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Cijanobakterije predstavljaju raznovrsnu grupu mikroorganizama poznatih po produkciji visoko potentnih prirodnih toksina koji predstavljaju ozbiljnu pretnju po zdravlje ljudi, životinja i životne sredine. Ovi cijanotoksini se razlikuju po svojoj hemijskoj strukturi i mehanizmu delovanja, a često se u istom okruženju mogu naći različite klase, što čini procenu njihovih toksičnih efekata izazovnom. Kontinuiran razvoj i usavršavanje testova za precizniju detekciju, karakterizaciju i procenu rizika od cijanotoksina je glavni imperativ, te se na globalnom nivou sve češće naglašava potreba za pristupom koji bi omogućio sveobuhvatno proučavanje cijanotoksičnosti i praćenje efekata na različitim biološkim nivoima. U cilju rešavanja ovih izazova, razvijaju se različiti alternativni testovi i primenjuju model organizmi, među kojima su u fokusu u pogledu njihove korisnosti u unapređenju našeg razumevanja ove oblasti, vodeni kičmenjaci i beskičmenjaci. U skladu sa tim, formulisani su primarni ciljevi ove doktorske disertacije koji su podrazumevali procenu akutne toksičnosti ekstrakata odabranih sojeva cijanobakterija in vivo, kod akvatičnih beskičmenjaka (Artemia salina i Daphnia magna) i kičmenjaka (Danio rerio) i identifikovanje faktora koji dovode do modulacije njihove toksičnosti, uključujući dostupnost hranljivih materija, fotoperiod i faze rasta. Dodatno, istraženi su toksični efekti koji se manifestuju na molekularnom nivou dva model organizma, analizom relativnih promena u ekspresiji gena koji reaguju na toksine, kako bi se definisali neki od mehanizama toksičnog delovanja odabranih sojeva. Pored toga, in vitro test bazirani na upotrebi ćelijske linije humanog neuroblastoma je korišćen kako bi se odredio citotoksični i neurotoksični potencijal ispitanih sojeva. Konačno, jedan od osnovnih ciljeva istraživanja je podrazumevao ispitivanje metaboličkog potencijala odabranih sojeva da produkuju određene tipove cijanobakterijskih toksina koristeći imunotest, genomsku i bioinf, Cyanobacteria are a diverse group of organisms known for producing highly potent cyanotoxins which pose significant threats to human, animal, and environmental health. These toxins have diverse chemical structures and toxicity mechanisms and several toxin classes can be present simultaneously, making it difficult to assess their toxic effects using physico-chemical methods. Continued development and refinement of assays is necessary for their improved detection, characterization, and risk assessment and emphasizes the need for a multi-level approach to studying cyanotoxicity. To address these challenges, alternative tests and model organisms among aquatic vertebrates and invertebrates are continually explored for their utility in advancing our understanding of this field. The primary objectives of this doctoral dissertation were to evaluate the acute toxicity of extracts of selected cyanobacterial strains in vivo, in aquatic invertebrate (Artemia salina and Daphnia magna) and vertebrate (Danio rerio) organisms and identify factors that lead to modulation of their toxicity, including nutrient availability, light period and growth phase. Furthermore, the study aimed to explore the toxic effects manifesting at the molecular level by analyzing the relative changes in the expression of toxin-responsive genes, in order to define some of the mechanisms of toxic action of the selected strains. Additionally, in vitro tests using a human neuroblastoma cell line were utilized to determine their cytotoxic and neurotoxic potential. Lastly, the research aimed to examine the potential of selected strains to produce the best studied groups of cyanobacterial toxins using an immunoassay and various bioinformatics tools. In a series of acute toxicity tests, cyanobacteria strains were evaluated using Daphnia magna, Artemia salina, and Danio rerio (zebrafish) as model organisms. The results showed that cyanobacterial strains exhibited dose- and time-dependent toxic effects, with variati

Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group (1-13) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3, 5, 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC50 values were in the range of 22.99-51.66 µM (HCT 116 cell line), 8.63-41.20 µM (BxPC-3 cell line) and 24.78-81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line (p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1, 2, 3 and 5) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.

The expanded prevalence of resistant bacteria and the inherent challenges of complicated infections highlight the urgent need to develop alternative antibiotic options. Through conventional screening approaches, the discovery of new antibiotics has proven to be challenging. Anti-infective drugs, including antibacterials, antivirals, antifungals, and antiparasitics, have become less effective due to the spread of drug resistance. In this work, we helped define the design of next-generation antibiotic analogs based on metal complexes. For this purpose, we used artificial intelligence (AI) methods, demonstrating superior ability to tackle resistance in Gram-positive and Gram-negative bacteria, including multidrugresistant strains. The existing AI approaches’ bottleneck relies on the current antibiotics’ structural similarities. Herein, we developed a machine learning approach that predicts the minimum inhibitory concentration (MIC) of Re-complexes towards two S. aureus strains (ATCC 43300 - MRSA and ATCC 25923 - MSSA). A Multi-layer Perceptron (MLP) was tailored with the structural features of the Re-complexes to develop the prediction model. Although our approach is demonstrated with a specific example of rhenium carbonyl complexes, the predictive model can be readily adjusted to other candidate metal complexes. The work shows the application of the developed approach in the de novo design of a metal-based antibiotic with targeted activity against a challenging pathogen.

Antimicrobial resistance (AMR) arises when bacteria and other microbes stop responding to medications. AMR is now recognized as one of serious global health threats, repeatedly appearing in the World Health Organization’s (WHO) lists of urgent global health challenges, including the 2024 list. It is taking a fatal toll – nearly 5 million deaths globally per year are associated with AMR, encompassing 1.27 million directly attributed to AMR. The COVID-19 pandemic paved the way for aggravation of bacterial AMR – primarily due to enhancement in unspecific and unjustified prescription and use of broad-spectrum antibiotics, resulting in what is now recognized as „silent pandemic of AMR“. Bacteriophages (phages) are natural and specific predators of bacteria - viruses that can infect, replicate inside and lyse arguably any bacteria. Their therapeutic potential is being hastily evaluated through different approaches: in silico, in vitro, ex vivo and in vivo – in laboratory animals as well as in human case and clinical studies. Although the results are promising,bacteria rapidly develop resistance against phages, which why the isolation and research of new phages is needed. Our work is concentrated on three bacterial species for which critical priority by WHO has been declared – carbapenem- resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Twenty distinct pathogenic strains of A. baumannii, 6 K. pneumoniae and 6 P. aeruginosa were used as targets for bacteriophage isolation, and total of 14, 22 and 8 potentially distinct phages were collected, respectively. All strains were nosocomial isolates obtained from various tissues, including from terminally ill patients. Six phages were characterized in detail. In particular, phage vB_AbaM_ISTD was applied against A. baumannii in zebrafish embryo model of systemic infection, and demonstrated powerful therapeutic potential, eradicating the infection. Interestingly, its DNA was characterized with hig

Fungal infections, once considered a rare disease, have become an everyday problem in modern societies, posing major challenges to global health. It is estimated that more than one billion people are affected by fungal infections and 1.6 million people succumb to these diseases every year. Of the 600 species of fungi capable of causing infections in humans, species of the genus Candida cause more than 85% of infections, especially C. albicans, which has become a serious threat to human health in immunocompromised and immunosuppressed individuals. Unfortunately, the current arsenal of clinical drugs relies on only four classes of approved drugs (polyenes, azoles, echinocandins and allylamines), which are only partially effective, resulting in incomplete eradication of the fungal infection. In addition, the serious side effects, ranging from systemic or organ-specific toxicity to poor bioavailability and low activity, significantly hamper the clinical use of antifungals. These problems call for new effective and safe antifungal agents,but also for appropriate preclinical models to accurately study potential adverse effects on the human population and test their efficacy against fungal infections. In this sense, zebrafish (Danio rerio) embryos have become one of the most powerful preclinical animal models in infection biology and drug discovery, offering the unique opportunity to simultaneously monitor the safety and efficacy of the applied molecule in real time. With the aim of providing a preclinical platform for the identification of new safe antifungal drugs to effectively control C. albicans infection, we comprehensively tested the toxicity of 13 clinical antifungal drugs in the zebrafish embryo model. The 21 toxicity endpoints, including survival, teratogenicity, cardiotoxicity and hepatotoxicity, were evaluated and compared with adverse effects described in rats and humans. Of the clinical drugs, the efficacy of fluconazole and voriconazole was evaluated in the

This study aimed to compare the biological properties of newly synthesized cements based on calcium phosphate with a commercially used cement, mineral trioxide aggregate (MTA). Strontium (Sr)-, Copper (Cu)-, and Zinc (Zn)-doped hydroxyapatite (miHAp) powder was obtained through hydrothermal synthesis and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectrometry (EDX). Calcium phosphate cement (CPC) was produced by mixing miHAp powder with a 20 wt.% citric acid solution, followed by the assessment of its compressive strength, setting time, and in vitro bioactivity. Acetylsalicylic acid (ASA) was added to the CPC, resulting in CPCA. Biological tests were conducted on CPC, CPCA, and MTA. The biocompatibility of the cement extracts was evaluated in vitro using human dental pulp stem cells (hDPSCs) and in vivo using a zebrafish model. Antibiofilm and antimicrobial effect (quantified by CFUs/mL) were assessed against Streptococcus mutans and Lactobacillus rhamnosus. None of the tested materials showed toxicity, while CPCA even increased hDPSCs proliferation. CPCA showed a better safety profile than MTA and CPC, and no toxic or immunomodulatory effects on the zebrafish model. CPCA exhibited similar antibiofilm effects against S. mutans and L. rhamnosus to MTA.