We treated non-growing stationary phase cultures with the drug candidates looking for those that cause post-incubation regrowth delay. A colony counting method optimized for high throughput was used to determine the bactericidal activity of the hit compounds. 38 drugs were previously identified to increase regrowth delay of nongrowing E. coli. They were further tested for their activity against nongrowing Pseudomonas aeruginosa and Staphylococcus aureus. Several drugs of multiple classes – fluoroquinolones, macrolides, and anticancer drugs – were found to heavily increase the regrowth time of P. aeruginosa. Additionally, these drugs exhibited strong bactericidal effect, killing multiple magnitudes of bacteria. Fewer drugs were as effective against S. aureus, but a couple – an antiseptic/disinfectant and an anticancer drug - were found to increase the regrowth delay significantly and showed bactericidal activity.
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    Characterization of gut microbiome composition in T2D patients under SGLT2i and GLP1RA treatment
    (Tartu Ülikool, 2023) Kazimova, Valida; Org, Elin, juhendaja; Krigul, Kertu Liis, juhendaja
    Type 2 Diabetes (T2D) is a globally recognized health concern that has garnered significant research attention. This thesis work focuses on gut microbiome analysis in T2D patients who start taking two medications antidiabetic drugs: Empaglifrozin (SGLT2i) and Semaglutide (GLP1RA). Microbiome composition was analyzed using 16S rRNA sequencing. The research aims to enhance our understanding of how the gut microbiome is related to SGLT2/GLP1 drug intake, potentially leading to the development of personalized treatment approaches for improved patient outcomes.
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    Characterization of non-native promoters in Saccharomyces cerevisiae
    (Tartu Ülikool, 2023) Bayón Buján, Paula; van der Hoek, Steven Axel, juhendaja
    Yeast has been a valuable model organism for both industry and research. However, until recently, the use of native promoters in scale-up production has led to strain instability and subsequently, loss of productivity. Previous studies have found TEF promoters to give stable expression levels across culture conditions in addition to being a gene very conserved among yeast strains. Therefore, we investigated the strength of TEF promoters from different organisms by analyzing their expression of GFP on different carbon sources and developed a synthetic biology toolkit containing different strength non-native promoters for Saccharomyces cerevisiae to avoid several copies of the same promoter in the genome. Kluyveromyces marxianus promoter sequence showed promising results, surpassing our positive control. Cyberlindnera jardinii and Candida parapsilosis TEF2 exhibited medium strength, and Candida parapsilosis TEF1, Scheffersomyces stipites, and Yarrowla lipolytica promoters demonstrated weaker strength but were still functional in the yeast, performing better than the negative control. These results show that the investigated promoters could be used to engineer cell factories in research and industry.
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    Susceptibility of nongrowing uropathogenic Escherichia coli to fluoroquinolones: killing and induction of the SOS response
    (Tartu Ülikool, 2023) Akulich, Anna; Kaldalu, Niilo, juhendaja; Hinnu, Mariliis, juhendaja
    Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections. UPEC is capable of entering the host cells and adapting to an intracellular lifestyle. This provides bacteria protection against host defensive mechanisms and antibiotics, leading to chronic and recurrent infections. Due to the limited nutrients inside host cells, bacteria are nongrowing. Nongrowing bacteria are not killed by most bactericidal antibiotics. However, some antibiotics of the fluoroquinolone group - gatifloxacin and ofloxacin – are known to be effective. According to an unpublished study, it was suggested that fluoroquinolones do not kill nongrowing bacteria but poison them. This study investigated the killing efficiency and in-duction of SOS response by a set of different fluoroquinolones in various growth conditions. Specifically, bactericidal effect on stationary phase culture before and after 1M NaCl incubation and induction of SOS response for growing stationary phase and regrowing cultures. The results of this thesis have supported the hypothesis of delayed bacterial death after fluoroquinolone poisoning.
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    Cdk1-regulated diphosphodegron tags for controlled protein expression
    (Tartu Ülikool, 2023) Strelchenko, Stepan; Faustova, Ilona, juhendaja; Örd, Mihkel, juhendaja
    Cyclin dependent kinases (CDKs) regulate the cell cycle by phosphorylating downstream proteins in an ordered manner. In yeast S. cerevisiae, Cdk1 regulates the cell cycle progression from G1 to M phase. Cdk1 phosphorylation can have various effects on the proteins, for example, they can be activated or targeted for degradation. The latter was used in this work to design a regulatory network for controlled protein expression that could be used in cell factories. For this, Far1-based diphosphodegron tags that are degraded in response to phosphorylation by Cdk1, were used to differentially regulate the levels of the transcriptional repressor TetR. Furthermore, as cell factories require dynamic control of protein levels over the cultivation process, the effect of cell culture density on cell cycle and the degron tags was studied. The work shows that Far1-based degron tags can be used as modular tags to regulate exogenous proteins by switching them on or off in an OD-dependent manner using the endogenous Cdk1 machinery. Finally, it was shown that more complex patterns of regulation can be achieved by inducing expression of cyclins at high OD.
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    Activity of macrolides against uropathogenic Escherichia coli
    (Tartu Ülikool, 2023) Shahpazir, Ana; Kaldalu, Niilo, juhendaja
    Azithromycin is a macrolide antibiotic extensively used to treat several infections attributa- ble to Gram-positive bacteria. Exceptionally, azithromycin has proven to be effective in clin- ical treatment of widespread chronic infections caused by Gram-negative bacterium, Salmo- nella. While the underlying mechanisms of azithromycin’s activity against this Gram-nega- tive bacterium remain enigmatic, its efficacy brings up the question of whether this macro- lide can be used in treatment of other Gram-negative bacterial infections as well. The goal of this work was to investigate the possibility of utilizing azithromycin against uropatho- genic Escherichia coli. For this purpose, we determined the minimum inhibitory concentra- tion of azithromycin in conditions resembling intracellular infection sites. Additionally, we validated the use of four macrolide bioreporters that were based on the regulatory leader peptide coding sequence of the macrolide resistance gene, ermCL.
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    Conjugation-driven horizontal gene transfer in bacteria on antimicrobial surfaces
    (Tartu Ülikool, 2023) Tomitšik, Levi; Ivask, Angela, juhendaja
    Conjugation is the main mechanism of horizontal gene transfer of antibiotic resistance genes between bacteria. There is evidence that the presence of trace amounts of antimicrobials in the environment may increase the conjugation frequency between bacteria and thus, potential spread of antibiotic resistance genes. However, to date the effect of stress conditions, including the presence of antimicrobials, on conjugation is relatively modestly studied and almost no studies are available about the effect of antimicrobial surfaces on conjugation frequency. This study first constructed an optimized conjugation protocol for solid surfaces. For that, a chromosomally marked strain of Pseudomonas putida KT2440 to be used as recipient strain in conjugation, was constructed. As a donor strain, Escherichia coli CSH26 with conjugative plasmid pKJK5 was used. The optimal experimental conditions: organic content, conjugation time, and donor-to-recipient ratio on conjugation frequency, were studied. For the conjugation experiments, a series of copper- silver- and quaternary ammonium- based surfaces that were expected to show an antimicrobial effect, were selected. Plastic and stainless-steel surfaces served as controls with no expected antimicrobial activity. Prior to conjugation, viability of donor and recipient on test surfaces was analyzed and the most toxic surfaces were not included in conjugation assay. On selected surfaces, conjugation experiments were carried out for 24 h and conjugation frequencies were calculated for each surface. The results showed that the presence of low level of copper on surface may promote conjugation while silver- and quaternary ammonium compounds-based surfaces did not significantly affect conjugation frequency.
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    Characterisation of Type II toxins discovered through iterative guilt-by-association search
    (Tartu Ülikool, 2023) Rustamova, Aytan; Brodiazhenko, Tetiana, juhendaja; Mets, Toomas, juhendaja; Hauryliuk, Vasili, juhendaja
    The classical toxin-antitoxin (TA) system is encoded as a bicistronic operon consisting of toxin and antitoxin genes. Toxin-antitoxin systems have been studied for more than 30 years and have been shown to play an important role in bacterial immunity and stress response. The most common and best-studied class of TA systems is type II, where both the toxin and the antitoxin are proteins, and the toxin is neutralized by the antitoxin via direct interaction. My thesis helps to uncover the molecular mechanisms of type II toxins found by the bioinformatics tool NetFlax (standing for Network of Flanking genes for toxins and antitoxins) via metabolic labelling assays. I characterize several toxins from different functional domains and show how the toxins affect the bacterial cell using Escherichia coli as the model organism.
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    Automating assessment of silver-enhanced in situ hybridization for evaluation of cancer properties
    (Tartu Ülikool, 2023) Kuklianov, Danila; Haamer, Rain Eric, juhendaja; Haamer, Sisi Carmen, juhendaja
    To correctly assess breast cancer properties, doctors have to compare and evaluate histopathological slides stained for the presence of certain proteins; in the event that the initial evaluation is inconclusive, additional assessment is done. The assessment is necessary to accurately determine the type of cancer and select fitting treatment that increases the likelihood of the patient’s recovery. In this paper, approaches to automate different steps of this assessment are explored with the immediate goal of implementing an end-to-end algorithm pipeline capable of performing this task with minimal human input, with the potential goal of incorporating this pipeline into an existing larger slide processing software.
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    Using and evaluating the real-time spatial perception system hydra in real-world scenarios
    (Tartu Ülikool, 2023) Hult, Carl Hjalmar Love; Kruusamäe, Karl, juhendaja
    Hydra is a real-time machine perception system released open source in 2022 as a package for Robot Operating System (ROS). Machine perception systems like Hydra may play a role in the engineering of the next generation of spatial AIs for autonomous robots. Hydra is in the preliminary stages of its existence and does not come with intrinsic support for running on custom datasets. This thesis primarily aims to find out whether the promised capabilities of Hydra can be replicated. As well as to establish a workflow and guidelines for what modifications to Hydra are needed to successfully run it.
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    Evaluating SNP detection in genomic data: a study on mutation patterns in the Estonian Biobank Data
    (Tartu Ülikool, 2023) Katsuba, Anton; Pankratov, Vasili, juhendaja
    Single nucleotide variants provide a rich source of information on cellular and genomic processes and phenomena. Rare variants are especially of interest, since they are likely to have experienced the least influence from evolutionary forces, which is important in esti- mating mutation rates. However, high quality SNV data is necessary for this. The goal of this thesis is to examine the quality of newly discovered SNVs in the Estonian Biobank dataset by looking at mutation patterns. The findings show that the subset of newly discov- ered SNVs has significant quality issues compared to the subset of already known SNVs. These issues are likely attributable to mapping errors.
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    Gamification of a medical learning application VRAna
    (Tartu Ülikool, 2023) Pisetskaya, Darya; Haamer, Rain Eric, juhendaja
    As the quality of anatomy education is declining worldwide, faculties need to come up with new ways to engage and motivate students. Gamification is a growing trend in various fields including education, and can be an efficient instrument to help students study theoretical information. This work focuses on the gamification of a medical learning application VRAna by introducing several quiz scenarios for memorizing anatomical structures on the bones. The created games can be used as a supplementary tool for learning skeletal anatomy.
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    Genome-wide association study for detecting autoimmune-disease-associated genetic pattern differences in specific HLA type carriers
    (Tartu Ülikool, 2023) Kukkonen, Arne; Abner, Erik, juhendaja
    The HLA locus variants are one of the strongest genetic predictors for most, if not all, human autoimmune diseases. The HLA locus genes include the antigen-presenting cell surface peptide encoding genes, which form an essential component in the maturation of the T-cell population in the thymus, and their subsequent activation in the periphery. Leveraging the modern population-wide genotype information that capture even the most polymorphic loci, this work sets the aim to design a case-control genome-wide association study (GWAS), that would result in the detection of non-HLA genetic variants that have a statistically different effect on an autoimmune disease in the carriers of certain HLA types, in comparison to the non-carriers. For the purpose of this aim, study groups are assembled based on specific HLA allele doses, so that for 42 HLA allele typesselected for this study there are 42 HLA-specific groups where every individual is a carrier of at least one copy of the HLA allele type. The effect sizes from the summary statistics of the HLA-specific GWASs are compared to a general population GWAS (which is done on all the participants of the Estonian Biobank in this case). The variants are considered relevant to this aim if their effect size is statisticallt different in the HLA-specific groups than they are in the general population GWAS.
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    Functional studies on the formation of Chikungunya virus replicase through the trans-complementation of defected replicase components
    (Tartu Ülikool, 2023) Claesson, Hanna Carolina; Lello, Laura Sandra
    Chikungunya virus has a positive-strand RNA genome and belongs to the Alphavirus genus. The viral replication complex includes four non-structural proteins (nsP1-4) which are needed for the virus replication. Several mutations in the non-structural region have been described which impair the formation and functioning of the replication complexes. The alphavirus replicase possesses trans activity which has enabled for the construction of trans-replication systems. These systems provide efficient tools for studying the replication of alphaviruses. The aim of this study was to explore the possibility of trans-complementing the impaired functions of CHIKV nsP1, nsP2 and nsP3 proteins by supplying a functional counterpart in trans. This thesis demonstrated that the inactivity of the CHIKV replicases can be, in some cases, trans-complemented.
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    Characterization of the high-risk human papillomavirus 18 genome replication and transcription in different cellular models
    (Tartu Ülikool, 2023) Babok, Sofiya; Piirsoo, Marko, juhendaja; Piirsoo, Alla, juhendaja
    Human papillomavirus (HPV) is responsible for approximately 30% of infection-caused cancers worldwide. The development of antiviral drugs requires a thorough understanding of the viral life cycle and its interactions with host cells. Replication of HPV genomes is restricted to a very limited number of human cell lines, and the reasons for this limitation are unknown. Human primary keratinocytes and osteosarcoma U2OS cells are widely used cellular model systems in HPV-related research. Conversely, it has been established that viral proteins, namely helicase E1 and transcription factor E2, can trigger the replication from the origin of replication if expressed from separate plasmids in almost all other cell types studied so far. This suggests that a deficiency in at least one of these viral factors may be a reason for the lack of replication of the viral genome in most cell lines. To test this hypothesis, the transcription, translation, and replication of the oncogenic HPV type 18 genome were studied in two different types of cells: embryonic kidney cells 293FT, which are not permissive for HPV replication, and well-described HPV host U2OS cells. The results obtained in the present thesis show that expression levels of all early viral genes, including E1 and E2, are very low in 293FT cells, and the absence of the E1 and E2 proteins causes the lack of viral replication. Additionally, it has been shown that in epithelial cells, such as keratinocytes HaCaT and squamous cervical cancer C33A cells, replication of HPV18 may be restored in the absence of the E8^E2 viral transcriptional repressor, which is expressed from an alternative viral promoter and counteracts E2 activities. However, this effect was not observed in 293FT cells and only exogenously added E1 protein was able to trigger replication of the HPV18 genome in these cells. These results suggest that different cell type-specific mechanisms may be employed to restrict replication of the HPV genome.
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    Cobalt, iron, and nitrogen co-doped graphene coated ceramic nanowire-based bifunctional oxygen electrocatalyst for Zn-air battery
    (Tartu Ülikool, 2023) Kapitulskis, Pavels; Tammeveski, Kaido, juhendaja; Mooste, Marek, juhendaja
    Structural and elemental composition of several graphene-coated Al2O3 nanowire-based cat- alyst materials prepared by functionalization with dicyandiamide and Fe and Co salts via pyrolysis were studied using several physical characterisation methods. Electrocatalytic ox- ygen reduction reaction (ORR) activity of the materials was studied using the rotating disk electrode method in a 0.1 M KOH solution. Using the same method, the oxygen evolution reaction activity was studied for the catalyst material with the highest ORR activity. Addi- tionally, the catalyst’s rechargeable Zn-air battery performance was assessed, showing su- perior power output and cycle life than those of a commercial Pt-Ru/C catalyst.
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    Effect of Escherichia coli growth activity on its susceptibility to Azithromycin
    (Tartu Ülikool, 2023) Nguyen, Ngoc Ha; Putrinš, Marta, juhendaja
    Due to the gradual decline in antibiotics discovery and the escalating emergence of antibiotic resistance, treating bacterial infections have become more challenging. This study examines the effect of bacterial physiology on the efficacy of Azithromycin, using fluorescent proteins as tools to track antibiotic inhibition and bacterial growth stage. We found that there is an inverse correlation between bacterial rate of growth and the efficacy of azithromycin to inhibit protein synthesis.