http://hdl.handle.net/11089/1309 2026-04-22T18:58:08Z http://hdl.handle.net/11089/58146 Charakterystyka kompleksów płytkowo-leukocytarnych i analiza ekspresji miRNA jako wskaźników stanu zapalnego i neurodegeneracji w rzutowo-remisyjnej i wtórnie postępującej postaci stwardnienia rozsianego Wasilewska, Karina Multiple sclerosis (MS) is a chronic neurodegenerative disease with an autoimmune background primarily affecting the central nervous system (CNS). The most common form of the condition is inflammatory relapsing-remitting MS (RRMS), whereas secondary progressive MS (SPMS), which typically develops after RRMS, is characterized by progressive neurodegeneration. However, recent reports indicate coexistence of inflammatory and neurodegenerative features in both phenotypes, complicating early differentiation, therapeutic decisions, and prognosis. The primary objective of the research was to identify markers enabling differentiation of disease phenotypes in patients with RRMS in remission and SPMS. In the first stage, platelet-leukocyte hetero-aggregates (PLAs) were characterized as elements linking vascular injury with inflammation. Increased leukocyte chemotaxis toward platelets and PLAs formation, predominantly involving B-cells, were demonstrated in MS. Next, screening of miRNA expression from extracellular vesicles (EVs) was conducted, followed by measurement of inflammatory cytokines and markers of neuronal/glial damage in plasma. A model combining miRNA expression with basic fibroblast growth factor achieved an AUC of 0.97, confirming its high ability to discriminate RRMS and SPMS. In the third stage, EVs presenting L1CAM, a protein used to enrich the neuronal fraction of EVs, were characterized in serum and cerebrospinal fluid to assess their usefulness as biomarkers for monitoring response to rituximab treatment, an anti-CD20 monoclonal antibody, in RRMS patients. In summary, the research expands understanding of MS pathophysiology, linking immune response with vascular injury, and identifies opportunities for non-invasive biomarkers to differentiate RRMS and SPMS as well as to monitor CNS immunopathology and treatment response. 2025-01-01T00:00:00Z http://hdl.handle.net/11089/58145 Aktywność biologiczna i toksyczność nanocząstek srebra pozyskanych na drodze mikrobiologicznej przy udziale grzyba strzępkowego Gloeophyllum striatum Tończyk, Aleksandra Silver nanoparticles (AgNPs) are among the most widely commercialized nanomaterials due to their versatile properties and strong antimicrobial activity, making them valuable in electronics, textiles, cosmetics, and biomedicine. The increasing demand for AgNPs highlights the need for synthesis methods that are cost-effective, rapid, and environmentally sustainable. Biological synthesis, particularly using filamentous fungi, offers an attractive alternative to conventional methods due to their ease of cultivation, high biomass production, and ability to produce metabolites influencing nanoparticle properties. This doctoral dissertation focused on the microbiological synthesis of silver nanoparticles using the brown-rot wood fungus Gloeophyllum striatum DSM 9592 under various process conditions, followed by evaluation of their antimicrobial, cytotoxic, ecotoxic, and synergistic activity with antibiotics. The results confirmed that G. striatum can efficiently synthesize silver nanoparticles, and that synthesis conditions, such as temperature and shaking, significantly affect their physicochemical and biological properties. The synthesized nanoparticles demonstrated antimicrobial activity against tested microorganisms, with fungi showing greater sensitivity than bacteria. The most sensitive organisms included the yeast Malassezia furfur and the bacterium Pseudomonas aeruginosa. Toxicity assessments revealed variable cytotoxic and ecotoxic effects depending on synthesis conditions. Nanoparticles synthesized at 4°C exhibited the lowest cytotoxicity while maintaining antimicrobial effectiveness. Ecotoxicity studies showed the highest sensitivity in the freshwater crustacean Daphnia magna and the lowest in tested crop plants. Overall, nanoparticles synthesized at 4°C without shaking showed the most favorable balance between antimicrobial activity and low toxicity. Additionally, these nanoparticles exhibited synergistic effects with antibiotics, enabling dose reduction. The findings demonstrate that optimization of fungal biosynthesis conditions enables the production of effective antimicrobial nanomaterials with reduced toxic potential. 2025-01-01T00:00:00Z http://hdl.handle.net/11089/57702 Rola kompleksu SWI/SNF-EP300 w powstawaniu oporności komórek nowotworowych na chemioterapię Gronkowska, Karolina Cancer remains a major global health problem and many tumours develop resistance to available therapies. Because drug resistance is often associated with genetic and epigenetic changes, targeting epigenetic regulators such as the histone acetyltransferase p300 and the SWI/SNF chromatin-remodelling complex may represent an effective therapeutic strategy.The aim of this thesis was to investigate the role of the SWI/SNF–p300 complex in the development of cancer drug resistance related to two mechanisms: overexpression of ABC transporters and activation of the DNA damage response pathway. In the first part of the study, I demonstrated that the chromatin remodelling ATPase BRG1 and the acetyltransferase p300 regulate the transcription of ABCC transporter genes (ABCC3, ABCC5 and ABCC10) in paclitaxel-resistant cancer cells. These lysosomal transporters were shown to sequester chemotherapeutic drugs such as doxorubicin and paclitaxel, reducing their cytotoxicity. Active promoters of these genes were enriched in BRG1, p300, HIF1A and activating histone marks. Pharmacological inhibition of SWI/SNF (PFI3), inhibition of p300 (C646), degradation of SWI/SNF ATPases, or silencing of HIF1A significantly increased drug toxicity by simultaneously reducing the expression of multiple ABC transporters. Bioinformatic analysis of clinical data further indicated that high expression of EP300, SMARCA4 and HIF1A may serve as prognostic markers of response to taxane-based chemotherapy in breast cancer. In the second part, I showed that cisplatin-induced activation of the ATM/ATR-Chk1/Chk2-p53 pathway enhances the association of p300 with chromatin and activates transcription of genes involved in DNA damage response. Inhibition of p300 or SWI/SNF reduced the expression of these genes and sensitised cancer cells to cisplatin. Overall, the results indicate that targeting SWI/SNF and p300 may reverse drug resistance and improve chemotherapy effectiveness. 2025-01-01T00:00:00Z http://hdl.handle.net/11089/57701 Właściwości biologiczne nanocząstek złota modyfikowanych polietylenoglikolem i ich zastosowanie jako potencjalnych nośników leczniczego siRNA w badaniach in vitro Okła, Elżbieta Drug carriers are systems designed to enhance the delivery of bioactive compounds to specific target sites in the body, including transport across biological barriers. Among the various nanomaterials investigated as potential carriers, gold nanoparticles (AuNPs) have gained considerable attention due to their favorable biophysical properties. They are characterized by high biocompatibility, low cytotoxicity, and the possibility of straightforward surface functionalization. One of the promising modification strategies is PEGylation, i.e., conjugation with polyethylene glycol (PEG), which improves the potential of AuNPs as carriers of drugs and nucleic acids, particularly for the delivery of therapeutic siRNA to tissues and cells protected by the blood–brain barrier. The aim of this study was to evaluate whether PEG-coated gold nanoparticles, AuNP14a and AuNP14b, differing in the ratio of carbosilane dendrons to PEG chains, can form complexes with siRNA and be internalized by cells in a blood–brain barrier model. The investigated siRNA targeted the apoE gene, whose ε4 allele is associated with an increased risk of Alzheimer’s disease. The results confirmed that AuNPs can successfully form complexes with siRNA (siApoE), with AuNP14a forming stable complexes at lower concentrations than AuNP14b. The nanoparticles interacted with plasma proteins without significantly altering their secondary structure and showed interactions with lipid membranes. Efficient uptake of AuNP/siRNA complexes by endothelial cells and their accumulation mainly in the cytoplasm were observed. The nanoparticles demonstrated low cytotoxicity toward endothelial cells, astrocytes, and pericytes, which was further reduced in the presence of siRNA. Both AuNPs and their siRNA complexes exhibited relatively low genotoxicity, while alterations in reactive oxygen species levels and mitochondrial membrane potential returned to baseline within 24 hours. Overall, the findings indicate that PEGylated AuNP14a and AuNP14b meet essential criteria for therapeutic siRNA carriers, with AuNP14a showing greater potential.; Nośniki leków są związkami umożliwiającymi transport substancji bioaktywnych do miejsca ich działania, w tym przez bariery biologiczne. Wśród potencjalnych systemów dostarczania leków szczególne zainteresowanie budzą nanocząstki złota (AuNP), które dzięki swoim właściwościom biofizycznym znajdują szerokie zastosowanie w biomedycynie. Cechują się one wysoką biokompatybilnością, niską cytotoksycznością oraz możliwością łatwej modyfikacji powierzchni. Jedną z obiecujących metod ich funkcjonalizacji jest pegylacja, czyli sprzęganie z glikolem polietylenowym (PEG), co zwiększa ich potencjał jako nośników leków i kwasów nukleinowych, w tym terapeutycznych siRNA, zwłaszcza w kontekście transportu przez barierę krew–mózg. Celem pracy było sprawdzenie, czy pegylowane nanocząstki złota AuNP14a i AuNP14b, różniące się stosunkiem dendronów karbokrzemowych do PEG, mogą tworzyć kompleksy z siRNA oraz przenikać do komórek modelu bariery krew–mózg. Badany siRNA był skierowany przeciwko genowi apoE, którego allel ε4 wiąże się ze zwiększonym ryzykiem rozwoju choroby Alzheimera. Wyniki potwierdziły zdolność AuNP do kompleksowania z siRNA (siApoE), przy czym AuNP14a tworzyły kompleksy przy niższych stężeniach niż AuNP14b. Wykazano także oddziaływania nanocząstek z białkami osocza bez istotnych zmian w ich strukturze drugorzędowej oraz interakcje z błonami lipidowymi. Kompleksy AuNP/siRNA efektywnie wnikały do komórek śródbłonka i akumulowały się głównie w cytoplazmie. Badane nanocząstki wykazywały niską cytotoksyczność wobec komórek śródbłonka, astrocytów i perycytów, która dodatkowo zmniejszała się w obecności siRNA. Zaobserwowana genotoksyczność była niewielka, a zmiany poziomu reaktywnych form tlenu i potencjału mitochondrialnego ulegały normalizacji w ciągu 24 godzin. Uzyskane wyniki wskazują, że pegylowane nanocząstki AuNP14a i AuNP14b spełniają podstawowe kryteria nośników terapeutycznego siRNA, przy czym bardziej obiecującą formulacją wydaje się AuNP14a. 2025-01-01T00:00:00Z