Pancreatic adenocarcinoma is the most common malignant neoplasm of the pancreas and represents one of the most lethal forms of cancer in the world, with no effective treatment options for advanced stages. It has an immunosuppressive tumor microenvironment that contributes to resistance to conventional therapy. These limitations have motivated the research for alternative therapies, for example use of supplements and natural compounds. Melatonin is emerging as a promising adjuvant therapy, not only alleviating the side effects of chemotherapy and radiotherapy, but also demonstrating antiproliferative effects in various types of cancer. In this context, this study aimed to characterize the effects of melatonin on the modulation of cellular parameters in human pancreatic adenocarcinoma cells (PANC-1). PANC-1 cells were stimulated with different concentrations of melatonin (0.625mM; 1.25mM; 2.5mM; 3.75mM and 5mM) for different periods. Carcinogenic parameters like proliferation, cell cycle, nuclear fragmentation, lipid droplets biogenesis and cell death were evaluated. Analyses to characterize cell death by pyroptosis were also carried out as the membrane pore formation, release of the enzyme lactate dehydrogenase (LDH), activation of caspase-1, generation of reactive species (ROS) as well as lysosomal acidification and mitochondrial morphology. Our results revealed that melatonin modulated carcinogenic parameters, reducing cell proliferation, arresting the cell cycle at the G1/G0 phase, decreasing the biogenesis of lipids droplets and inducing cytotoxicity in a dose- and time-dependent manner. Cell death by pyroptosis was also observed, evidenced by reduced plasma membrane integrity, LDH release and caspase-1 activation, as well as an increase in ROS production and lysosomal and mitochondrial damage. This study highlights the potential antitumor effect of melatonin in vitro, unveiling new perspectives for its use as an adjuvant in the treatment of pancreatic cancer.

Ionic channels are transmembrane proteins that possess properties such as the opening and closing movement mediated by electrical, chemical, or mechanical signals, and the conduction of ions through the membrane. Potassium channels play a role in regulating cellular excitability and can undergo modulations in their response patterns in the presence of peptidic toxins from venoms of various animals, including arthropods. Spider venoms are widely studied due to their complex mixture of bioactive molecules capable of modulating or inhibiting ion channels, having membranolytic activity, among other pharmacologically interesting properties. Ap6 is a 31-amino acid peptide previously described in the venom of the tarantula Acanthoscurria paulensis. Its effects were evaluated on NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1, and CaV2.2 channels at a concentration of 1 µM. Among these ion channels, Ap6 showed activity only on CaV2.1 channels, leading to its classification as a κ-toxin. Since Ap6 shares sequence similarity with other Theraphosidae spider peptides that have shown activity on potassium channels, and considering that it is common for such peptides to exhibit promiscuity regarding the molecular target, this study aimed to assess its modulation capacity on KV1.1, 1.2, 1.3, and 3.1 channels. The Ap6 peptide was purified from the venom of the Acanthoscurria paulensis spider through two stages of reverse-phase liquid chromatography (RP-HPLC). Mass spectrometry using MALDI-ToF confirmed the purity and experimental monoisotopic molecular mass of 3717.9 Da. The activity on K+ channels was evaluated using the patch-clamp technique in the whole-cell system. HEK 293T cells expressing Kv 1.1 and Kv 1.2 channels, L292 cells expressing Kv 1.3 and Kv 3.1 channels, and CHO cells expressing Kv 1.4 were used, with Ap6 applied at a concentration of 500 nM. Under the experimental conditions used, Ap6 did not demonstrate the ability to modify potassium currents in the studied channels. Despite sequence similarity analysis and structural arrangement in the ICK motif indicating potential action on potassium channels and, therefore, a possible classification as κ-toxins, Ap6 was not able to modify the conductance of KV1.1, 1.2, 1.3, and 3.1 channels. Thus, it is suggested that Ap6 continues to be classified as a κ-toxin.

Cryptococcosis is an infectious disease caused by fungi from the genus Cryptococcus, the infection with C. neoformans being the most common and globally disseminated. It is estimated that 152 thousand new cases of cryptococcosis appear annually, which 73% of these cases lead to death. C. neoformans is able to produce melanin and now it is known that it acts as a virulence factor, facilitating infection. Melanin production is possible due to the presence of the enzyme laccase 1(LAC1) in the fungi. However, the role of the melanin and laccase 1 in the inflammation and death of the host cells still remain unclear. Therefore, in this work we investigate whether the presence of melanin and laccase 1 could interfere in inflammatory mediators produced by human macrophages and, consequently, in their death. We could notice that the presence of melanin as the lack of LAC1 provoked less phagocytosis by macrophages of C. neoformans but not change the number of yeast per cell. Moreover, C. neoformans infection caused more cell death, and melanin and LAC1 absence induce even more death after 24 hours. Investigating which type of death is trigged by etiologic agent, we discovered that in early infection (5 h.p.i) is induced apoptosis, whereas in late infection, apoptosis is greatly reduced and lytic death is the predominant type of death. The presence of melanin and the lack of LAC1 caused more lytic death. Since C. neoformans infection and its melanin provoked more death, we evaluated wether this death could be trigged by inflammasomes activation and despite we barely saw activation until 2 h.p.i, in early infection (5 h.p.i) we could notice there was cleavage and activation of caspase 1 (CASP1) and gasdermin D (GSDMD), which was more pronounced in late infection (24 h.p.i). However, it is visualized a greater caspase 8 activation in early infection when LAC1 is absent, compared to CASP1, while in late infection showed a massive activation of CASP1. This could explain the apoptosis increased at 5 h.p.i and the prevalence of lytic death in human macrophages at 24 h.p.i interacted with the yeast. Therefore, we conclude that C. neoformans is able to induce cell death in human macrophages during infection, which is trigged by inflammasome activation and its virulence factors could change this process.

Every year there are more than 1.5 million deaths worldwide from invasive fungal infections. Candida auris is becoming the main fungus causing nosocomial invasive infections. The emergence of secondary resistance to azoles in strains of A. fumigatus has caused worldwide concern. Ergosterol is an essential lipid for cell viability as it is responsible for membrane fluidity and permeability. The erg6 gene encodes a sterol 24-C-methyltransferase (SMT) that acts by transferring the C-24 methyl group to zymosterol and alternatively to lanosterol. CauSMT (C. auris) and AfSMT (A. fumigatus) were produced, purified, and characterized, including determination of substrate preference. CauSMT appears to be pentameric and AfSMT tetrameric. The affinity constants show that both enzymes have more affinity for the substrate S- adenosyl-methionine (SAM) than for sterols. The catalytic efficiency of CauSMT is slow and tomatidine has been identified as an inhibitor. The secondary structure of AfSMT and CauSMT corresponds to the percentages predicted for the three-dimensional structures. Thermal stability data demonstrated that CauSMT and AfSMT are more stable at acidic and neutral pH. Molecular docking data showed a site for each substrate. Analysis of sequence and structural alignments shows the conservation of important residues in the active site of both enzymes. CauSMT and AfSMT crystals with the SAM substrate were obtained under different crystallization conditions. Diffraction data collected at the Manacá LNLS/Sirius beamline with CauSMT crystals co-crystallized with the SAM substrate showed resolutions of 7 to 8 Å.

 Komagataella phaffi (formerly known as Pichia pastoris) is considered one of the most important platforms available for large-scale production of proteins and metabolites today, due to its high productivity and easy control of fermentative parameters. Its most distinctive physiological feature is the ability to use methanol as a sole carbon source, achieved through the hyperexpression of the AOX1 gene encoding the peroxisomal alcohol oxidase enzyme. The PAOX1 promoter allows high expression with finely regulated control by methanol concentration, making it the most widely used recombinant expression system in this organism. However, the main limitation of this system in the context of industrial bioprocesses is the highly toxic and flammable nature of methanol. The development of a gene expression induction system based on a non-toxic inducer agent, preferably not consumed by the cell, is necessary to reduce production costs on a large scale. In this work, two expression control mechanisms were tested: at the transcriptional level we used the GAL system from S. cerevisiae to induce the production of green fluorescent protein (GFP) under the control of the PGAL1 promoter regulated by galactose and; at the translational level, expression control was achieved using a tetracycline-controlled riboswitch aiming to inhibit the production of β-galactosidase and Ku70p proteins. The results demonstrated success in using the riboswitch as a mechanism for controlling protein production in K. phaf with easy application and significant potential impact on the application of this system on a large scale. 

SARS-CoV-2, the virus causing COVID-19 was first described in Wuhan, China in 2019, from where it quickly spread worldwide, causing the pandemic announced in March 2020. Several studies have found viral mechanisms of immune escape that disrupt the development of the response generated by the innate immune system and delay the response of the adaptive immune system. These mechanisms include the ability of the virus to repress antiviral response caused by interferons (IFNs), and the ability to interfere with splicing processes. Both mechanisms are accomplished with the aid of viral proteins expressed in the host cell and generate a complex response, requiring large-scale analysis to elucidate the consequences of pathogen-host interactions. In this work, modulation of gene expression, alternative splicing patterns and RNA editing were evaluated from the immune cell transcriptome available in public databases. Transcript expression analyses in peripheral blood mononuclear cells (PBMC) from patients with moderate-stage COVID-19 showed little immune system response and enriched terms related to viral replication, operations between set of differentially expressed genes in COVID-19 and dengue showed similar NF-kB-induced inflammatory response, indicating a possible inflammatory response, with diminished antiviral, host-damaging response, in COVID-19. Signature related to IFN response was enriched in genes with differential alternative splicing. CD74 and MX2 genes related to IFN-1 pathways showed differential alternative splicing with loss of function. Expression analysis of circulating monocytes from infected patients in severe state showed decreased IFN-1 response compared to mild cases, correlation with hypoxic state, increased inflammatory response via NF-kB and RNA editing patterns that indicate decreased IFNs response and may help to understand downregulation and infection of this cell type.

Scorpions are well-adapted arachnids and have been around for approximately 400 million years. The Buthidae family is notable for the clinical importance of its species, which possess neurotoxic venoms. In Brazil, there are over 82 species, with a focus on the genus Tityus. The venom of these scorpions contains various compounds, such as peptides, enzymes, and inorganic salts, with diverse activities under study. In the central region of Brasília, near the University of Brasília, approximately 30 specimens of both male and female Tityus fasciolatus were collected. The animals were kept alive in the Animal Facility of the Institute of Biology at the University of Brasília, and through the process of electrostimulation, 46 milligrams of crude venom were extracted. The material underwent purification through two stages of High-Performance Liquid Chromatography (HPLC) to obtain 86.2 µg of pure TfTx peptide. The neurotoxin TfTx, a peptide isolated from the venom of Tityus fasciolatus, has a molecular mass of 3,583.64 Da [M+H]+ and its sequence revealed 31 amino acid residues stabilized by four disulfide bridges. In comparisons made in databases, it was observed that this peptide has a high degree of identity with peptides acting on potassium channels, Bmkk4 isolated from Mesobuthus martensii venom and pMeKTx7 isolated from Mesobuthus eupeus venom, in addition, a high degree of identity was also observed with two other peptides: CllNtx isolated from the venom of Centruroides limpidus and 'Peptide A' isolated from Centruroides hirsutipalpus. This second group of peptides has no regulated activity, and studies carried out with these recommendations show that they comprise a not yet described family of neurotoxins with possible activity on potassium channels. This work had as main objective the elucidation of the primary structure and the evaluation of the activity of the toxin in selective ion channels for potassium (KV1.4, KV3.1 and hERG1) and selective channels for calcium (CaV1.2, CaV1.3, CaV2 1, CaV2.2 and CaV2.3), however, no significant current or voltage change was observed to determine the activity of the peptides in these channels.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB -which converts chorismate to trp-was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

The severe acute respiratory syndrome coronavirus 2019 (SARS-CoV-2) is the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic. The adoption of public policies, particularly mass vaccination, were essential in fighting and mitigating the negative impacts of the pandemic on public health. In this context, vaccine platforms based on messenger RNA (mRNA) were revolutionary, demonstrating advantages in terms of speed of production and efficacy. Despite the availability of data characterizing innate and adaptive immune responses to different vaccines against COVID-19, molecular dynamics and processes in mRNA vaccines still require further investigation, which, in turn, could help improve vaccine formulations against possible emerging coronaviruses. In this work, using public single cell RNA sequencing (scRNA-seq) data from individuals vaccinated by BNT162b2, it was possible to characterize adaptive immunity dynamics induced, longitudinally, by the immunizer with a complete vaccination schedule. The present work demonstrated particular cellular dynamics after immunization, associated with a profound but transient specific T lymphopenia, with expansion of B cell populations, such as plasmablasts and memory B cells. Differential gene expression analysis showed expression of cellular activation, proliferation and cytotoxicity markers in B and T lymphocytes, in addition to progressive expression of INF-I over the post-vaccination time periods. Notwithstanding, gene ontology analysis highlighted terms that corroborate to the transcriptional profile findings. Furthermore, the determination of B and T subpopulations enabled trajectory inference analyses, deepening findings of post-vaccination cellular dynamics and, finally, cell-cell communication analyzes could establish interactional and signaling patterns between cell groupings indicating non-exclusive, however crucial, participation of pathways such as FTL3, MIF and BAFF/APRIL promoted by the immunization. The results obtained in the present work highlighted cellular and molecular mechanisms that propels the development of more efficient, effective and safer mRNA vaccines against future pandemics associated with coronaviruses and other emerging infectious diseases.

The COVID-19 pandemic began in Wuhan province, located in China, and was caused by the SARS-CoV-2 coronavirus. Within a few months, the virus had already spread throughout the world, leading to a major global public health concern. To date, more than 770 million cases of infection and more than 6.9 million deaths have been recorded worldwide. These high infection numbers demonstrate the importance of diagnostic methods capable of identifying the presence of the virus in the first days of infection to guide public policies on containment measures to prevent its further spread. For this initial phase, the recommended method is RT-qPCR, currently considered the gold standard. However, this methodology presents some limitations such as high production cost, need for refrigeration, long execution time of the technique in addition to the need for specialized labor and equipment. Consequently, the need for refrigeration during transport and storage limits its wide distribution and use in various environments, especially those far from urban centers that do not have an adequate public health structure. Therefore, this study aims to develop a sensitive and easy-to-use diagnostic method, free from enzymatic activity, specialized equipment, labor and refrigeration. Relying on ribozymes for the recognition and cleavage of viral RNA and metastable DNA hairpin molecules with fluorophores at their ends, capable of performing hybridization chain reaction followed by fluorescence resonant energy transfer (FRET-HCR) for identification of the virus. The results indicated that two engineered ribozymes were able to identify and cleave the in vitro transcribed target of viral RNA and the DNA hairpin molecules were efficient in HCR reactions and the fluorophores in FRET reactions, with the best results showing fluorescence intensity 1.34 ± 0,11 times higher than the negative control, thus highlighting the potential of the proposed method. Therefore, this study served as a proof of concept for the joint use of ribozymes and DNA hairpin with fluorophores in FRET-HCR reactions to detect SARS-CoV-2.

An endo-1,4-beta-xylanase from Humicola grisea var. thermoidea belonging to the GH11 family was obtained by expression in Pichia pastoris and named HXYN2. This enzyme has 227 amino acids and a molecular mass of 23 kDa and is thermally stable, with greater activity in the range of 40-50 °C and pH 5.0-9.0, and optimum temperature and pH of 50 °C and 6.0, respectively. In this pH range, HXYN2 presents a stable structure with slight conformational changes, indicated by fluorescence emission spectra with slight displacement of the emission bands, consistent with tryptophan residues exposed to the solvent, and changes in the side chains of tryptophan residues and/or of amino acid residues that are in the tryptophan microenvironment. Circular dichroism (CD) spectra of the enzyme at pH 4.0, 6.0, 7.0 and 9.0 showed typical bands at wavelengths that correspond to proteins with β-sheet structures. The thermal denaturation curves obtained at pH 6.0, 7.0 and 9.0 showed two states of the protein, native and unfolded, with a melting temperature (Tm) between 50 and 60 °C. At pH 4.0, this point was identified between 65 and 70 °C, which suggests that HXYN2 is more structurally stable at acidic pH. HXYN2 activity was increased by 75% in the presence of the phenolic compound ferulic acid (FA), without altering its affinity for the beechwood xylan substrate. However, FA increased the Vmax, the catalytic efficiency and the turnover number of the enzyme. Data from ITC, fluorescence quenching and molecular docking showed that AF forms a complex with HXYN2 in the aglycone region, interacting with solvent-exposed tryptophan residues and other residues in this vicinity, through hydrogen bonds, hydrophobic and ionic interactions. Binding constant values for the HXYN2:FA complex are pH dependent and indicated moderate (at pH 7.0 and 9.0) to strong (at pH 4.0) affinity. In the presence of FA, the fluorescence spectra of HXYN2 showed pH-dependent changes in the position and intensity of the emission bands. The secondary structure of HXYN2 in the presence of FA was altered with a decrease in the α-helix and an increase in the β-turn and disordered structures. The thermal denaturation curve in the presence of FA showed a Tm of 54 °C, indicating lower structural stability of HXYN2 in the presence of this compound. HXYN2 crystals were obtained under different crystallization conditions in the screening step. The refinement of these conditions resulted in crystals of different shapes and sizes. Two of these crystals were taken to the National Laboratory of Synchrotron Light – Sirius for the collection of X-ray diffraction data. The diffraction data were processed, indicating that the space group P212121 (orthorhombic) and the presence of a dimer in the asymmetric unit. The crystallographic structure of HXYN2 was obtained by molecular replacement using the coordinates of the structure of endoxylanase from Thermomyces lanuginosus (PDB 1YNA) as a model.

The ability to remain dormant or in a latent state is an adaptation observed in several cells and organisms, in which cells reduce their metabolism, transcription and translation, to remain alive in conditions not ideal for their growth. Thus, they resume active growth when the environment returns to favorable conditions. Such adaptation is also observed in Cryptococcus neoformans yeasts, the fungus that causes cryptococcal meningitis in immunocompromised patients. In this work we analyze the mechanisms involved in the reactivation of C. neoformans during macrophage infection and whether yeasts induce changes in the cellular metabolism of this phagocytes. Furthermore, we infected immunosuppressed and immunocompetent WT and KO mice to analyze whether the presence of IFN-γ or NO prevented C. neoformans reactivation also in an in vivo context, as this pattern was previously demonstrated in vitro by our research group. We observed that it is not necessary for the fungus to have phagocytosis for it to reactivate, with only contact with extracellular compounds released by macrophages being sufficient, although yeasts reactivate at more expressive rates when there is phagocytosis. We also observed that the extracellular vesicles released by macrophages are important in this process of fungal reactivation. Furthermore, we observed that our model of dormant C. neoformans (DCn) was only able to reactivate in mice immunosuppressed with dexamethasone (C57Bl/6) deficient in IFN-γproduction or in immunodeficient mice (Balb/c nude), in which the fungus maintained its virulence and migrated to the CNS, where it resumed its proliferation. For analysis of cellular metabolism, we infected macrophages with DCn, Stat and heat-killed (HK) + 1% Stat for 24h. Next, we analyzed mitochondrial mass, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, glucose and fatty acid uptake by flow cytometry. Furthermore, we analyzed the respiratory profile of infected cells by oximetry and performed RT-qPCR to quantify important genes in cell metabolism. We observed that DCn doesn’t significantly alter most of the aspects analyzed regarding mitochondrial metabolism, characterizing a virulence strategy to prevent activation of the immune system, favoring its survival in the phagolysosome. However, all fungi induced extracellular acidification, indicating an increase in the performance of glycolysis by macrophages, while Stat prevents the increase in glucose uptake induced by treatment with LPS and IFN-𝛾, possibly representing an active strategy to avoid pro-inflammatory activation. Interestingly, DCn induces a greater uptake of fatty acids by macrophages pre-treated with LPS and IFN-𝛾, despite reducing the ability and dependence of macrophages to use this energy source. This demonstrates the preference of DCn for the use of lipids as an energy source and the ability to modulate the metabolism of the host macrophage in its favor. Finally, DCn modulated the expression of genes related to glucose and fatty acid metabolism, although to a lesser extent than Stat, showing that for phenotypic expression, a longer infection time should be necessary.

Many biological processes are regulated by interactions with small ligands. As occupancy states and sites number grow, it becomes harder for nowadays available methods to characterize them. This problem can be solved by considering a new approach that molecule interaction is a partition to a phase that corresponds to the protein interface. In this work, the two-site approach is used for obtaining a partition coefficient from flooding simulations. From the partition coefficient, many properties in different concentrations can be reconstructed, e.g. titration curves and tridimensional ligand densities. Furthermore, the partition process is separated into steps from a thermodynamical cycle and each step and concentration influence are analyzed carefully. The results obtained were satisfactory close when compared to independent systems. The development of the tools in this work has a very promising application potential, being very useful to a myriad of small molecules interactions with proteins.

Voltage-gated sodium channels (Nav) are responsible for the initiation and propagation of the action potential. These channels are distributed throughout the body and have nine different subtypes (Nav1.1-Nav1.9). Structurally, they have four transmembrane domains, with each domain having six α-helices (S1-S6) and two or three β subunits. Changes in the kinetics of these channels can occur due to mutations in the genes that encode ion channels, resulting in channelopathies (eg epilepsy and arrhythmia) and the effect of animal toxins. Epilepsy is a disease caused by abnormal electrical activity in the brain. It can be focal, generalized, focal and generalized combined, or unknown. Infections (viral or bacterial in the brain), autoimmune diseases, acquired causes and genetic mutations are the main etiologies. Among the mutations, we can mention those caused in the genes that express voltage-dependent ion channels (Na+ , K+ , Ca2+ and Cl- ), such as those in the SCN2A gene that encodes the Nav1.2 channel. The I1596S variant has been associated with epilepsy by causing benign neonatal infantile seizure (BFNIS); the V1627M variant is associated with childhood epilepsy with focal migratory seizures (EIMFS) and the L1650P variant has been described to be related to early infantile epileptic encephalopathy (EIEE). Furthermore, the scorpions of the Buthidae family are of greater medical importance due to the number and severity of accidents caused by their species in humans. The venom of these animals has also been shown to be important for its biotechnological potential due to the presence of ion channel modulators. Tst2 is a peptide obtained from the venom of the scorpion Tityus stigmurus that has a high percentage of identity with peptides that act on voltage-gated sodium channels (NaScTxs). The objectives of this work were to evaluate the effect of the pathogenic variants I1596S, V1627M and L1650P, associated with epilepsy, on the kinetics of mutated human Nav1.2 channels and to carry out the electrophysiological characterization of the Tst2 peptide in Navs channels. As a result, it was found that the channel kinetics with the I1596S variant had the probability of opening upon activation altered for more hyperpolarized potentials and the probability of opening upon inactivation altered for less hyperpolarized potentials, and the V1627M mutation caused the Nav1. 2 had a slower recovery from inactivation that was faster than the control. The electrophysiological characterization (Nav1.1-Nav1.7) of the peptide Tst2 (100 nM) was performed. For the probability of opening on activation, the most affected channel was Nav1.1 (with pre-pulse) and Nav1.7 (without pre-pulse). Nav1.3 was most affected in the probability of opening on inactivation and Nav1.4 in the recovery of slow channel inactivation. No changes were observed in the rapid inactivation of any subtype tested. The Nav1.2 channel is expressed in the central nervous system (predominance in glutamatergic neurons). That said, mutations in the SCN2A gene that generate gain of function can lead to abnormal brain electrical activity. Finally, the Tst2 peptide has a high percentage of identity with both alpha and beta peptides. After the electrophysiological characterization, only the beta toxin activity could be observed, with the observed effect of shifting the probability of channel opening due to the entrapment of the domain II voltage sensor in a pre-activated state. In addition, a reduction in the macrocurrent was observed due to the passage of some channels from the closed state directly to the inactivated state. The electrophysiological characterization of the kinetics of the L1650P variant has not yet been carried out and the partial sequencing of the Tst2 peptide is being carried out in collaboration with Prof. Lourival Possani's group from the Institute of Biotechnology at UNAM, Mexico.

Lignin is one of the main components of lignocellulose biomass corresponding to ~35% of its composition. This macromolecule is considered the main source of aromatic compounds in nature. In addition to natural lignin, this structure is generated as a residual by-product of industrial processes, such as pulping, and is normally destined for combustion for energy production. Lignin utilization as a raw material to produce products of industrial interest such as vanillin, guaiacol, and phenol could contribute to the implementation of biorefineries. Lignin needs to be deconstructed before it can be transformed into bioproducts with highadded value. White rot fungi and different genera of aerobic bacteria are known as the main degraders of lignin in nature. Bacteria are also able to deconstruct lignin anaerobically, however, the process is still poorly understood. In this sense, the present work explored the potential of bovine rumen bacteria to anaerobically deconstruct lignin. For this, rumen liquid samples were inoculated in culture media containing kraft lignin as a carbon source with or without the addition of yeast extract at 37 ºC for four days, under an anaerobic atmosphere for five passages. The consortia obtained through these passages were able to decolorize the culture media, and the maximum decolorization for the consortium cultivated with kraft lignin and yeast extract as a carbon source (KLY) was 40% and 29% for the consortium using lignin kraft (KL) as a carbon source. SEM, FTIR, and GC-MS analyses indicated changes in the lignin structure and the presence of aromatic compounds related to lignin degradation such as phenol, hydrocinnamic acid, homovanyl alcohol, and vanylmandelic acid. Diversity and taxonomy analyses based on barcoding sequencing (16S rRNA gene) indicated a decrease in bacterial diversity and enrichment of members of the genus Dickeya in the consortia KLY and KL compared to the rumen microbiota. Furthermore, a remarkable number of bacterial ASVs classified as Unknown were identified suggesting the presence of yet undescribed bacterial genera related to lignin degradation. Functional prediction analysis inferred the presence of eight metabolic pathways related to lignin metabolism in the KLY and KL consortia. Bacteria were isolated from these consortiums on solid media containing kraft lignin as a carbon source, though, they were unable to grow and discolor liquid culture media in the absence of glucose.

This study evaluated the prospection of bacterial strains with potential for the production of antimicrobial compounds from the enrichment of culture media with the supernatant of Paenibacillus elgii, which is abundant in antimicrobial peptides and other signal molecules. Soil samples were inoculated in these media in order to select only bacterial strains resistant to that supernatant and, therefore, with potential for the production of similar compounds. The search for strategies aimed at obtaining strains with biotechnological potential is necessary due to the limitations of classic cultivation techniques in prospecting new species that produce antimicrobials. It is also known that the repertoire of bioactive compounds established for known microorganisms has not yet been fully unveiled. There is also concern that antibiotic resistance developed by some bacteria, a fact that has been a growing threat to the effective treatment of infections caused by these microorganisms. Therefore, there is a demand for the discovery of new antibacterials to replace those that have become ineffective. The development of antimicrobials requires a series of steps, the first of which is the discovery and characterization of potential producers. The bacterial strains obtained through this protocol were evaluated for their antibacterial activity against Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa and were previously identified by Sanger sequencing of the 16S rRNA molecular marker gene. All strains showed antibacterial activity for B. subtilis and E. coli and only two of them for P. aeruginosa. Analysis of the 16S rRNA showed that the selected strains belong to genera already known for their biotechnological potential. They are: Paenibacillus, Pseudomonas, Enterobacter, Kitasatospora, Sphingomonas, Xanthobacter, Burkholderia and Methylobacterium. Bacterial strains isolated and with antibacterial activity against P. aeruginosa were named K002 strain and K003 strain and had their genotypes and phenotypes characterized. Their genomes were sequenced on the Illumina and Oxford Nanopore Technologies platforms. The taxonomic classification based on genomes showed that the species closest to the K002 strain is Kitasatospora xanthocidica (dDDH 32.8-37.8% and ANI 86.86%) indicating that it may be a new species. The K003 strain was confirmed to be a Methylobacterium radiotolerans (dDDH 90.3-94% and ANI 97.51%). The functional annotation of the genomes indicated the presence of sixty biosynthetic gene clusters related to secondary metabolism for the K002 strain and ten for the K003 strain. Most of them can be related to the antimicrobial potential of the strains. The presence of certain biosynthetic gene clusters in common with P.elgii may indicate the production of similar compounds between it and the K002 and K003 strains selected from its supernatant. The methodology described in this work allowed the isolation of distinct bacterial genera. The results show that K002 and K003 strains harbor several genes responsible for the biosynthetic production of secondary metabolites confirming their potential as a valuable source of bioactive compounds. Thus, this study showed the efficiency of using P. elgii supernatant in selecting microorganisms with potential for biotechnological application. Furthermore, the fact that the K002 strain is a species not yet described may indicate that the methodology favors the isolation of new bacterial species.

Proteomics is a research field that employs methodologies capable of identifying, quantifying and characterizing proteins and their post-translational modifications (PTMs). There are two main proteomic approaches based on mass spectrometry (MS), known as bottom-up (BU) and top-down (TD). The former analyzes peptides derived from proteolytic digestion, and the latter directly examines intact proteins. Recently, the integration of these two approaches has enabled improvements in the identification and characterization of proteoforms, defined as different molecular forms produced by a single gene. This study applied BU and TD proteomic approaches to investigate Corynebacterium glutamicum, an important industrial workhorse for amino acid production, and Trypanosoma cruzi, the causative agent of Chagas disease. In C. glutamicum, the TD proteomic analysis revealed new proteoforms of OdhI, an important regulator of glutamate production, and mepB, a peptidase related to cell envelope metabolism. Additionally, quantitative BU proteomic analysis of C. glutamicum, comparing control and glutamate-induced production conditions, unveiled uncharacterized proteins involved in the early stages of this process. Further, a study was initiated to integrate BU and TD approaches for C. glutamicum under the same conditions, suggesting the influence of proteoforms related to nitrogen assimilation and branched-chain amino acid metabolism in the glutamate production process. Moreover, using the BU approach for PTM identification and subsequently employing this information to TD proteomics resulted in a significant increase in the number of identified proteoforms. In the case of T. cruzi, the BU approach was applied for quantitative proteomic analysis of axenic and intracellular amastigote forms, revealing a large number of regulated proteins between conditions. Among the regulated proteins were trans-sialidases, Leishmanolysin-like peptidases, and proteins associated with kDNA, suggesting the importance of these proteins in the replication process of T. cruzi. Lastly, the TD proteomic analysis was performed for T. cruzi epimastigotes, suggesting the identification of proteoforms caused by amino acid residue substitutions in proteins related with responses to environmental stress. The preliminary analysis also indicated methodologies that can be optimized for a better characterization of T. cruzi proteoforms, such as the identification of PTMs using the BU approach and the use of these data for proteoform identification via TD

Gastric cancer is a global health problem, being among the 10 most common types of cancer worldwide. Despite recent advances in cancer therapy, over the years there has been little change in cure and survival rates in patients suffering from gastric cancer. Docetaxel (DTX), one of the most used drugs for the treatment of adenocarcinomas, has a mechanism of action in inhibiting mitosis and cell division, which may cause hypersensitivity, nephrotoxicity, fluid retention and neutropenia. However, its encapsulation in solid lipid nanoparticles (NLS) could reduce these problems, improving its effectiveness and directly transporting the drug to interact with specific tumor cells. NLS-DTX showed greater cytotoxicity against cancer cells (AGS), demonstrating an IC50 value lower than the concentration of free DTX, after 24 h of treatment, evidencing the efficiency of nanoparticles. Through morphological analysis, it was observed that the cells assumed a rounded shape and decreased cytoplasmic projections after treatment. NLS-DTX and DTX induced damage to microtubules, binding proteins and nucleus fragmentation, in addition to impairing cell adhesion, proliferation and migration. It also showed changes in tests involving cell organelles (mitochondria and lysosomes) and cell metabolism. The NLS did not show significant toxicity in the AGS tumor lineage in any of the assays, behaving similarly to the untreated control. Therefore, with the results of this work, it was possible to conclude that the association of DTX with NLS was efficient, presenting cytotoxic action in gastric adenocarcinoma cells, and favoring the use of this formulation in drug administration.

In recent decades, the number of cancer cases has been increased every year, as has the trend in the number of deaths. Among the types of cancer, breast cancer has become the one with the highest number of diagnoses in 2021. In addition, the classic therapies used for neoplasms are generally associated with various systemic toxic effects, which compromise the well-being of patients, especially in triple-negative breast cancers. In that context, docosahexaenoic acid (DHA), which is a polyunsaturated fatty acid from the omega-3 family and is obtained significantly from fish oil, emerges as a potential molecule for the treatment of neoplasms. The consumption of adequate amounts of this lipid is related to several benefits in neurological, cardiovascular, and metabolic pathologies, as well as cancer. This compound has shown promise for its protective effects against the development of neoplasms, inhibition of tumor growth, angiogenesis, and metastasis formation, as well as a decrease in inflammation. In addition, it has also demonstrated synergistic actions with some chemotherapy drugs, such as, decreased unwanted effects of this therapeutic approach. The action of different doses of DHA is still controversial in the literature and it is quite scarce regarding the metabolic alterations, due to supplementation, in tumors. Thus, the aim of this study is to evaluate the effects of omega-3 (DHA) supplementation at two different doses (500 mg/Kg and 2 g/Kg) in mice with breast cancer: carcinogenic parameters, metabolic and inflammatory profile. The results suggest that DHA has antitumor effects, reducing tumor growth, decreasing the levels of pro-inflammatory cytokines in white and brown adipose tissue (IL-6,TNF-a and IL-33), in blood plasma (1L1b) and in the tumor (IL-6), as well as was able to reduce the level of cytokines that act in the immune evasion process of the tumor (TGF-b), but without significant changes in tumor metabolism at the high dose. This was not the case with the low dose, which showed an increase in cytokines (IL-6, IL-33, and TGF-b) and did not slow tumor growth. The high dose in the animals without tumor, led to a reduction in serum triglyceride levels, an increase in some pro-inflammatory cytokines (IL-6 and IL-33) in the adipose tissues and a reduction in TGF-b in the breast, as well as a metabolic modulation with an increase in oleic acid and vaccenic acid in the breast tissue, which have antitumor and protective actions against the development of breast cancer. In contrast, the low dose led to a decrease in these fatty acids. Both doses showed no systemic toxicity, as well as no liver or kidney damage. Thus, this work characterized the impact of DHA supplementation at two different doses on carcinogenic, inflammatory and metabolic parameters, in which the differential role of the doses was evident, and the antitumor role of the high dose was manifest. In addition, the metabolic profile of the mammary tissue showed significant differences in response to the doses of DHA, in which an increase in fatty acids that can act against tumor development, such as oleic and vaccenic acids, was observed.

More than 90 genus are part of the Solanaceae family, with emphasis on the Solanum, considered one of the great branches of this taxonomic family with about 2,000 described species, distributed in tropical and subtropical regions of the world. The wide diversity of the genus Solanumrelates species with high nutritional, biotechnological, as well as medicinal value, among them, model plants for biotechnology, widely used in genomic and/or metabolic characterization studies, such as: the potato (Solanum tuberosum L.), eggplant (Solanum melongena), as well as tomato (Solanum lycopersicum). The tomato, in turn, is characterized by a fruit rich in vitamins and minerals, in addition to other important phytochemicals, standing out as one of the five largest commercial crops in the world, as well as for its biotechnological value, largely due to its lineage Micro-Tom, considered a model plant characterized by an optimized life cycle and to reduced physical stature, favoring its use in biotechnological investments. The susceptibility of tomato cultivation in the most diverse environmental conditions does not cancel out the damage caused by events of environmental instabilities, as well as by the continuous action of predators, a process that can reach the mark of up to 50% of total production losses in underdeveloped countries, being that, among the main predatory agents of tomato is the Traça-do-tomateiro (Tuta absoluta), native of South America and widely described as a generalized predator in different cultures, however, with a wide predatory target in tomato crops. In recent decades, frequent reports of T. Absoluta have been observed in regions not previously occupied by this pest, such as Asia, Africa, Europe and North America, with predatory damage caused by T. absoluta responsible for about 5% of total tomato losses worldwide. In turn, due to the challenges associated with the conventional control of T. absoluta, a process that limits its population expansion worldwide, the management strategies used, sometimes, are summarized in the repeated use of agrochemical agents, opening the way for environmental risks and public health associated with them. On the other hand, recent population monitoring studies have pointed to an increase in the emergence of pesticide resistance in populations of T. absoluta against the active ingredients commonly used in agriculture, emphasizing the need to search for alternative molecules with safety and efficacy. In this sense, among the main biomolecules of technological importance described in the last decades are the Bt protoxins, an integral part of the secondary metabolism of the bacterium Bacillus thuringiensis and holders of a vast literary content about their toxicity mediated by the dissolution of the toxin in intestinal pH in different ways insect larvae (pH 8.0). Among the widely studied Bt families are the Cry biomolecules, with high toxicity reported against diptera, coleoptera, nematode, as well as lepidoptera, and studies have frequently evidenced their cytotoxic capacity of these toxins against T. absoluta, with emphasis on α- Cry10Aa endotoxin, described in the literature with a focus on its toxicity against members of the Lepidoptera family, especially against moths. Based on these data and on the economic and cultural importance associated with the management of T. absoluta, one of the main predatory tomato pests in expansion worldwide, we propose here to obtain and validate the toxicity of a tomato carrying the cry10Aa transgene for the management of T. absoluta.

Chagas disease is a neglected disease caused by the protozoan Trypanosma cruzi, that is mainly transmitted by the insect vector commonly known as kissing bug, affecting over 6 million people worldwide. The symptoms are generally mild and acute, but approximately 30% of the cases evolve to the chronic phase where organs such as the heart and intestine are compromised. To this moment, there is no effective vaccine and the treatment is based on two drugs of small efficacy that are not capable of treating the chronic phase of the disease. The development of less toxic chemotherapeutic alternatives requires the search for target molecules that are vital for the protozoan and can be inhibited in a specific manner, such as enzymes. Thimet oligopeptidase (TOP) is a zinc-dependent metallopeptidase belonging to the M3 family that cleaves bradykinin, a vasoactive peptide that increases vascular permeability. In humans, TOP is one of the main intracellular oligopeptides processor, selecting antigens presented by the MHC-I in cases of viral infections and cancer. Due to scarce information on TOP in protozoans, little is known about the role of TOP in T. cruzi (TOPTc). However, TOPTc has been identified in the secretome and in the surface of the parasite in different phases of its life cycle, indicating a possible interaction with the host cell. In this context, the objective of this work was to express recombinant TOPTc in E. coli, perform its biochemical characterization and elaborate a structural analysis through molecular modelling, providing, thus, relevant information for the study of this enzyme, such as optimal pH, temperature and buffer, optimal concentration of ions, inhibitory effect and kinetic aspects of the enzyme. Among the main results, we observed that the enzyme is active towards the substrate Mca-Pro-Leu-Gly-Pro-DLys(Dnp), with Km of 24,89 µM in 50mM phosphate buffer pH 7.0. TOPTc activity is dependent on zinc and the enzyme is thermophilic. Inhibition is not affected by EDTA, but the enzyme is inhibited by Cpp-Ala-Ala-Phe-pAb, with an IC50 of 54,29nM. The 3D model generated by AlphFold showed the best parameters such as smaller MolProbity score and larger Ramachandran Favored score. Finally, anti-TOPTc specific antibodies colocalized in the flagellar pocket, where molecules are released to the extracellular milieu. This work represents the first study of TOP in protozoans and presents new observations related to this class of enzymes that will contribute to the development of new inhibitors molecules capable of disrupting the parasite life cycle and aid in the search for a treatment for Chagas’disease.

Many animals endure seasonal conditions associated with oxidative stress when oxygen is limited or unable to be extracted by organisms due to environmental conditions. Examples include frogs that freeze during the winter and stay in a hypometabolic state, and marine invertebrates from coastal environments that face aerial exposure during the tide cycle. Some well-known mechanisms that can explain hypoxia tolerance are (i) high constitutive levels of antioxidants, (ii) tolerance to oxidative damage and (iii) preparation for oxidative stress (POS). The latter has been demonstrated in 9 different animal phyla and consists in the upregulation of endogenous antioxidants during environmental stress, so the animal can be in a favorable redox balance during reoxygenation, when a high production of oxyradicals is expected. With the antioxidant system working consistently, oxidative damage (including DNA damage, protein oxidation, and lipid peroxidation) can be minimized. Currently, one of the biggest challenges related to POS’s research is its evolutionary origin, in which the most basal animals proved to be POS positive are two species of cnidarians. The Porifera phylum, whose origin is estimated to be more than 800 million years ago, is a good biological model for the evolutionary analysis of the POS mechanism. Therefore, this research aimed to verify whether the sponge Hymeniacidon heliophila, which daily goes through tidal aerial exposure, fits the POS criteria. The sponge H. heliophila is commonly found in the southern region of Brazil, inhabiting coastal environments, rarely seen submerged throughout the whole day. The species developed several adaptations that allow it to survive the functional hypoxia caused by the low tide, and one of such adaptations could be POS. Moreover, besides the hypoxic stressor, ultraviolet radiation (UV) has been recently suggested to be a trigger of POS. In this line of work, our research group analyzed POS in mussels Brachidontes solisianus, which also inhabit coastal environments. The findings indicate that B. solisianus uses POS in response to air exposure and UV radiation. Considering these two stressors, expeditions were made along the coast of São Sebastião (SP) to collect sponges H. heliophila. To verify the occurrence of POS in the field under totally natural environmental conditions, day and night samplings were made, with submerged and exposed sponges (high and low tides), on 3 different days. The sponges were scraped from the rocks, cleaned with water, and frozen in liquid N2, preserving the animal's biochemistry at the time of sampling. Then, whole-body homogenates were used to measure the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, total and selenium-dependent glutathione peroxidase, and glutathione transferase), levels of glutathione (total GSH, GSSG, and GSH/GSSG), and oxidative damage (TBARS and carbonyl proteins) of sponges under different environmental conditions (UV radiation, temperature, and relative humidity). We also compared the redox effect of air exposure during the day and night. This dissertation discusses the role of redox metabolism and POS as part of the biochemical adaptations of H. heliophila sponges to survive and cope with tidal and solar radiation stresses.

The use of microorganisms for the production of biomolecules has been attracting more and more commercial interest. During the development of this work, three microbial processes were evaluated for the production of lovastatin and Hyaluronic Acid and 2'Fucosyllactose. Lovastatin is a biomolecule naturally produced by some fungi, especially Aspergillus terreus. It is a molecule that acts as a competitive inhibitor of a key enzyme in the cholesterol pathway, 3-hydroxy3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase, contributing to the reduction of hypercholesterolemia. As Brazil has a vast biodiversity, the production of lovastatin in fungi of the genus Aspergillus isolated in Brazilian territory was evaluated, as well as the genomic diversity of these fungi. Thus, a strain was identified, URM 5961, which presented a production of 0.6 g/L and a high degree of genomic identity with the reference strain, ATCC 20542, which produces about 1.0 g/L of lovastatin. Hyaluronic acid (HA) is a biopolymer naturally produced by animals and some microorganisms. Due to its rheological characteristics such as high viscosity, hygroscopicity and biocompatibility, HA is widely used in ophthalmological treatments, facial fillers and in the formulation of cosmetics. Obtaining HA from natural sources was originally from animal tissue, such as rooster comb or, by bioproduction by pathogenic bacteria of the genus Streptococcus, but these sources have been replaced by heterologous production in other microorganisms, such as Bacillus subtilis. As Ogataea polymorpha has been gaining importance as an organism for heterologous expression, this work aimed to investigate its potential as a host for the production of HA. For this purpose, three strains were generated, EMB 102, EMB 103 and EMB 104, which differ in terms of the promoters used to guide the introduced genes. We confirmed the ability of O. polymorpha to produce HA, in which strain EMB 103 showed a production of 0.8 mg/mL. These results open the way for future investigations to improve the production of these compounds. The 2'Fucosyllactose (2'FL) is a human milk oligosaccharide (HMOs) consisting of three monosaccharides: glucose, galactose and fucose, corresponding to 80% of the HMOs. The 2'FL has immunomodulatory activity acting on inflammatory processes, decreasing interactions between pathogens and the gastrointestinal mucosa in newborns. The K2WΔSacII strain was constructed using the Kluyveromyces lactis yeast where the gmd and fcl genes from Escherichia coli and the WcfB gene from Bacteroides fragilis were inserted so that it was able to produce 2'FL using lactose and hydrolyzed açaí seed as a substrate. mostly by mannose. The objective of this work was to characterize the K2WΔSacII strains in terms of 2'FL production and improve this production by disrupting the lac4 gene that encodes β-galactosidase. Possible reasons for this not to have occurred, in addition, the disruption of the lac4 gene was performed, confirmed by the expected phenotype of the yeast losing the ability to grow on lactose as the only carbon source. This opens the way to proceed with the project, aiming at the production of 2'FL in K. lactis.

Lignocellulosic biorefineries are sustainable industrial facilities that use agricultural waste as raw material to produce higher value-added productssuch as biofuels and chemical “building-blocks”. One of the major costs of biorefineries is the enzymes needed to break down the plant cell wall into fermentable sugars. These enzymes are obtained from microbial sources such as filamentous fungi. The current industry standard for enzyme production are fungal monocultures. This technique produces limited cocktails, as no organism is capable of secreting all the necessary enzymes in sufficient quantities. An alternative to be explored is to simulate the degradation of organic matter that occurs in nature and to use more than one filamentous fungus in the enzymatic production, a practice called cocultivation. In this work, the enzymatic characterization of Aspergillus brasiliensis was performed and its co-cultivation with the fungus Trichoderma reesei RUT-C30 was evaluated using sugarcane bagasse as carbon source. The resulting enzymatic cocktails were characterized regarding the effect of strain inoculation time and the effects of pH and temperature on enzymatic activities. The results show that the profile of each enzymatic extract is highly dependent on the type of culture and the order in which the participating fungi were inoculated. Some of the co-cultures, under certain conditions, reached higher activities than their respective monocultures for enzymes such as CMCase, pectinase, β-glucosidase and β-xylosidase.