Biotehnoloogia magistritööd - Master's theses
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Item Identification of inhibitors of the Human Papillomavirus type 5 replication using high-throughput screening and machine learning(Tartu Ülikool, 2023) Ibragimov, Ruslan; Piirsoo, Marko, juhendaja; Piirsoo, Alla, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutHuman papillomaviruses (HPVs) have been known to cause a wide variety of health complications from warts to cancer. Although vaccination against several high-risk types of HPVs is available, there is currently no treatment method that would target already established infections. The focus of this study is to perform high-throughput screening of 1584 randomly selected chemicals in order to identify potential inhibitors of the HPV type 5 replication, and then use machine learning to predict interactions between those compounds and proteins expressed in basal keratinocytes, the only cell type that supports HPV replication. At the end of this study, several potential inhibitors were discovered and connections were made to proteins and pathways absolutely necessary for the replication of the viral genome or occurrence of the cancer.Item Investigation of Excitatory Ion Channels In Par- kinsonian Sensory Neurons(Tartu Ülikool, 2023) Eltalb, Imane Muhammad Higazy; Hickey, Miriam Ann, juhendaja; Jakobson, Maili, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutBackground: Parkinson’s disease (PD) is associated with tremor, slowness of movement and stiffness, the latter two of which are caused by the loss of dopaminergic neurons of the substantia nigra pars compacta. Interestingly, although olfactory impairment is now well recognised in PD, skin symptoms differentiate individuals who go on to develop PD even better than olfactory impairment. However, little is known of the cause of this skin pathology, which includes proteinaceous aggregates and loss of peripheral neurites. Recently, several excitatory ion channels were found to be upregulated in PD patient skin. Here, we have examined the effect of overexpression of these channels in a sensory neuronal cell model of PD. Methods: The 50B11 cell line, which is an immortalised DRG rat sensory neuronal cell line (kind gift from Dr Höke, Johns Hopkins University), was used. Familial PD was modelled by transfection with eGFP-alpha synuclein. Sporadic PD was modelled by treating with rotenone, a well-validated risk factor for PD and inhibitor of complex I. In our first series of experiments, cells overexpressing eGFP-alpha-synuclein or GFP (control plasmid) were treated with rotenone or DMSO and were then fixed for imaging and cytotoxicity analysis. In our second series, cells were co-transfected with alpha synuclein and HCN1 and then treated with rotenone or vehicle, then fixed for imaging and analysis of cytotoxicity. In ourfinal series,cells were transfected with HCN1 or control plasmid, treated with rotenone or vehicle and then imaged live for mitochondrial membrane potential (MMP) and cell morphology. Results: In our first series of experiments,the density of alpha synuclein-overexpressing cells was increased following rotenone exposure. However, in our second series, co-transfection of HCN1 with alpha synuclein resulted in lowered density of cells treated with rotenone. In our final series of experiments, rotenonecaused a small reduction in MMP. HCN1 overexpressionalone reducedMMP to a much greater degree. HCN1 overexpression exacerbated the effect of rotenone on MMP. This combination of HCN1 overexpression with rotenone changed the morphology of sensory neurons to a smaller, more rounded shape. Conclusions: HCN1 is an important regulator of activity in sensory neurons and its overactivity is involved in several types of neuropathic pain. PD patients show loss of peripheral neurons and recently, HCN1was found to be overexpressed in skin samples of PD patients. Although the normal function of alpha synuclein is unclear, it may induce proliferation and also may increase expression of anti-oxidant genes, and here, we found that alpha synuclein protected against rotenone toxicity. However, co-expression of HCN1 prevented this effect and resulted in increased cell death. In separate experiments, the combination of overexpression of HCN1 and rotenone resulted in exacerbated loss of mitochondrial membrane potential, and a change in cell shape indicative of toxicity. Thus, HCN1 overexpression may precipitate toxicity in parkinsonian sensory neurons and contribute to peripheral neuropathy in PD.Item 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. TehnoloogiainstituutAlphaviruses 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.Item Physiological heterogeneity of uropathogenic Escherichia coli in bladder epithelial cell infection model(Tartu Ülikool, 2023) Hasanov, Turgay; Putrinš, Marta, juhendaja; Kerkez, Ivana, juhendaja; Tenson, Tanel, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutUropathogenic Escherichia coli (UPEC), which invades host cells, is the primary agent of urinary tract infections. Antibiotics are commonly used to treat UTIs. During the long treatment period, bacteria are getting resistant to antibiotics. To increase understanding of antibiotic treatment efficiency of urinary tract infections an in vitro model of intracellular and extracellular infections was needed. Within these, an infection model with HTB-9 human bladder cells and UPEC CFT073 strain has been established. Specific experiments were designed to compare the different conditions to see what the ideal way would be to get the infection to a level where we can use the model to follow infection and antibiotic treatment. To test the viability of the cell, we have compared the CFU results with flow cytometry-based cell counting. The data suggests that bacteria that specifically attach to bladder epithelial cells are targeted by host antimicrobial peptides.Item 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. TehnoloogiainstituutMunicipal 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.Item 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. TehnoloogiainstituutCell 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.Item 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. TehnoloogiainstituutThe 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.Item Generation of flaviviral replicon expressing stable cell lines(Tartu Ülikool, 2023) Konno, Melis Nur; Zusinaite, Eva, juhendaja; Koit, Sandra, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutFlaviviruses are a group of arboviruses that are considered re-emerging pathogens which can cause severe illnesses in vulnerable populations. Although a significant issue for the healthcare system, there are no effective treatments available, and flavivirus countermeasures rely on preventative actions like the control of vector spread and vaccination. The majority of flaviviruses are biosafety level 3 (BSL3) agents, which necessitate special precautions when working with infectious viruses. This complicates the development of antivirals and vaccines. To address this issue, when investigating the antiviral mechanism of inhibitors at lower biosafety levels, subgenomic replicons can be used to study viral replication events without virus entry or assembly. In this work, we generated stable cell lines based on flavivirus replicon RNAs, including reporter marker genes and the puromycin N-acetyltransferase (PAC) gene. The resulting puromycin-resistant stable cell lines persistently expressed non-structural proteins and fluorescent or luminescent reporter proteins. These stable cell lines can be used for high-throughput screening of potential replication inhibitors for the development of novel antiviral therapies against flaviviruses.Item Regulation of the cell cycle to cytoskeleton remodelling: the diverse functions of N6AMT1(Tartu Ülikool, 2023) Serova, Evgeniia; Mutso, Margit, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutN6AMT1, also known as HEMK2, is an evolutionally conserved protein which belongs to the human TRMT112-methyltransferase network. It is involved in the methylation of various substrates. Previous studies have shown that this significant methyltransferase is engaged in the majority of vital processes like regulating of cell proliferation, cell cycle factors, etc. Therefore, in the present research, it was studied how the depletion of N6AMT1 affected the viability of U2OS cells. Using CRISPR/Cas9 genome editing system we obtained an N6AMT1 knock-out cell line that showed stunted growth, delayed cell cycle progression, and alterations in morphology. Moreover, it was noticed that N6AMT1 reduction had an impact on cytoskeleton remodelling, namely the β-tubulin level. In conclusion, human N6AMT1 was considered as a potential target responsible for cell cycle and cell cytoskeleton remodelling.Item 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. TehnoloogiainstituutThe 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.Item 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. TehnoloogiainstituutPhosphate-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.Item Investigating the genetic basis of obstetrical diagnoses related to miscarriage and placental biology using genome-wide association study meta-analysis(Tartu Ülikool, 2023) Jelisaveta, Džigurski; Laisk, Triin, juhendaja; Pujol Gualdo, Natàlia, juhendaja; Mägi, Reedik, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutThe triad of genetic, environmental and lifestyle factors contribute to adverse pregnancy outcomes. While the recent work investigating maternal genetic susceptibility to miscarriage has identified several genes involved in placental biology, the specific genetic risk factors of pregnancy complications remain poorly understood. To elucidate the maternal genetic basis of seven distinct obstetrical diagnoses related to miscarriage and placental biology, we conduct a case-control genome-wide association study meta-analysis using population-based data from the Estonian Biobank and the FinnGen study. We identify novel genomic risk loci and propose the most likely associated genes for diagnoses “haemorrhage in early pregnancy” and “premature rupture of membranes”. In addition, we estimate SNP- heritability, provide evidence of shared genetic background between the studied traits, and describe which other diagnoses are more common in women diagnosed with pregnancies with abortive outcomes and placenta-associated pregnancy complications.Item 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. TehnoloogiainstituutPlants 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.Item 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. TehnoloogiainstituutMultisite 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.Item The role of ATR in G1 phase DNA damage response(Tartu Ülikool, 2022) Matiyevskaya, Frida; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutItem The Response of Microbial Community to Dispersed Oil Contamination in Arctic Sea-ice(Tartu Ülikool, 2022) Fathima, Farheen; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutItem 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. TehnoloogiainstituutItem Bioelectrochemical system for oil pollution remediation in marine sediments(Tartu Ülikool, 2022) Ugoezue, Samuel Tochukwu; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutItem Familial hypercholesterolemia: enhancing actionability through the recall-by-genotype experience(Tartu Ülikool, 2022) Nurm, Miriam; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. TehnoloogiainstituutItem In vitro and in silico epitope-paratope mapping(Tartu Ülikool, 2022) Jermakovs, Klavs; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut