Biotehnoloogia magistritööd - Master's theses. Kuni 2025

Selle kollektsiooni püsiv URIhttps://hdl.handle.net/10062/72745

Sirvi

Nüüd näidatakse 1 - 20 69

Characterization of Antibacterial Drugs Against Non-Growing Bacteria
(Tartu Ülikool, 2025) Berzinš, Normunds; Kaldalu, Niilo, juhendaja; Vind, Kristiina, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Non-growing bacterial cells, such as those found in chronic infections, are highly tolerant to antibiotics and contribute to treatment failure and relapse. A high-throughput screen of over 6,000 drugs and drug candidates from repurposing libraries identified 39 with activity against non-growing uropathogenic Escherichia coli. These hit compounds were subsequently tested against non-growing Pseudomonas aeruginosa and Staphylococcus aureus, and the 24 most active candidates were selected for further evaluation. Dose-dependent effects on regrowth delay and bacterial killing were assessed across all three species. Statistical analysis confirmed that the observed regrowth delays were significant and reproducible, with p-values below 0.0001 for the most effective treatments. The impact of treatment duration, testing conditions, and efflux pump activity on drug efficacy was also investigated. Most compounds exhibited rapid activity, with significant effects detected within the first hour of exposure. The composition of the growth medium—particularly phosphate buffering—strongly influenced drug activity, whereas bacterial strain and regrowth conditions had minimal effect. Inhibition of efflux pumps during treatment or regrowth had no clear effect. Finally, delafloxacin—a fluoroquinolone not included in the original libraries—was tested due to its structural similarity to sitafloxacin, one of the most potent hit compounds. Despite its enhanced halogenation, delafloxacin showed inferior activity against non-growing bacteria compared to sitafloxacin.
Peptide-Lipid Nanoparticle Platform for Cas9-Mediated Gene Editing
(Tartu Ülikool, 2025) Talgre, Iris Robyn; Sork, Helena, juhendaja; Lehto, Tõnis, juhendaja; Lehto, Taavi, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
This thesis explores cell penetrating peptides as delivery vehicles for gene therapy. The primary objective of this work is to characterize the impact of PepFect14 saturated fatty acid modifications on the peptide’s ability to deliver CRISPR/Cas9 ribonucleoprotein into cells and mediate gene editing. Through physicochemical characterization, membrane activity, cell viability and gene editing efficiency studies this thesis identifies the length of saturated fatty acid modification of PepFect14 as an important parameter in its efficiency to facilitate gene editing. It was found that longer saturated fatty acid tail is beneficial for stable nanoparticle formation, cargo encapsulation and gene editing efficiency.
Interactions between Saccharomyces cerevisiae YEATS Domain-Containing Yaf9 and Swc4 Proteins
(Tartu Ülikool, 2025) Mehanikova, Krista; Jürgens, Henel, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
The YEATS domain is a conserved histone mark reader implicated in the regulation of chromatin structure and transcription. In Saccharomyces cerevisiae, the YEATS domain-containing protein Yaf9 is a subunit of SWR1 and NuA4 chromatin remodelling complexes. In both, it directly interacts with a shared subunit Swc4. This study investigated the Yaf9-Swc4 interaction and possible Yaf9 role outside of its complex-associated functions. A series of Yaf9 deletion mutants truncated at different regions of the protein were constructed using overlap extension PCR and were introduced into yeast. Protein interactions were examined using co-immunoprecipitation, and mutant strains were phenotyped via stress assays involving formamide exposure. The results revealed that deletions in the C-terminal region of Yaf9, as well as within the 160-168 amino acid region – located in the YEATS domain, but outside of the domain’s primary reader region – disrupted interaction with Swc4 and phenocopied a full YAF9 deletion under stress. Additionally, results of this work show that Yaf9 likely does not act independently of both complexes. These findings highlight regions of Yaf9 required for Swc4 association and enhance understanding about the protein’s function. Moreover, these data provide a basis for protein studies in higher eukaryotes.
Validating Three Anti-Phage Defense Systems in Pseudomonas putida
(Tartu Ülikool, 2025) Polekauskaite, Ilze; Ainelo, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Bacteriophages (phages) are viruses that infect bacteria, causing significant mortality. Due to this selective pressure, bacteria contain anti-phage defense systems, proteins which help to abrogate or reduce the effects of infection. As biotechnology employs genetic editing techniques to optimize microbial strains, understanding their genome is necessary to guide these decisions. Aside from efficient performance or production, anti-phage defense systems are an important consideration for minimizing risks to bacterial survival. The soil bacterium Pseudomonas putida prominently figures in synthetic biology research due to its favourable characteristics. Despite this, the anti-phage defense systems of P. putida, and the biotechnological PaW85 strain, are not studied extensively due to the lack of isolated phages. In this study, we use the CEPEST P. putida phage collection to verify three predicted anti-phage defense systems, PD-T7-1, HerA/DUF4297 and RMII. Mutant strains lacking the respective systems were constructed and phage susceptibility assays were conducted to test the function of each system in anti-phage defense. Ultimately, the roles of PD-T7-1 and RMII could not be verified with the phages available, while HerA/DUF4297 was seen to have defensive function. Further, using a strain with an active site mutation in DUF4297 showed that the enzyme likely functions as a nuclease in the complex. In the future, the expansion of the CEPEST collection will enable to further test the potential anti-phage defense roles of PD-T7-1 and RMII. Additionally, the validation of defensive function initiates future research in characterizing the activation mechanisms of HerA/DUF4297 in P. putida. Together, the findings expand the current body of knowledge of the anti-phage defense systems in P. putida and may improve future biotechnology applications.
Development of a Novel Claudin 6-targeting CAR-T Cell Therapy
(Tartu Ülikool, 2025) Shahpazir, Ana; Mahlakõiv, Tanel, juhendaja; Teesalu, Tambet, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Chimeric antigen receptor (CAR)-T cell therapies have demonstrated remarkable success in B-cell malignancies but have yet to show comparable outcomes in solid tumors. A major obstacle is the scarcity of tumor-specific antigens that enable precise and effective targeting. Claudin 6 (CLDN6), an oncofetal tight junction protein, serves as a promising target due to its aberrant upregulation in several solid tumors and absence in healthy adult tissues. In this study, we identified two CLDN6-specific antibodies and leveraged them to generate two second-generation CAR-T cells. Both constructs selectively eliminated CLDN6-positive tumor cells in vitro, while exhibiting distinct profiles in terms of efficacy and specificity. These findings present a promising avenue for developing next-generation immunotherapies for CLDN6-expressing solid tumors and provide a foundation for further development of CAR-T therapy in the treatment of solid tumors.
Genetic and Environmental Interactions in Human Obesity
(Tartu Ülikool, 2025) Strelchenko, Stepan; Eriksson, Jon Anders, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Obesity is a rising global problem, clinically defined as having a body mass index (BMI) > 30 or a waist-to-hip ratio >0.9 for males and >0.85 for females. It is a complex disease with both genetic and environmental factors affecting its development and progression. However, although BMI is highly heritable (60-90% according to twin studies), only ~6% of the variance is explained by additive genetic variation discovered in large genome-wide association studies. Possible sources of unexplained BMI variance are gene-gene (GxG) and gene-environment (GxE) interactions, but to date, only a few studies have addressed this question, and mostly focused on the UK biobank and data from smaller longitudinal studies This study used data from the Estonian biobank to investigate the potential of using the Brown-Forsythe (BF) test to detect heteroscedasticity in BMI based on the genotype and phenotype groups. Moreover, in this work, the BF test was used to select genotypes and environmental variables that were significantly associated with BMI and WHR variance. The study demonstrated sex and age-dependent differences in heteroscedasticity, with higher levels of heteroscedasticity in females compared to males and decreasing heteroscedasticity with increasing age for both sexes. Next, the gene-environment interactions for the outcome variables BMI and WHR were modeled and studied for two genes (FTO and MC4R) and environmental factors, such as physical activity, smoking, and education level. The study found evidence for significant interaction of FTO with smoking for the age group of 31-50 years for males, along with significant interaction of FTO with physical activity for Females (18-30 age group) and males (31-50 age group) in the case of BMI as the outcome variable. Moreover, the evidence for significant interaction of FTO and physical activity was found for females (age group 18-30) for the WHR as the outcome variable. Furthermore, the significant interaction was observed in the case of MC4R and physical activity for BMI in males (18-30 year old group). Additionally, it was shown that the significant interaction between MC4R and education level for males (31-50 age group) was present for BMI. Finally, the study outlined the ground for future research work in the area of gene-environment interactions and statistical modeling, indicating potential environmental factors that can be studied for possible gene-environment interactions.
The Role of the Viral E1 and E2 Proteins in the Stable Replication of Human Papillomavirus Type 5 Genome
(Tartu Ülikool, 2025) Babok, Sofiya; Piirsoo, Alla, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Beta human papillomavirus (HPV) infections are associated with cutaneous squamous cell carcinomas in immunocompromised individuals. There is currently no treatment or vaccines targeting beta HPVs. The oncoprogression is dependent on persistent infections, and despite their clinical relevance, the mechanisms of the persistent beta-HPV replication remain poorly understood. In this study, we aimed to investigate the role of viral replication proteins E1 and E2 in the establishment and long-term maintenance of the episomal HPV5 genome that represents the best-studied beta HPV type. In a transient system, E1 and E2 RNA interference led to a time-dependent decrease in viral genome replication. Cell cycle and viability were unaffected by the E1 and E2 silencing. These results confirmed that E1 and E2 are essential for establishing viral genome replication. Further, we created and characterized a stable cell line bearing the HPV5-E1HA-Nluc-E2Flag genome (H5-Nluc+ cells), as well as compared it to a previously described cell line bearing the HPV5 WT genome (H5+ cells). Southern blot analysis confirmed the episomal nature of viral genomes in both cell lines. H5-Nluc+ cells carried approximately 16-fold more viral genome copies than H5+ cells. During investigation of the physical state of the viral genomes, we observed a previously described dominant oligomeric replicon in H5+ cells that replicates in an E1 and E2-independent manner, while no such replicon was present in the H5-Nluc+ cells. These findings suggest that there are two modes of HPV5 replication, one dependent on the E1 and E2 expression and another supported by host cell machinery, with the latter developing after long-term maintenance of the HPV5 genome. However, whether the E1/E2-independent mode of replication occurs during natural HPV infection remains to be elucidated.
Exploring the Role of the HT1-MPK12 Module in Guard Cell CO2 Sensing
(Tartu Ülikool, 2025) Yadlos, Oleksii; Wang, Yuh-Shuh, juhendaja; Kollist, Hannes, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
To optimise the balance between CO₂ uptake for photosynthesis and loss of water by transpiration, plant stomatal pores regulate their aperture in response to changes in CO₂ concentration. The HT1–MPK12/4 complexes have been implicated as key CO₂ sensors in stomatal guard cells, but the molecular basis of MPK12 and MPK4 function in CO₂ signalling, as well as the exact CO₂-sensing mechanism, remain unclear. MPK12 is specific to the guard cells, whereas MPK4 plays diverse roles in many cell types. Interestingly, MPK11, as a close homolog of MPK4 and MPK12, is not involved in stomatal regulation by CO₂. This thesis aimed to determine which regions of MPK12 are required for its CO₂-specific function and whether the HT1–MPK12 module can directly sense CO₂ via lysine carbamylation. We constructed MPK12/MPK11 chimeras and MPK12 truncation mutants and tested their ability to bind HT1 and restore normal CO₂-induced stomatal responses in plant lines in which native MPK12 was deleted. We also identified potential CO₂-binding lysines on MPK12 and HT1 and evaluated their functional importance in planta. Chimeric and truncation analyses revealed key features of MPK12 domains: the C-terminal lobe is essential for interaction with HT1, while the N-lobe is required for a wild-type CO₂ response. Several amino acids in the N-lobes of MPK12 and MPK4 were proposed to contribute to CO₂ signalling and HT1 inhibition, warranting further investigation. Preventing potential carbamylation on selected lysine residues in MPK12 and HT1 did not abolish stomatal CO₂ responses. These findings suggest that non covalent HCO₃⁻ binding, rather than lysine carbamylation, serves as the primary CO₂-sensing mechanism within the HT1–MPK12/4 module.
A Comparative Study of Mayaro Virus TRVL and BeAr Strains
(Tartu Ülikool, 2025) Rutmane, Anna; Varjak, Margus, juhendaja; Merits, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Mayaro virus (MAYV) is an arthritogenic alphavirus and the causative agent of Mayaro fever, which is characterized by myalgia, arthralgia, high fever, and maculopapular rash. Currently, the distribution of MAYV is limited to Central and South America, where it causes sporadic outbreaks. However, in recent years, there have been concerns about its potential emergence into urban transmission cycles. Phylogenetically, MAYV is divided into three distinct genotypes: D, L, and N. In this study, we investigate two MAYV strains: TRVL (genotype D) and BeAr (genotype L). To compare the replication and transcription efficiencies between the two strains, we implement a trans-replication system, in which the expression of alphavirus replicase and RNA replication are uncoupled. Our study indicates that the replicase of the TRVL strain is more active in human cells, whereas the replicase of the BeAr strain is more active in mosquito cells. The differences in replicase activity between the strains are driven by determinants located both within the replicase itself and within conserved sequence elements. Lastly, our experiments demonstrate that TRVL and BeAr strains are capable of co-infection.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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