http://hdl.handle.net/11089/1 2026-08-26T11:48:10Z 2026-08-26T11:48:10Z Skibiński, Jakub Mikołaj http://hdl.handle.net/11089/59161 2026-08-21T02:16:25Z 2026-01-01T00:00:00Z

Identyfikacja niskocząsteczkowych związków hamujących aktywność wybranych enzymów zaangażowanych w metabolizm RNA u Helicobacter pylori Skibiński, Jakub Mikołaj The increasing antibiotic resistance of Helicobacter pylori (H. pylori) significantly reduces the effectiveness of eradication therapies and highlights the urgent need to identify novel molecular targets for potential therapeutic substances. This study aimed to characterize selected components of the H. pylori RNA degradosome - polynucleotide phosphorylase (PNPase), the RNA helicase RhpA, and the protein PapS with particular emphasis on evaluating PNPase as a potential therapeutic target. Additionally, the function of PapS in RNA metabolism and its interaction with RhpA were investigated. Recombinant PNPase, RhpA, and PapS were successfully purified. Enzymatic assays for PNPase activity, including RNA polymerization and degradation reactions, were optimized to enable downstream inhibitor screening. The spatial structure of PNPase was resolved, followed by structural-functional analyses and interaction studies of the RhpA-PapS complex. PNPase was shown to form a homotrimeric assembly and to retain enzymatic activity under conditions compatible with high-throughput screening applications. PapS did not exhibit polyadenylation activity toward single-stranded RNA; however, it catalyzed the sequential addition of the 3′-terminal CCA sequence to tRNA, consistent with its role as a CCA-adding enzyme. A transient and moderate interaction between PapS and RhpA was detected. Transcriptomic analysis revealed limited changes in gene expression in the H. pylori G27 Δpnp and ΔpapS strains, whereas deletion of rhpA resulted in extensive transcriptomic remodeling. Although pnp deletion did not abolish bacterial viability, it markedly impaired adaptive capacity. The Δpnp strain displayed reduced tolerance to temperature stress (slower growth at 39°C and decreased survival at 4°C), increased sensitivity to UV-C radiation and oxidative stress, reduced motility and urease as well as catalase activity, while enhanced ability to biofilm complex formation, which needs further study to elucidate the potential mechanism. A four-stage screening strategy for PNPase inhibitors was implemented, integrating in vitro enzymatic assays, qualitative selection steps, and evaluation using bacterial cells. From a library of 8,562 compounds, 23 candidates were selected for further validation. Among them, niclosamide exhibited the most pronounced PNP-dependent activity profile. Collectively, these findings indicate that although H. pylori PNPase is not essential for viability, it represents a central regulatory node in RNA metabolism that governs stress response and adaptive phenotypes. Therefore, PNPase constitutes a rational and promising target for the development of novel antibacterial strategies.; Narastająca lekooporność Helicobacter pylori (H. pylori) na stosowane obecnie antybiotyki ogranicza skuteczność eradykacji, co wiąże się z koniecznością poszukiwania nowych celów molekularnych dla potencjalnych leków. W pracy scharakteryzowano wybrane komponenty degradosomu RNA H. pylori, biorąc pod uwagę ich strukturę i funkcję, a także wzajemne oddziaływania: fosforylazę polinukleotydową PNP (PNPazę), helikazę RhpA oraz białko PapS, ze szczególnym uwzględnieniem roli PNP jako potencjalnego celu terapeutycznego. Określono funkcję PapS w metabolizmie RNA i zbadano występowanie interakcji PapS-RhpA. Oczyszczono rekombinowane białka PNP, RhpA i PapS. Zoptymalizowano testy aktywności PNP (reakcje polimeryzacji i degradacji RNA). Wykazano, że PNPaza występuje w formie trimeru i zachowuje aktywność enzymatyczną w warunkach umożliwiających dalsze badania przesiewowe. PapS nie wykazywało aktywności poliadenylacyjnej wobec jednoniciowego RNA, natomiast katalizowało sekwencyjną dobudowę końca 3′ CCA do tRNA, co wskazuje na funkcję enzymu dobudowującego CCA. Stwierdzono przejściowe, umiarkowane oddziaływanie PapS-RhpA. Analiza transkryptomiczna ujawniła ograniczone zmiany ekspresji w szczepach delecyjnych H. pylori G27 Δpnp i ΔpapS oraz rozległą przebudowę profilu transkryptomicznego w ΔrhpA. Delecja pnp nie wiązała się z obumieraniem komórek, lecz powodowała istotne ograniczenie zdolności adaptacyjnych: obniżenie tolerancji na stres temperaturowy (wolniejszy wzrost w 39°C i słabsze przeżycie w 4°C), wzrost wrażliwości na UV-C i stres oksydacyjny, zahamowanie ruchliwości i aktywności ureazy oraz katalazy. Jakkolwiek delecja pnp wiązała się z nasileniem tworzenia obszaru biofilmu, czego wyjaśnienie wymaga dalszych badań. Zastosowano czteroetapową strategię przesiewową użytych w badaniach inhibitorów PNP łączącą testy in vitro, selekcję jakościową i ocenę działania wobec komórek bakteryjnych. Z biblioteki 8562 związków wyłoniono 23 kandydatów do dalszej weryfikacji, a najbardziej obiecujący profil działania zależny od obecności PNP wykazał niklozamid. Uzyskane wyniki wskazują, że PNPaza H. pylori, mimo że nie jest niezbędna do przeżycia bakterii stanowi kluczowy węzeł regulacji metabolizmu RNA determinujący odpowiedź stresową i fenotypy adaptacyjne bakterii oraz racjonalny cel dla poszukiwania inhibitorów PNP w celach terapeutycznych.

2026-01-01T00:00:00Z Studzian, Maciej http://hdl.handle.net/11089/58796 2026-07-08T02:10:21Z 2026-06-01T00:00:00Z

Yeast display for directed evolution of amadoriase, an enzyme capable of cleaving protein glycation products - MINIATURA 8 (2024/08/X/NZ1/01781) (dataset) Studzian, Maciej This dataset was generated as part of the Miniatura 8 project, which focused on amadoriases. These FAD-dependent enzymes catalyze the breakdown of glycated amino acids, producing a free amino acid, glucosone, and hydrogen peroxide. Naturally occurring in fungi and bacteria, they are of considerable practical importance due to their applications in medical diagnostics and their potential therapeutic use, particularly in the treatment of diseases such as diabetes. Previous attempts to improve amadoriase I from Aspergillus through targeted amino acid substitutions increased the enzyme's thermostability but did not enhance its catalytic activity. Similarly, directed evolution of the related enzyme amadoriase II resulted in only marginal improvements. The aim of this project was to develop methods for the in vitro evolution of amadoriases to increase their activity using a non-directed, ultra-high-throughput approach based on yeast surface display. This technique enables the generation of random enzyme variants displayed on the surface of yeast cells, which can then be screened for catalytic activity. A biotin- and azide-modified fructoselysine analogue, designed to mimic a glycated substrate, was successfully synthesized and immobilized on the yeast surface to serve as a substrate for activity screening. Upon catalysis by an amadoriase, the substrate was expected to lose its fluorescence, allowing fluorescence-activated cell sorting (FACS) to isolate yeast cells displaying enzyme variants with higher catalytic activity. A previously engineered variant of amadoriase II from Aspergillus fumigatus (AmaII_SII_82) was used as the starting point for the evolution experiments. Unfortunately, because the initial enzyme exhibited only negligible activity toward the biotin-modified substrate, the variants obtained after three rounds of sorting also failed to show the expected improvement in activity. Nevertheless, the techniques developed during this research provide a strong foundation for future studies aimed at improving other classes of deglycating enzymes, such as FN3K and FrlB/FrlD.

2026-06-01T00:00:00Z Juszczak, Michał http://hdl.handle.net/11089/58757 2026-07-07T02:10:31Z 2024-12-10T00:00:00Z

Ochronna rola CORMs w łagodzeniu stresu oksydacyjnego i stanu zapalnego indukowanego doksorubicyną w modelu prawidłowych komórek skóry - MINIATURA 8 2024/08/X/NZ7/01320 (dataset) Juszczak, Michał Doxorubicin is a potent anticancer drug that unfortunately damages healthy cells. Researchers from the University of Łódź investigated whether carbon monoxide could protect against these side effects. They used CORMs (carbon monoxide-releasing molecules)—specifically CORM-2 and CORM-3—which safely deliver the gas to cells. The team tested these on healthy human fibroblasts exposed to doxorubicin, alongside inactive, gas-free molecules as a control. While pretreating cells with CORMs did not prevent the drug's overall toxicity, it effectively combated oxidative stress. Both CORM-2 and CORM-3 significantly reduced destructive oxygen free radicals induced by doxorubicin. Surprisingly, the inactive, gas-free molecules were equally effective at this. The researchers also assessed programmed cell death by measuring caspase activity. Active CORM-3 inhibited this process, showing a protective effect, whereas CORM-2 unexpectedly accelerated cell death. Additionally, CORM-3 reduced the activity of certain oxidative stress response genes without affecting inflammatory genes or final defensive protein levels. This research highlights the complex relationship between carbon monoxide releasers and chemotherapy. Although these molecules are not a cure-all for doxorubicin's toxicity, they possess valuable antioxidant properties. Crucially, the study reveals that the positive intracellular effects often stemmed from the molecule’s structural backbone itself rather than the released carbon monoxide. This discovery is a significant step toward understanding cellular mechanisms and designing better protective therapies for cancer patients. Dane dotyczą wyników z oznaczeń: cytotoksyczności, reaktywnych form tlenu, apoptozy, ekspresji genów i białek zaangażowanych w stan zapalny i stres oksydacyjny.

2024-12-10T00:00:00Z Mietlińska-Sauter, Joanna Battaglia, Sebastiano Lorkiewicz, Wieslaw Fruciano, Carmelo http://hdl.handle.net/11089/58462 2026-06-11T09:31:05Z 2024-06-26T00:00:00Z

Morphological structure of the vertebra and predisposition to fatigue spondylolysis in the human population - geometric morphometrics studies on skeletal material - MINIATURA 8 (2024/08/X/NZ8/00212) dataset Mietlińska-Sauter, Joanna; Battaglia, Sebastiano; Lorkiewicz, Wieslaw; Fruciano, Carmelo Mietlińska-Sauter, Joanna Fatigue spondylolysis is commonly interpreted in bioarchaeology as a direct skeletal marker of increased axial loading, particularly in pre-industrial populations engaged in strenuous manual labour. However, clinical studies in contemporary populations suggest that vertebral morphology may also play an important role in fracture susceptibility. The present project examined whether anatomical predisposition contributed to fatigue spondylolysis in historical human populations from the Brześć Kujawski microregion (Kuyavia, central Poland), dating from the early Middle Ages to the mid-nineteenth century. Fifth lumbar vertebrae (L5) representing individuals with and without spondylolytic fractures were selected from osteological collections housed at the University of Lodz. Following cleaning and, where necessary, recomposition, the vertebrae were scanned using structured-light 3D surface scanning. Their morphology was analysed using landmark-based geometric morphometrics, including fixed landmarks and sliding semilandmarks, and the resulting data were statistically evaluated in MorphoJ, R software environment and Statistica. The analyses revealed statistically significant differences in L5 anatomy between spondylolytic and non-pathological vertebrae. Fractured vertebrae displayed morphological features suggestive of functional predisposition to injury. In addition, previously undescribed differences in fluctuating asymmetry were identified between the two groups, indicating that developmental instability may also have contributed to fracture susceptibility. These findings suggest that fatigue spondylolysis in pre-industrial populations should not be regarded solely as a simple indicator of increased spinal loading. Instead, its occurrence appears to reflect an interaction between mechanical stress as well as individual anatomical and developmental predispositions. The study demonstrates the utility of geometric morphometrics for investigating vertebral variation in archaeological populations and provides a more nuanced framework for interpreting spondylolysis in bioarchaeological research. 1) Folder 1: representative scans in .STL format (one non-pathological vertebra, SBK4_59_L5, and one vertebra affected by fatigue spondylolysis, SBK4_250_L5) 2) Folder 2: reference landmark configuration used in the study (including a .vtk file containing the vertebral model, 26 .mrk.json files with landmarks assigned to individual landmark sets, an Excel file defining all landmarks, and a readme.txt file with instructions for opening the files) 3) Folder 3: digitization results for all 36 analysed vertebrae - 10 affected by spondylolysis and 26 controls (including two folders corresponding to the first and second digitization rounds, respectively; each folder contains files in four formats: .mrk.json, .json, .fcsv, and .csv). The folder also includes an Excel file defining all landmarks, presented without subdivision into individual vertebral elements, but distinguished as fixed landmarks and sliding semilandmarks on curves and surfaces. 4) Folder 4: output models (including two .ply files representing the mean shape of a spondylolytic vertebra and the mean shape of a non-pathological vertebra and a readme.txt file with instructions for opening the files)

2024-06-26T00:00:00Z