Biotehnoloogia magistritööd - Master's theses

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A preclinical trial of Bisdemethoxycurcumin in a mouse model of Alzheimer's disease
(Tartu Ülikool, 2022) Mansouri, Seyedeh Elnaz Sadat; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
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.
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.
Analysis of different substrate docking pockets on Clb5- and Clb4-Cdk1
(Tartu Ülikool, 2023) Kiselev, Viacheslav; Faustova, Ilona, juhendaja; Örd, Mihkel, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Cell cycle is coordinated via temporally resolved protein phosphorylation by cyclin-dependent kinases (CDKs). CDKs form cell-cycle-stage-specific complexes with cyclins. Cyclins in turn recruit substrate proteins through specific binding motifs and direct the kinase to phosphorylate different proteins to trigger cell cycle events. While most of the cyclin-substrate interactions take place via a hydrophobic patch on the cyclin, recent studies have found novel pockets that could participate in substrate recognition. This work investigates the im- portance of two novel substrate binding pockets on Saccharomyces cerevisiae S and G2 phase cyclins Clb5 and Clb4. Structural and conservational analysis supported the presence of multiple substrate pockets on the cyclin surface. Kinase assays revealed that the phosphate-binding pocket and an NPFF motif pocket are critical in mediating phosphorylation of Kar9 by Clb4-Cdk1 complex. A comparison between Clb4-, Clb3-, and Clb5-Cdk1 revealed distinct substrate preferences for these complexes within a panel of 10 Cdk1 substrates of S/G2 phase. Further, assays using mutant cyclin-Cdk1 complexes suggested that the NPFF pocket is used by a small number of substrates. Overall, these findings highlight the im- portance of cyclins as substrate recruitment modules rather than just activators of CDKs.
Analysis of the docking interaction between Sld2 and Clb5 in budding yeast
(Tartu Ülikool, 2021) Mikhailova, Valentina; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
The cell cycle of Saccharomyces cerevisiae is governed by cyclin dependent kinases (Cdks), which are activated by periodically synthesized and degraded cyclins. The substrate target-ing by a cyclin-Cdk complex is mediated by the active site on the Cdk and the cyclin docking pocket. Docking interactions between short linear motifs (SLiMs) in target proteins and cy-clin-dependent kinases are shown to be critical regulators of different cell cycle events. SLiMs function in almost every pathway due to their role in regulatory function and signal transduction.
Autotrophic nitrogen removal for low organic wastewater treatment
(Tartu Ülikool, 2023) Anwuta, Kelvin Ugochukwu; Zekker, Ivar, juhendaja; Rikmman, Ergo, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Municipal wastewater can be directly anaerobically treated to recover energy in the form of biogas. By using an autotrophic process, such as anaerobic ammonium oxidation (anammox) process, to remove the ammonium from the anaerobic effluent, one can further reduce the energy needed for wastewater treatment. Up until now, anammox has primarily been utilized to treat waste streams at elevated temperatures (>25°C) for concentrated (>500 mg N/L) flows. The challenges of anammox process are its application of the water line of municipal wastewater treatment include lower nitrogen concentrations (100 mg N/L), high organic carbon (pharmaceutical presence) and lower temperatures (20 °C). Two 20-liter moving bed biofilm reactors were used for this study, and they were run at 22 °C for 918 days after being injected with a significant amount of anamox bacteria coming from anaerobic digester suspended biomass. The specific nitrogen removal rate (NRR) and removal efficiency increased significantly, reaching values of 0.642 g N/L/d and 92% volume, respectively. Additionally, five pharmaceuticals—ciprofloxacin, norfloxacin, ofloxacin, sulfamethaxazole, and sulfadimethoxine were employed in this investigation to assess their impact on anammox activity. The diversity of anammox bacteria was impacted by the presence of the targeted pharmaceutical chemicals, but these microorganisms were able to withstand and effectively eliminate nitrogenous substances. Throughout the course of the experiment, several conditions, including anoxic and aerobic conditions, starvation and non-starvation phases, and other conditions, were observed.
Bioelectrochemical system for oil pollution remediation in marine sediments
(Tartu Ülikool, 2022) Ugoezue, Samuel Tochukwu; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
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.
Cell cycle independent signaling by modified Clb5- Cdk1 complex
(Tartu Ülikool, 2021) Agerova, Alissa; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
A eukaryotic cell contains a robust regulatory network that controls the ordered sequence and timing of distinct cell cycle events. In budding yeast this is mainly regulated by the growing activity of cyclin-dependent kinases (Cdks) during the cell cycle. Post-translational modifications, particularly protein phosphorylation is a powerful mechanism regulating stability, localization, activity and interactions of proteins. Building synthetic circuits based on protein phosphorylation has remained a riddle due to the overlapping specificity of protein kinases. To tackle this issue it was aimed to obtain a cell cycle independent cyclin-Cdk1 input that could target substrates created specifically for this particular cyclin-Cdk1 complex. By manipulating the Cdk1 specificity of phospho-degron and -localization modules it was anticipated to design orthogonal Cdk circuits that respond to the modified Clb5-Cdk1 complex.
Characterization of Excitatory Ion channels in Parkinsonian Model Sensory Neurons
(Tartu Ülikool, 2022) Obidi, Ojochide; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Characterization of ligand binding to Dopamine D3 receptor using fluorescence anisotropy and radioligand binding
(Tartu Ülikool, 2021) Thoondee, Lakshmi; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
There are five subtypes of dopamine receptors that play a role in the dopaminergic system. Due to their limited distribution and involvement in cognitive and emotional functions, Dopamine D3 receptors are attractive pharmacological targets for treatment of drug addiction and neuropsychiatric disorders. D3 receptor ligands have been labelled with a fluorescent dye or a radioisotope for direct monitoring of ligand binding to the receptor. However, there are not many fluorescent ligands that are available for studying D3 receptor. A2-TAMRA is a novel fluorescent ligand with high affinity for D3 receptor. Binding of [3H]-methylspiperone to D3R was studied in parallel to validate results from A2-TAMRA binding to D3R. The D3 receptor ligands had similar affinities in inhibiting A2-TAMRA and [3H]-methyl spiperone binding to D3 receptor, since a very good correlation (R2 = 0.94) was obtained between both methods. The affinities for the known antagonists had a good correlation with previously published data. Selectivity of A2-TAMRA towards two different subtypes was also studied and we found that A2-TAMRA prefers D3 receptor over D1 receptor.
Construction and functional analysis of stable cell lines expressing nsP1 of alphaviruses
(Tartu Ülikool, 2023) Mirzajani Sarvandani, Mandana; Wang, Sainan, juhendaja; Merits, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Alphaviruses are positive-strand RNA viruses that are transmitted by mosquitoes and cause various (often serious) diseases in their vertebrate hosts, including humans. Similar to other positive-strand RNA viruses, alphaviruses encode for RNA replicase. RNA replicase of alphaviruses consists of four virus-encoded subunits, called non-structural proteins 1-4 (nsP1-4). nsP1 is a membrane anchor or the replicase complex. It forms a dodecameric ring that holds other components in their place, its guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities are essential for the capping of viral RNA genomes. In this study, tetracycline-inducible stable cell lines expressing nsP1 or its mutants of different alphaviruses were generated. Trans- complementation assay was performed by supplementing homologous or heterologous wild-type nsP1 or its mutant version to functionally defective replicase of Chikungunya virus (CHIKV) in which the defect was caused by mutations of nsP1. We found that expression of wild-type nsP1 of matching or closely related alphaviruses can significantly compensate for capping defects of CHIKV replicase. The compensation was less effective, or not observed at all, for replicase mutants where the defect was due to its compromised membrane attachments. Adding the defective nsP1 to the CHIKV replicase which harbours the same mutation worsens the activity of replicase; however, activities of mutated replicase were restored when it was supplemented with nsP1 harbouring different functional defects. Furthermore, expression of these findings expands our knowledge about alphavirus RNA replication and trans-complementation of replicase-associated defects. Besides, tetracycline-inducible stable cell lines can be used as tools to trans-complement alphaviruses with lethal defects in nsP1 allowing obtaining conditionally infectious alphaviruses virions which can be used at low biosafety level laboratories for high-throughput neutralization and antiviral testing.
Controlling protein levels through Grr1- dependent synthetic degrons
(Tartu Ülikool, 2023) Sethi, Jhalak; Valk, Ervin, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
The cell cycle control system, governed by cyclin-dependent kinases (Cdks), orchestrates the precise timing and coordination of cell cycle events. Ubiquitination-mediated protein degradation, facilitated by the Skp1-Cullin-F-box protein (SCF) complex and one of its F- box proteins, Grr1, is pivotal in regulating cell cycle progression. This study explores using phosphodegron tagging while incorporating various modules to control protein expression and degradation. The development of phosphodegron through synthetic biology has great potential for practical uses, including improving production yields for valuable compounds.
CRISPR/nCas9 deletion of genes linked to the C1-fixing gene cluster in a gas-fermenting acetogen
(Tartu Ülikool, 2022) Nwaokorie, Ugochi Jennifer; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Deciphering the interaction of clustered phosphorylation sites in a multisite phosphorylation network – example of Ndd1 protein
(Tartu Ülikool, 2023) Leoshko, Valeriia; Valk, Ervin, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Multisite phosphorylation is a vital cellular regulatory process. Cdk1, the master cell cycle regulator, forms a complex with cyclin and Cks1, activating Cdk1 and directing it to specific target proteins. Phosphorylation by Cdk1 occurs at serine or threonine residues followed by proline, with enhanced specificity in the presence of arginine or lysine at the +3 position. Cks1 and the Clb2 phosphate-binding pocket facilitate secondary phosphorylation in phosphorylation clusters that tend to form in intrinsically disordered protein regions, generating signalling specificity. This thesis focuses on Ndd1, a transcriptional co-activator essential for nuclear division. The involvement of Cks1 and the recently discovered Clb2 phosphate-binding pocket is critical for Ndd1 multisite phosphorylation. By investigating a specific phosphorylation cluster within Ndd1, this thesis aims to unravel the complex phosphorylation networks mediated by clustering, Cks1, and the Clb2 phosphate-binding pocket.
Deciphering the two dominant interfacial types of tryptophans in photosynthetic membrane proteins using PFAST
(Tartu Ülikool, 2022) Jafarov, Ali; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Deploying Pseudomonas putida for the conversion of aromatic compounds from fractionated industrial hydrolysis lignin
(Tartu Ülikool, 2023) Morehead, Philip Arthur; Bottoms, Scott, juhendaja; Salmar, Siim, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
The world’s most abundant natural aromatic polymer is paradoxically one of its most un- derutilized feedstocks. Lignin is the pulp and paper industry’s primary by-product, and it shows great potential as a renewable and carbon-neutral source of industrially relevant chemicals. The utilization of Pseudomonas putida, a robust bacterium known to catabolize aromatic compounds natively, was explored in this study as a promising approach for lignin valorization. To this end, dry hydrolysis lignin (HL) was used as the feedstock, and various HL fractionation techniques were applied, including alkaline extraction, to obtain lignin monomers, dimers, and trimers in solution for use as a growth medium. The composition of the fractionated HL media was ascertained using a range of analytical techniques. Changes in composition during flask cultivations helped inform the selection of target genes for deletion to direct compounds in the benzoate degradation pathway towards the production of cis,cis-muconic acid, an intermediate of nylon 6,6, which finds applications in industrial components, textiles, automotive parts, and electronics.
Determination of phosphate accumulating organism accumulation in sewage sludge of wastewater
(Tartu Ülikool, 2023) Furkan, Karadeniz; Kisand, Veljo, juhendaja; Tenno, Taavo, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Phosphate-accumulating organisms (PAOs) are diverse bacteria that retain phosphate as polyphosphate. These bacteria play an essential role in various water treatment plants beca- use they are used effectively to remove a proportion of the phosphorus in wastewater. Was- tewater treatment plants (WWTPs) are vital in today's world, and the significance of phosp- hate in our lives and its relationship with these bioorganisms formed the main idea of this project. These plants use physical, chemical or biological methods to treat wastes in the water, and the abundance of these organisms will differ as each treatment plant has different management and environmental conditions. Therefore, in this study, biological detection of these organisms and a comparison of their abundance in several different wastewater treat- ment plants were carried out. Comparisons using a confocal microscope showed the same results trend when samples that had already been chemically measured were observed under the microscope stained with DAPI (4',6-Diamidino-2-phenylindole). The confocal micros- cope was successfully utilized to detect these organisms in the sludge in the wastewater. The comparison of these treatment plants regarding the abundance of these organisms and flow cytometry data was successfully performed.
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.
Dominant-negative mutations in small G-protein ROP6 impair stomatal closure
(Tartu Ülikool, 2023) Ponamareva, Ekaterina; Yarmolinsky, Dmitry, juhendaja; Kollist, Hannes, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Plants exchange gases with surrounding environment through stomata, specialised pores that open and close in response to various stimuli. Stomatal movements are regulated by a complex network of signalling pathways, some components of which have not been yet identified. A forward genetic screen identified an Arabidopsis line carrying mutation in small G-protein ROP6that was sensitive to ozone, had high stomatal conductance, and displayed impaired stomatal responses. In this work, we showed that the ROP6 mutation was dominant negative and caused impairment of stomatal responses to several environmental factors. By using gas exchange experiments, we also demonstrated that ROP6 acts antagonistically with another G-protein ROP2 in stomatal regulation.

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