Bioengineering - Master's theses

Selle kollektsiooni püsiv URIhttps://hdl.handle.net/10062/112353

Sirvi

Nüüd näidatakse 1 - 10 10

listelement.badge.access-status Embargo ,
Timing of bactericidal activity of fluoroquinolones against nongrowing uropathogenic Escherichia coli
(Tartu Ülikool, 2026) Akulich, Anna; Kaldalu, Niilo, juhendaja
Urinary tract infections (UTIs) are among the most common bacterial infections, especially in women, and are often caused by uropathogenic Escherichia coli (UPEC). These bacteria have the ability to invade bladder cells, making them difficult to eliminate completely. In many cases, UPEC can persist in the body even after antibiotic treatment and later cause recurrent infections. Fluoroquinolones (FQs) are antibiotics that kill bacteria by damaging their DNA. Some fluoroquinolones are of particular interest because standard killing assays have shown that they can eliminate a substantial proportion of non-growing bacteria, unlike many antibiotics whose activity is largely restricted to actively growing cells. However, bacteria in a nongrowing state are often more tolerant to these drugs. Recent studies suggest that DNA damage does not occur during FQ treatment when cells are nongrowing. Instead, it occurs after the bacteria are placed in fresh, drug-free medium and begin to regrow. This suggests that fluoroquinolones may not kill nongrowing bacteria immediately but instead poison them, leading to damage and death later during recovery. This study measured the timing of SOS response activation in the uropathogenic E. coli strain CFT073 using a fluorescent reporter system. The bacteria were treated with four fluoroquinolones—ofloxacin, gatifloxacin, sitafloxacin, and norfloxacin—under growing, nongrowing, and regrowing conditions. A dual-fluorescent plasmid tracked bacterial presence and DNA damage in real time. The results showed that the SOS response was low during the treatment of nongrowing cells but increased significantly after cells resumed growth. These findings support the idea that fluoroquinolones poison nongrowing E. coli without causing immediate DNA damage, which only appears during regrowth. This has important implications for improving treatment strategies.
listelement.badge.access-status Avatud juurdepääs ,
The effect of loss-of-function of adaxially or abaxially enriched genes on stomatal patterning in Arabidopsis thaliana
(Tartu Ülikool, 2026) Dievinš, Karlis; Hõrak, Hanna, juhendaja
Stomatal pores regulate CO₂ uptake for photosynthesis and water loss via transpiration. In amphistomatous species such as Arabidopsis thaliana, stomata occur on both lower and upper leaf surfaces, yet the mechanisms that control their distribution between leaf surfaces and potential surface-specific functions remain unclear. Here, I analysed stomatal patterning and conductance in loss-of-function mutants of genes enriched in the stomatal lineage cells and preferentially expressed in specific leaf surfaces. Most mutants did not differ from wild type plants in stomatal density or stomatal ratio. However, stomatal index was increased in el19y-2, prx34, and el21x-1 on the abaxial surface, and in el19y-2 on the adaxial surface too, indicating effects of respective genes on stomatal lineage progression. In a subsequent experiment the scrm mutant showed reduced stomatal density on both leaf surfaces. Despite this, scrm exhibited increased adaxial stomatal conductance and higher conductance per individual stoma on both leaf surfaces, suggesting more open stomata. These results show that the studied genes have limited effects on stomatal distribution, but some of them can influence stomatal development. This work supports partially independent regulation of stomatal traits between leaf surfaces.
listelement.badge.access-status Avatud juurdepääs ,
Removal of nitrogen and pharmaceutical compounds in a biofilm system
(Tartu Ülikool, 2026) Ekwebe, Blessing Ambang; Zekker, Ivar, juhendaja; Mamun, Faysal-Al, juhendaja
As global wastewater generation reaches an estimated 380 billion cubic meters annually, the primary challenge for sanitation infrastructure has shifted from volumetric management to the mitigation of chemical matrices, specifically pharmaceutical active compounds (PhACs). Conventional treatment systems often fail to sequester theses recalcitrant pollutants, which propagate antimicrobial resistance and threaten aquatics ecosystems. This thesis investigates the synergistic potential of an anaerobic ammonium oxidation (anammox) consortium cultivated within a moving bed biofilm reactor (MBBR) for the simultaneous removal of nitrogenous compounds and five target PhACs – marbofloxacin, ofloxacin, enrofloxacin, sulfamethoxazole and carbamazepine. The experimental results demonstrated that the MBBR system successfully decoupled biomass retention from hydraulic retention time (HRT), allowing for the enrichment of slow-growing anammox bacteria on K1-shaped biofilm carriers. The system achieved nitrogen removals, maintaining stability despite fluctuations in total nitrogen loading. Batch kinetic studies revealed that while nitrogen removal followed a first order model (R2 up to 0.8299), PhAC attenuation was highly compound specific. The findings from this project suggest that, while anammox-mediated systems are highly efficient for energy-neutral nitrogen removal, the attenuation of PhACs relies on maintenance of diverse and stratified microbial community. This research provides a fundamental framework for designing next-generation wastewater infrastructure capable of addressing both nutrient and emerging micropollutants in a single-stage process.
listelement.badge.access-status Embargo ,
Evaluation of PF14-derived cell-penetrating peptides for functional delivery of plasmid DNA
(Tartu Ülikool, 2026) Geekiyanage, Isuri Anuththara Perera; Pooga, Margus, juhendaja; Arya, Geeta, juhendaja
Plasmid DNA (pDNA) represents a promising platform for gene therapy due to its substantial coding capacity and capability to support sustained gene expression. However, its intracellular delivery remains a significant challenge attributed to its large molecular size, negative charge, and multiple biological barriers. Cell-penetrating peptides (CPPs) serve as promising non-viral delivery vectors, adept at forming nanoscale complexes that facilitate the intracellular transport of nucleic acids. Among the class of amphipathic CPPs, PepFect14 (PF14) has exhibited remarkable efficacy in nucleic acid delivery, characterized by efficient nanoparticle (NP) formation and cellular uptake. Based on the success of PF14, a series of structural modifications has been systematically investigated to optimize NP characteristics and improve the delivery efficiency. The study evaluates a series of PF14-derived RNA delivery peptides featuring distinct amino acid substitutions and hydrophobic modifications for their efficacy in pDNA delivery. The CPP–pDNA NPs were characterized for complexation efficiency, physicochemical properties, stability, and post-transfection functional delivery and cell viability. The functional delivery of a luciferase-encoding plasmid was assessed in CHO-K1 cells, as well as heparan sulfate-deficient CHO 2242 cells, to evaluate how peptide modifications impact delivery performance and to elucidate the role of cell-surface heparan sulfate in CPP-mediated pDNA delivery. Most peptides efficiently condensed pDNA and formed nanoscale NPs with generally favorable physicochemical characteristics. Notably, peptides with histidine substitutions or specific hydrophobic modifications demonstrated enhanced functional delivery, while the performance of other hydrophobic variants displayed variability, underscoring the necessity for a careful balance of hydrophobicity to optimize NP formation, stability, and intracellular release. Reduced transfection efficiency in CHO 2242 cells further emphasized the critical role of cell-surface heparan sulfate in the functional delivery of pDNA. Overall, the findings of this study elucidate the significant influence of structural modifications on the performance of PF14-derived CPPs and contribute to the rational design of advanced CPP-based non-viral gene delivery systems.
listelement.badge.access-status Embargo ,
Characterization of stress responses in pseudomonas putida using plasmid-based fluorescent reporter systems
(Tartu Ülikool, 2026) Grigorjeva, Julija; Jõesaar, Merike, juhendaja; Viggor, Signe, juhendaja
The rising interest in implementing microbial biosynthesis strategies for production of high-value compounds, as an alternative to traditional chemical manufacturing, has facilitated the development of microbial bioengineering. However, numerous valuable target substrates and products, such as C1 compounds and terpenoid-related molecules, exhibit considerable toxicity to microbial hosts, which limits their industrial application. Consequently, for the construction of more reliable strains the stress response pathways are aimed to be thoroughly studied. Nonetheless, well-characterized stress response reporter systems remain scarce for the recently emerged Pseudomonas putida as a model organism. This study aimed to evaluate the performance of engineered stress-response reporter constructs in P. putida and to investigate the stress responses induced by different valuable compounds. A series of fluorescence-based experiments was conducted in order to assess reporter systems’ performances in terms of sensitivity, response kinetics and strain stability during the chemical exposure to methanol, formate, isoprenol and lycopene. The results suggested that P. putida can tolerate relatively high concentrations of methanol, which mainly caused membrane and growth stress, formate that triggered mild stress from the DNA damage, and membrane and growth stress after the prolonged exposure, and lycopene, inducing the stress from the DNA integrity disruption. In contrast, even low concentrations of isoprenol induced moderate genotoxic effects and severe membrane and growth stresses.
listelement.badge.access-status Avatud juurdepääs ,
Establishing phosphorylated human protein kinase N3 generation via PDPK1 Co-expression in Sf9 cells
(Tartu Ülikool, 2026) Gushchyna, Yelyzaveta; Veikšina, Santa, juhendaja; Kopantšuk, Sergei, juhendaja; Viht, Kaido, juhendaja
Protein Kinase N3 (PKN3) is an AGC family serine/threonine kinase implicated in cancer due to its elevated expression in tumour cells. Its activation requires phosphorylation (Thr718) and regulation by upstream factors, notably by phosphoinositide-dependent kinase 1 (PDPK1), which increases the amount of active PKN3. This work utilises the MultiBac baculovirus system to co-express PKN3 and PDPK1 in Sf9 cells, enabling production of phosphorylated PKN3. The protein is purified via Strep-tag affinity chromatography using Strep-Tactin. Furthermore, immobilisation of PKN3 on magnetic beads is explored to develop fluorescence-based assays, providing a platform for future high-throughput screening of PKN3 inhibitors.
listelement.badge.access-status Embargo ,
Modelling Parkinson’s Disease pathology using MITF mutant mice
(Tartu Ülikool, 2026) Mammadzada, Nargiz; Hickey, Miriam Ann, juhendaja; Faisal, Mahvish, juhendaja
Background: In Parkinson’s disease (PD), dysfunctions in the autophagy-lysosomal system are well known and indeed, several genes related to familial PD also regulate lysosomal function. The MiT/TFE transcription factor family, which includes MITF, TFEB, TFE3, and TFEC, has been shown to play a significant downstream role in this regulation of lysosomal biogenesis and the autophagy-lysosomal system. However, only TFEB has been studied in the context of PD. This study investigates whether neuropathology in MITF mutant mouse models Parkinson’s pathology. Methods: Cryosections of post-fixed brain from male wildtype and MITF mutant mice aged 3 months and 12 months were examined (Mitf mi-enu22(398)/Mitf mi-enu22(398); Mitf Mi wh/+; N=3-8 per group). Fluorescence intensity of tyrosine hydroxylase (TH) in striatum, and density of dopaminergic (TH-positive) neurons in substantia nigra (SN) was analyzed, based upon immunostaining. Immunostaining for glial fibrillary acidic protein (GFAP, a marker of astrocytosis) and for ionized calcium-binding adaptor molecule 1 (IBA1, a marker of microglia) was also completed in several brain regions, including striatum and substantia nigra. Morphology of astrocytes and microglia in these regions was also completed using standardised analyses with software, including ImageJ. Results: Although there was no change in density of TH-positive neurons in substantia nigra, TH content was significantly reduced in both Enu and Mi-wh mutant mice in striatum at both 3 and 12 months of age. GFAP expression was increased in SN, nucleus accumbens and hippocampus by 12 months, in particular in Mi-wh mice, with strong trends observed in Enu mice. Size of individual astrocytes was also increased in Mi-wh mice, suggesting a change in morphology to a more activated state. The size of individual microglia was also increased in SN in Mi-wh mice. Conclusions: MITF mutant mice show neuropathology reminiscent of early PD, including a significant loss in striatal TH, with evidence of astrocytosis, and microgliosis in SN. However, we did not observe a loss in dopaminergic neurons of the SN. We note that many mouse models that carry mutations that lead to familial PD in humans similarly show striatal pathology with no evidence of TH loss in SN. Findings of this study indicate that mutant, dysfunctional MITF may be associated with early dopaminergic impairment and pro- Page 3 of 51inflammatory alterations, supporting further investigation of the role of the MITF transcription factor in PD.
listelement.badge.access-status Avatud juurdepääs ,
A comparative life cycle assessment of hydrogel and polydimethylsiloxane (PDMS) actuators
(Tartu Ülikool, 2026) Morozov, Vjacheslav; Torop, Janno, juhendaja; Tkaczyk, Alan Henry, juhendaja; Özcan Kilcan, Cansu, juhendaja
This thesis compares the environmental performance of a cellulose-based hydrogel actuator and a polydimethylsiloxane (PDMS) actuator. A cradle-to-gate life cycle assessment (LCA) was conducted for the production of a single 10 g actuator. Both systems were modeled in OpenLCA 2.4.1 using the Ecoinvent database version 3.11 and assessed with the Environmental Footprint (EF) v3.1 midpoint LCIA method. The results showed greater impacts from the PDMS actuator across most impact categories; for example, PDMS had about 166% higher climate change impact and about 118% higher non-renewable energy demand than the hydrogel actuator, while hydrogel generally showed a lower environmental profile.
listelement.badge.access-status Avatud juurdepääs ,
Molecular crosstalk at the endometrium-embryo interface
(Tartu Ülikool, 2026) Rodriguez Morales, Brayan Stiveen
Embryo implantation relies on tightly regulated molecular interactions between the endometrium and the developing embryo, however the relative contributions of each remain poorly understood. The main objective of this Master Thesis is to understand the molecular and genomic crosstalk between endometrium and embryo during implantation using a human in vitro model consisting of endometrial organoids derived from healthy fertile women and blastoids (embryo-like structures). To unravel the maternal and embryonic contributions, bulk RNA sequencing was integrated with genotyping, and a workflow was developed to assign the origin of expressed variants. Differential expression analysis identified 264 upregulated and 172 downregulated genes associated with blastoid attachment under hormonally induced conditions. Integration of variant profiles enabled classification of the assignment of gene expression to maternal, embryonic, or shared origin, revealing key pathways related to immune response, cell adhesion, and extracellular matrix remodeling. Consistently origin-assigned genes across donor-derived models were identified from implantation-related interactions. These findings demonstrate that integration of genomic data enhances the resolution of embryo-endometrium crosstalk and provides novel insights into mechanisms underlying implantation success and failure.
listelement.badge.access-status Embargo ,
Modified cell-penetrating peptides for delivery of splice-correction oligonucleotides and small interfering RNA
(Tartu Ülikool, 2026) Wartabidian, Mariza; Pooga, Margus, juhendaja; Porosk, Ly, juhendaja
The therapeutic potential of nucleic acids (NA) is often constrained by their poor intracellular uptake and subsequent loss of activity. Cell-penetrating peptides (CPPs) offer a promising strategy for enhancing the delivery and resulting effect of splice-correcting oligonucleotides (SCO) and small interfering RNA (siRNA) into cells. This study aims to evaluate the delivery efficiency of novel CPPs designed for short nucleic acid delivery. The characterization of the nanoparticles formed between these CPPs, and their nucleic acid cargo was performed using dynamic light scattering analysis. Moreover, the functional delivery was evaluated using reporter cell lines and CPP/siRNA and CPP/SCO nanoparticles. The results demonstrated that several peptide-NA formulations mediated efficient intracellular delivery of siRNA and SCO, leading to significant levels of reporter rescue through splice correction in HeLa pLuc705 cells and efficient gene silencing in U87 MG-Luc2 cells. Notably, one of the CPPs, RDP18, formed nanoparticles with the hydrodynamic diameter of ~70 nm and induced ~100-fold reporter rescue in the cells, when nanoparticles were formed at a CPP/SCO molar ratio 10:1, while maintaining cell viability. Additionally, RDP7, regardless of larger particles with hydrodynamic diameter around 205 nm, also led to similar levels of reporter rescue. The observed variations in delivery efficacy were attributed to the differences in CPP sequence and used formulation conditions. These findings highlight the importance of optimization of peptide characteristics, and formulation conditions to achieve suitable nanoparticle for specific applications.

Sirvi

Viimati lisatud