PPGBIO - Doutorado em Biotecnologia
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Doctoral Thesis Trans-desidrocrotonina livre e encapsulada em sistema coloidal SNEDDS e avaliação farmacológica em modelos experimentais in vivo de diabetes mellitus tipo 1 e inflamação pulmonar(Universidade Federal do Rio Grande do Norte, 2025-01-30) Lima, Laís Rocha; Maciel, Maria Aparecida Medeiros; Arcanjo, Daniel Dias Rufino; https://orcid.org/0000-0001-7021-2744; http://lattes.cnpq.br/0537823822525075; http://lattes.cnpq.br/5360188002708095; http://lattes.cnpq.br/2665364140542291; Rizzo, Márcia dos Santos; https://orcid.org/0000-0003-4276-3113; http://lattes.cnpq.br/9049791992923270; Lima, Aurea Echevarria Aznar Neves; http://lattes.cnpq.br/1879077396134052; Medeiros, Caroline Addison Carvalho Xavier de; https://orcid.org/0000-0001-9224-2434; http://lattes.cnpq.br/2982271986555450; Lima, Waldenice de Alencar Morais; http://lattes.cnpq.br/2575667613995470Croton cajucara Benth is used in folk medicine in the Amazon region of Brazil to treat diabetes, diarrhea, fever, jaundice, hepatitis and malaria, among other indications. In this study, the diterpene 19-nor-clerodane trans-dehydrocrotonin (t-DCTN) isolated from the stem bark of C. cajucara was coencapsulated with the oil-resin from Copaifera reticulata Ducke (OCPR), into a self-nanoemulsifying drug delivery system (SNEDDS) to evaluate its efficacy in an experimental model of type 1 diabetes mellitus (T1D) as well in the pulmonary inflammation by lipopolysaccharide (LPS) from Escherichia coli O55:B5, on biochemical and oxidative stress parameters. For this purpose, hematological and biochemical parameters, oxidative stress markers and histopathological analysis were determined. Rattus norvegicus Wistar animals were kept under temperature conditions (24±1 ºC), light/dark cycle (12 h), with free access to food. After fasting (12 h), they received 45 mg kg-1 (i.p.) of Streptozotocin (STZ) diluted in citrate buffer pH 4.5. For the lung inflammation (PI) model, the animals received LPS instillation (4 mg kg-1) (i.t.). The tested samples were: solution of t-DCTN (15 mg kg-1) solubilized in DMSO, which is free t-DCTN (unencapsulated compound, t-DCTN-L), and the nanoproducts SNEDDS-OCPR-DCTN resulting from the co-encapsulation of oil OCPR (0,5%) and t-DCTN (1 mg). Diabetic animals were treated (v.o.) with SNEDDS-OCPR-DCTN administered at doses of 0.1 mL 100 g-1 and 0.05 mL 100 g-1, as well as with solution of t-DCTN-L administered in a single dose (0.1 mL 100 g-1), and also with the SNEDDS-OCPR carrier system (containing 0.5% OCPR) administered in a single dose (0.1 mL 100 g-1). In the T1DM model, the groups treated with SNEDDS-OCPR-DCTN, tDCTN-L, and SNEDDS-OCPR showed weight variation, increased water and food intake, as well as increased relative weight of the liver and kidneys. A reduction in glycemic indices was observed in the groups treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1) and t-DCTN-L. Increased AST, ALT, CPK, CK-MB and urea were observed in all groups. The group treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1) showed elevated albumin and reduced LDH. Leukocytosis and neutrophilia were observed in the group treated with t-DCTN-L, and lymphocytosis in the group treated with SNEDDSOCPR. Histopathological changes were evidenced in the liver tissue of the group treated with SNEDDSOCPR-DCTN (0.05 mL 100 g-1), with Kupffer cell hypertrophy, and for the group treated with t-DCTNL, slight microvacuolization was observed in some hepatocytes, mild passive congestion and moderate Kupffer cell hyperplasia. Regarding the renal tissue, changes were observed in the group treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1) with moderate passive congestion, and mixed inflammatory infiltrate moderate. For SNEDDS-OCPR group it was observed mild nephrosis and moderate passive congestion. In the IP model, there was no variation in the weight of the animals, however, a lower leukocyte index was observed in the group treated with t-DCTN-L. Differential leukocyte analysis showed neutrophilia, as well as macrophages, more evident in the SNEDDS-OCPR-DCTN group (0.05 mL 100 g-1) and less evident in t-DCTN-L. Eosinophils were more evident in the goup treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1) and lymphocytes were less evident in the goup treated with SNEDDS-OCPR-DCTN (0.05 mL 100 g-1). There was an elevation of cardiac markers in the goup treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1), but it was less evident in the goup treated with t-DCTN-L. The group treated with SNEDDS-OCPR-DCTN (0.1 mL 100 g-1) showed reduced stress oxidative stress in cardiac (MDA) and pulmonary (CAT) tissue. Pulmonary histopathological analysis confirmed the occurrence of acute bronchiolitis, presence of neutrophilic and macrophagic inflammatory infiltrate, and a picture of serous interstitial pneumonia ranging from acute to moderate. The colloidal nanoproduct SNEDDS-OCPR-DCTN is suitable for oral ingestion, showed hypoglycemic results and pulmonary antioxidant activity. Based on these findings, it is being considered as an advanced colloidal system (SNEDDS) to optimize the bioavailability of t-DCTN co-encapsulated with copaiba oil (OCPR).Doctoral Thesis Caracterização da microbiota de uma amostra de petróleo do pré-sal(Universidade Federal do Rio Grande do Norte, 2024-08-30) Freitas, Júlia Firme; Lima, Lucymara Fassarella Agnez; Lanza, Daniel Carlos Ferreira; Dalmolin, Rodrigo Juliani Siqueira; Ramos, Pablo Ivan; Abrahão, Jônatas SantosThe COVID-19 pandemic has highlighted the need to develop pathogen surveillance systems for the early detection and management of zoonotic diseases and pandemic threats. In the context of genomic surveillance, some studies have shown that wastewater treatment plants are critical points for the dissemination of genes, such as antimicrobial resistance genes (ARG), due to the high density and diversity of microbial communities and the presence of mobile genetic elements that facilitate horizontal gene transfer. Thus, sewage can reflect the population's health, and understanding the microbiota present in this waste can provide essential information for public health authorities. In this context, the present study aimed to evaluate the virosphere and bacteriosphere of wastewater from the city of Natal to identify the main pathogens present, antimicrobial resistance genes, and virulence factors and their correlations to obtain biomarkers for monitoring and provide subsidies for public policies. For this purpose, the study was conducted longitudinally, with weekly collections over a year (June 2021 to May 2022) at three of Natal's main water treatment plants. A flocculation protocol was used to concentrate viral particles, and viral DNA and RNA extraction was performed using commercial kits to obtain the virosphere. The water samples were also subjected to total DNA extraction to evaluate the microbial community. The weekly samples were combined by month and sequenced via the NextSeq 1000 platform. Several bioinformatics tools were used to access the taxonomic profile, assemble contigs and genomes, and identify resistance and virulence genes. Co-occurrence networks were obtained via Bray-Curtis dissimilarity analysis. The virosphere was stable throughout the year, mainly in viruses infecting microorganisms or plants. An alternation in the representation of viruses that infect animals was observed. Bacteriophages associated with genera Escherichia, Pseudomonas, and Caulobacter bacteria were among the most abundant. The Odontoglossum ringspot virus was identified as a potential biomarker of RNA viruses of agricultural importance. Among the DNA viruses that infect animals, members of the Poxviridae family were observed in the samples. Co-occurrence network analysis identified potential biomarkers such as the Volepox virus, Anatid herpesvirus 1, and Caviid herpesvirus 2. Among RNA viruses that affect animals, the genera Mamastrovirus, Rotavirus, and Norovirus were the most abundant pathogens. Additionally, members of the Coronaviridae family, including SARS-CoV-2, exhibited high centrality in the co-occurrence network, connecting even with unclassified viruses. Furthermore, we confirmed the presence of SARS-CoV-2 by qPCR. We observed an association between Coronaviridae sequences, rainfall, and the number of reported COVID-19 cases. The rarefaction curve showed that all samples reached species stability regarding the metagenome. Phylogenetic diversity analysis did not reveal significant differences in the richness and evenness of communities over the months, suggesting a similar taxonomic composition throughout the year. However, differences in the proportion of some taxa were observed between samples. The genus Aliarcobacter was the most predominant in all samples. Non-metric multidimensional scaling (NMDS) analysis revealed four distinct groups indicating seasonality. Canonical correlation analysis (CCA) allowed the evaluation of the relationship between the taxonomic profile and variations in rainfall and temperature, indicating the same groupings. However, these environmental variables individually did not have a statistically significant impact on microbial composition. Among the viral communities identified in the metagenome, the genus Paundivirus predominated in most samples. Between January and March, crAssphage was the predominant virus. 221 antimicrobial resistance genes (ARG) classified into 16 categories were identified, with multidrug resistance genes being the most abundant. The genes msrE, mphE, sulI, and tetC were more abundant, with significant enrichment in January, July, and December. Virulence factors genes (VFG) analysis revealed 213 genes, classified into 14 categories, with the most prevalent adhesion factors. The genes tapT and mrkC were the most abundant, with significant enrichment in January and April. Co-occurrence networks revealed that ARG showed significant co-occurrence with viruses, while few ARG co-occurred with VFG. CrAssphage was the only virus that co-occurred with VFG. NMDS analyses confirmed the direct proportional relationship between ARG and viruses and the inversely proportional relationship between ARG and VFG. A total of 95 MAG (metagenomeassembled genomes) were obtained, among which 69 resistance genes, mainly related to multidrug resistance, were identified. Additionally, 33 MAG are those with the highest number of ARG. The most recurrent gene was the adeF gene (associated with tetracycline and fluoroquinolone resistance). Of the 33 MAG obtained, the genera Tolumonas and Rivicola were the most represented. In conclusion, this study's findings highlight the microbial community's complexity and stability in wastewater environments and the presence and dynamics of antimicrobial resistance genes and virulence factors over time. These advances will significantly contribute to our preparedness and response to future threats. Additionally, our study contributes to the knowledge of microbial dynamics, offering insights that can contribute to the direction of future public health policies and interventions and identifying potential monitoring biomarkers.Doctoral Thesis Integração de técnicas biotecnológicas para diagnóstico endometrial na infertilidade: videohisteroscopia, imunohistoquímica e análise molecular(Universidade Federal do Rio Grande do Norte, 2025-07-23) Torres, Mychelle de Medeiros Garcia; Lanza, Daniel Carlos Ferreira; https://orcid.org/0000-0002-1341-4814; http://lattes.cnpq.br/6851351991421755; http://lattes.cnpq.br/4362932674420752; Sousa Júnior, Francisco Canindé de; https://orcid.org/0000-0003-2042-4348; http://lattes.cnpq.br/3721560802857426; Moreira, Susana Margarida Gomes; Rego, Amalia Cinthia Meneses do; Cobucci, Ricardo Ney OliveiraThis thesis highlights the significance of inflammatory alterations and endometrial bacteriome imbalance in the pathophysiology of female infertility, particularly in cases of unexplained infertility. Employing an innovative approach, the research investigates the relationship between bacterial dysbiosis and endometrial inflammation through a combination of hysteroscopic, immunohistochemical, and molecular methods, with an emphasis on the application of advanced biotechnological technologies. The analysis of the endometrial bacteriome was enhanced by standardized protocols that considered essential factors such as aseptic collection, menstrual cycle phase, DNA integrity, and updated databases, thereby ensuring greater reproducibility and accuracy of the results. The observational study revealed a significantly higher prevalence of chronic endometritis in infertile women, underscoring the role of inflammatory components in infertility. Furthermore, the use of sensitive molecular techniques, such as nested PCR, demonstrated a high detection rate of multiple pathogens associated with dysbiosis, even in cases deemed negative by traditional methods. These biotechnological advances enable a deeper understanding of the uterine environment, facilitating earlier diagnoses and personalized treatment strategies. Thus, the integration of molecular and immunohistochemical tools represents a promising advancement in reproductive medicine, contributing to more precise and effective assessment of uterine alterations related to infertility.Doctoral Thesis Nanoformulações SNEDDS à base de Copaifera reticulata Ducke e trans-desidrocrotonina: caracterizações físico-químicas e avaliação do efeito antioxidante(Universidade Federal do Rio Grande do Norte, 2024-12-20) Oliveira Netto, José Robério de; Maciel, Maria Aparecida Medeiros; http://lattes.cnpq.br/5360188002708095; https://orcid.org/0009-0001-2249-203X; http://lattes.cnpq.br/6576018161315499; Nascimento, José Heriberto Oliveira do; https://orcid.org/0000-0001-6804-2854; http://lattes.cnpq.br/7033735079037677; Lima, Waldenice de Alencar Morais; http://lattes.cnpq.br/2575667613995470; Lima, Aurea Echevarria Aznar Neves; http://lattes.cnpq.br/1879077396134052; Magalhães, Nereide Stela Santos; https://orcid.org/0000-0002-2576-4213; http://lattes.cnpq.br/1063790618901917Among the herbal resources of Brazilian biodiversity, stand out the natural products copaiba oil extracted from species of the genus Copaifera and the bioactive 19-nor-clerodane transdehydrocrotonin (t-DCTN), obtained from Croton cajucara Benth. The main objective of this research was to develop a colloidal SNEDDS formulation based on Copaifera reticulata Ducke copaiba resin oil (OCPR), as a carrier system of the bioactive t-DCTN. Chromatographic analyses were performed for C. reticulata (GC-MS) and the hydroalcoholic extract from stem bark of C. cajucara, for isolation of t-DCTN. The chemical structure of t-DCTN was analyzed by spectroscopic methods (IR and 1H NMR). The SNEDDS-OCPR system, free of cosurfactants containing copaiba oil (C. reticulata) and Tween 80, was prepared using a ternary phase diagram based on the determination of the maximum solubility of the active matter (surfactant) in the aqueous and oily phases, by means of mass titrations. The diterpene tDCTN (1 mg) solubilized drop by drop with SNEDDS-OCPR by mechanical stirring under heating (40 oC to 55 oC), formed the nanoproduct SNEDDS-OCPR-DCTN (1 mg/mL). The formulations were evaluated in in vitro experimental models for antioxidant [reducing activity equivalent to ascorbic acid (CAT), reducing power, chelating activity of copper ions and hydroxyl radical scavenging. The encapsulation efficacy of t-DCTN was evaluated by the minimum and maximum solubility method, and quantification was performed by UV-visible (λmax 238 nm). Transmission electron microscopy (TEM) analysis of the SNEDDS-CO carrier showed the presence of spherical nanoparticles with an average size between 80 nm and 70 nm. The particle size, PDI and Zeta potential analyses showed, respectively, mean values of 11.66 nm, 0.17 and -3.85 for SNEDDS-OCPR, and 11.29 nm, 0.10 and -3.44 mV for SNEDDSOCPR-DCTN, indicating that there was a small and uniform distribution of the nanodroplets in both systems. The in vitro kinetic profiles of encapsulated t-DCTN (SNEDDS-OCPR-DCTN) were evaluated using the dialysis technique, providing t-DCTN release profiles according to the Fickian model, in which t-DCTN reached a maximum cumulative sustained release of 90.33 ± 0.01% (360 min.). The antioxidant effect of both SNEDDS-OCPR and SNEDDS-OCPRDCTN systems showed potential antioxidant results. Comparatively, SNEDDS-OCPR-DCTN showed strong CAT activity, as well as reducing power and sequestration of superior hydroxyl radicals. The SNEDDS-OCPR system showed better activity in the chelating activity assay. Under specific conditions, the drug delivery systems SNEDDS-OCPR and SNEDDS-OCPRDCTN formed fine nanoemulsions with significant efficacy for the bioavailability of copaiba oil co-encapsulated with t-DCTN, being considered as potential antioxidants. In this sense, the new nanobioproducts are bioavailable for future in vivo pharmacological investigations.Doctoral Thesis Bebidas probióticas de aveia enriquecidas com antocianinas de jambolão (Syzygium cumini): desenvolvimento, caracterização e efeitos da suplementação em um modelo animal com diabetes tipo 2(Universidade Federal do Rio Grande do Norte, 2025-03-14) Nascimento, Sara Sayonara da Cruz; Sousa Júnior, Francisco Canindé de; Passos, Thais Souza; https://orcid.org/0000-0003-2042-4348; http://lattes.cnpq.br/3721560802857426; https://orcid.org/0000-0002-9419-9027; http://lattes.cnpq.br/0415189674400748; Rezende, Adriana Augusto de; Serquiz, Alexandre Coelho; Porto, Ana Lúcia Figueiredo; Oliveira, Emanuel Neto Alves deThe aim of this study was to develop and evaluate the bioactive properties, stability and microbial prediction during storage, as well as the bioactive effects in an animal model with type 2 Diabetes mellitus (T2DM) of oat-based probiotic beverages enriched with anthocyanins from jambolan (Syzygium cumini). Initially, a narrative review of the literature was carried out, searching for in vitro and in vivo studies, with the aim of presenting the functional properties of probiotics in plant matrices. The scientific results compiled in the review indicated evidence related to the benefits of probiotics in plant food matrices and their association with bioactive compounds, showing adjuvant potential for the treatment of several diseases such as cancer, liver disease, obesity and chronic and inflammatory diseases. In addition, probiotic beverages based on water-soluble oat extract were developed, containing grape pulp, Lactobacillus acidophilus LA14, and enriched with 3% and 5% of the anthocyanin-rich extract of jambolan. Over 28 days of storage, probiotic observations above 9 Log CFU/mL were observed in all beverages, with higher prediction results in the formulations that used the anthocyanin extract (10.8–12.59 log CFU/mL), in addition to presenting predictions of 10 Log CFU/mL after simulated gastrointestinal digestion. The antioxidant activity by scavenging ABTS radicals improved from 8.08 to 134.75 µmol Trolox/mL after storage, while αglucosidase inhibition increased from 16.24% to 52.37% with the addition of EA. The accessible sensory of the probiotic beverage enriched with 3% anthocyanin-rich jambolan extract was 67.7%. Finally, a beverage enriched with 3% of anthocyanin-rich jambolan extract was evaluated in the treatment of DM2 in an animal model, and the results indicated improvement in DM2 symptoms, such as reduction in blood glucose, glycated hemoglobin, diuresis, and water intake, in addition to promoting attenuation of pancreatic and intestinal damage and modulating the intestinal microbiota of animals, with increased production of short-chain fatty acids. These results indicate the bioactive potential of the developed beverages, which may be an option as a plant probiotic vehicle and adjuvant in the treatment of type 2 Diabetes mellitus.Doctoral Thesis Desenvolvimento e avaliação de um biscoito nutracêutico à base de folhas de moringa oleifera para cães(Universidade Federal do Rio Grande do Norte, 2024-11-26) Silva, Maria Vitória Serafim da; Padilha, Deborah de Melo Magalhães; http://lattes.cnpq.br/8944056115237337; http://lattes.cnpq.br/9593347648288673; Molina, Wagner Franco; Lima, Adley Antonini Neves de; Guzen, Fausto Pierdona; Cobucci, Ricardo Ney OliveiraCurrently, the relationship between the human and canine species has changed. Previously, the domestic animal was seen as a hunting companion and guard dog. Nowadays, it is a member of the family, creating a great emotional and affective bond between the person responsible and the pet. This fact has encouraged veterinary science professionals to invest in the Pet industry, focusing on making the lives of these animals healthier and longer-lasting. Therefore, one of the main ways to make dogs' lives healthier is by exploring food as an alternative to promoting health, thus creating nutraceuticals. Resistance to pathogens has been a challenge in veterinary medicine, and, as a result, there has been an increasing use of nutraceuticals to assist in clinical treatment. In this context, Moringa oleifera has stood out for being a medicinal plant abundant in nutrients and presenting some studies that demonstrate its antiinflammatory action, antidiabetic and antioxidant effects, potentially emerging as a new nutraceutical possibility in veterinary medicine. Therefore, the objective of this thesis was to develop and evaluate the effects of a nutraceutical biscuit based on Moringa oleifera leaves on anti-inflammatory responses in dogs. The biscuit was prepared and the nutrients evaluated according to the attached patent. For the experiment, 45 privately owned dogs (n=45) were used, regardless of breed and sex, aged over 1 year, with 25 dogs being used for the biscuit acceptance test and 20 for evaluating hematological and biochemical parameters. The blood of these animals for evaluation of blood counts and biochemical aspects was collected before the start of treatment with the nutraceutical biscuit and after 30 days of daily feeding with the biscuit (1 biscuit/day). In the present experiment, it can be observed that the levels of TGO and TGP were lower (p<0.05) after 30 days of treatment with the cookies (TGO: 28.7; TGP: 30.3) when compared to these levels before treatment (TGO: 45.8; TGP: 52.7). In view of the above, it is concluded that biscuits based on Moringa oleifera showed excellent palatability for dogs, with an adequate level of protein, 30.51%, and can be used in dog food, making pet food products with greater nutritional quality and nutraceutical function to increase immunity. Furthermore, the use of Moringa oleifera biscuits had an effect on the biochemical parameters TGO and TGP, before and after supplying the biscuits, suggesting a nutraceutical effect.Doctoral Thesis Transcriptômica da Covid-19: identificação de potenciais biomarcadores e mecanismos moleculares da gravidade da doença(Universidade Federal do Rio Grande do Norte, 2024-09-30) Oliveira, Thaís Teixeira; Lima, Lucymara Fassarella Agnez; https://orcid.org/0000-0003-0642-3162; http://lattes.cnpq.br/1083882171718362; https://orcid.org/0000-0001-9397-7061; http://lattes.cnpq.br/8383930401109308; Medeiros, Silvia Regina Batistuzzo de; Araújo, Joselio Maria Galvão de; Franco, Glória Regina; Santos, Ândrea Kely Campos Ribeiro dosDuring the COVID-19 pandemic, transcriptome studies were conducted to investigate potential mechanisms of immune response to SARS-CoV-2 and identify possible treatment targets, virus-host interactions, and disease biomarkers. However, much of this data remains underutilized, and some studies involve a limited number of evaluated patients. This has led to a wide variety of observed results, with few translating into future research or clinically applicable markers. The objective of this study is to identify differentially expressed genes, molecular mechanisms, transcriptional regulators, and potential biomarkers involved in the pathogenesis of severe COVID-19. To achieve this, the methodology was divided into three main approaches: (1) To enhance the identification of molecular targets and biomarkers, this work reviewed the literature on transcriptomics published during the pandemic and its key findings. Specifically, 203 articles reporting transcriptomic experiments conducted on SARS-CoV-2-infected patients and uninfected controls were analyzed. (2) Furthermore, multiple datasets from patients with severe and mild COVID-19 were analyzed in an integrated manner to identify potential targets or pathways associated with disease progression and discover biomarkers of severity. This analysis included three whole blood datasets and one dataset of leukocyte samples. Additionally, data from nasal swabs and peripheral blood mononuclear cells (PBMCs) were examined. (3) Finally, the highlighted genes were evaluated in the serum of patients with mild and severe COVID-19 to validate testable serum biomarkers. The literature highlights the dysregulation of interferon-stimulated genes (ISGs), cytokines, chemokines, and genes related to neutrophil activation. In contrast, integrated reanalysis revealed that patients with severe COVID-19 exhibit overexpression of genes encoding extracellular exosome proteins, endomembrane system proteins, and neutrophil granules. Key targets such as S100A9, LY96, GAPDH, RAB1B, NFKBIA, and CD63 were identified as playing significant roles in the cellular response to infection. Conversely, severe COVID-19 patients show downregulation of genes encoding components of the T-cell receptor complex and nucleolar proteins, including TP53, IL2RB, and NCL. On the other hand, patients with severe COVID-19 present downregulation of genes encoding components of the T-cell receptor complex and nucleolar proteins, including TP53, IL2RB, and NCL. Furthermore, prediction analysis of master regulators—considering the sets of up- and downregulated genes—identified SPI1 as a transcription factor associated with upregulated genes, while TP53, MYC, and MAX were found to play critical roles in regulating the expression of downregulated genes during COVID-19. The validation of testable RNA targets in serum revealed that S100A9, GAPDH, and NFKBIA RNAs are promising biomarkers for assessing the prognosis of severe COVID-19. Notably, several of these genes had not been cited in prior studies, underscoring the novel contributions of this work. These findings offer valuable insights into the molecular pathophysiology of SARS-CoV-2 infection. Additionally, identifying transcriptional factors that regulate the expression of genes involved in infection and the inflammatory response may be pivotal for advancing our understanding of the disease and for the development of new therapeutic targets.Doctoral Thesis Nanobioformulações do tipo SNEDDS à base de Anacardium occidentale L. e avaliações farmacológicas(Universidade Federal do Rio Grande do Norte, 2024-01-29) Araújo, Leonardo Bruno Aragão de; Maciel, Maria Aparecida Medeiros; http://lattes.cnpq.br/5360188002708095; http://lattes.cnpq.br/6033875033893673; Padilha, Deborah de Melo Magalhães; Lima, Aurea Echevarria Aznar Neves; Arcanjo, Daniel Dias Rufino; Paiva, Weslley de SouzaColloidal systems increase the bioavailability of drugs and are largely used as an important strategy in the protection and directed delivery of bioactives compounds from synthetic or natural origin. In this sense, the therapeutic efficacy of the medicinal species Anacardium occidentale Linn. (AO) was evaluated in a nanobiotechnological study in which in vitro (antioxidant) and in vivo (pulmonary anti-inflammatory evaluation) experimental models were accessed. In that, the hydroalcoholic extract (EHA-AO) and a fraction rich in water soluble tannins (FT-AO), obtained from the stem bark of this vegetable, were loaded into a colloidal carrier SNEDDS-type system (selfnanoemulsion drug delivery system). In this context, stands out the reduction of administered therapeutic doses, reduction of toxicological risks, maintenance of the constant level of the drug in the blood (by controlled release), and prolonged efficacy of therapeutic effects, widely attributed to the colloidal systems. The choice of the carrier SNEDDS-type, consist in the fact that SNEDDS systems shows stability under water dilutions, variations of temperature and/or pH, and are effective in the controlled release (slow and prolonged), samples loaded at low concentrations, for example, can be administered orally and keep its activities. The experimental part of the study was carried out in different stages, such as: i) bromatological and microbiological analyses of the regional raw material (stem barks of A. occidentale); ii) phytochemical procedures for obtaining the EHA-AO and FT-AO samples; iii) solubility analyses and development of the polar colloidal nanosystem O/A (oil in water) with characteristic SNEDDS, cosurfactant free; iv) application of the formulations SNEDDS-EHA-AO and SNEDDS-FT-AO in the experimental in vitro oxidation tests, as well as in an in vivo experimental model of inflammation (pulmonary emphysema in Rattus norvegicus through orotracheal papain instillation), via oral administration. The SNEDDS carrier was prepared with Tween 80 (surfactant phase), a food-based vegetable oil containing the following constituents: vitamin E, polyunsaturated, saturated fat, and a mixture of the acids oleic, linoleic and linolenic. The SNEDDS-white system (free from the samples of the vegetable A. occidentale), after physicochemical characterization (pH, surface tension, CMC, viscosity and droplet diameter), satisfactorily solubilized the samples EHA-AO and FT-AO. The antioxidant potential of the nanoproducts SNEDDS-EHA-AO (10 mg mL-1 ) and SNEDDS-FT-AO (2.5 mg mL-1 and 5.0 mg mL-1 ) was evaluated by using in vitro experiments with significant results observed by the following tests: reducing activity equivalent to the ascorbic acid, chelating activity of the ions iron and copper, as well as the sequestration of hydroxyl and superoxide radical. The SNEDDS-FT-AO system did not show cytotoxicity to healthy cells of the RAW, Vero and L-929 strains. The in vivo pharmacological study of the SNEDDSEHA-AO and SNEDDS-FT-AO systems, in an experimental model of lung inflammation, as well as subsequent evaluation of histopathological analyses, showed that the SNEDDS-FT-AO formulation presents the best anti-inflammatory result. These findings, together with preliminary results of the medicinal potential of Anacardium occidentale Linn., which has proven immunomodulatory, anti-inflammatory, and antiviral action, will enable the phytotherapeutic validation of the SNEDDS-EHA-AO and SNEDDS-FT-AO formulations against the Sars-CoV-2 virus, and, therefore, may contribute against the therapy against COVID-19.Doctoral Thesis Abordagens moleculares e citogenéticas na validação taxonômica de espécies da tribo Amphiprionini (Pomacentridae: Teleostei) comercializadas no Brasil(Universidade Federal do Rio Grande do Norte, 2023-09-28) Carvalho, Marcelo Moreira de; Molina, Wagner Franco; https://orcid.org/0000-0002-6695-0952; http://lattes.cnpq.br/8437464961129518; https://orcid.org/0000-0003-2173-6968; http://lattes.cnpq.br/7343103413719345; Padilha, Deborah de Melo Magalhães; Teixeira, Dárlio Inácio Alves; Motta, Jonas Henrique de Souza; Soares, Rodrigo XavierClownfish (Tribe Amphiprionini), originating from the Indo-Pacific and Red Sea, are among the taxonomic groups of greatest commercial interest in the marine ornamental industry. In Brazil, basic information about marine ornamental fish species, as well as genomic and cytogenetic characterizations, is limited, making it challenging to estimate the current diversity and origin of commercially traded species and hybrids, thus hindering their traceability and proper management. This work aimed to make a quantitative and qualitative survey of the main marine ornamental fish species traded in Brazil and contribute to the prospection of molecular and cytogenetic biodiversity of the main species of Amphiprionini in the national’s market. Samples from the most representative commercial species were obtained in specialized stores and ornamental fish husbandry in Brazil. Species and interspecific hybrids were ranked according to the volume of individuals sold, establishing their origin and taxonomic designation or popular names. The species were taxonomically validated through the sequencing of mitochondrial DNA strands (COI) and subsequent comparisons with sequences deposited in GenBank. Additionally, the seven most representative species were submitted to cytogenetic analysis using conventional methodologies (Giemsa, Cbanding and Ag-RONs staining). The market analysis from the year 2015 to 2021, gathered with genetic information from clownfish species provided a biotechnological basis for taxonomic identification, fisheries management and genetics’ improvement. Data indicated this fish group is the most popular among ornamental marine species in the Brazilian market. Data generated contribute to strengthening of national marine ornamental fish farming. Additionally, the results have an impact on biological conservation, through the expansion of captive production and the consequent mitigation of extractive exploitation in natural environments.Doctoral Thesis Identificação de sequências da subfamília Orthocoronavirinae em metagenomas e metatranscriptomas e análise da diversidade genômica do coronavírus humano NL63(Universidade Federal do Rio Grande do Norte, 2023-09-29) Silva, Giovanna Melo Martins; Oliveira, Riva de Paula; Rodrigues, Thiago Bruce; https://orcid.org/0000-0002-6092-6142; http://lattes.cnpq.br/6790385939188325; https://orcid.org/0000-0002-4584-6961; http://lattes.cnpq.br/1182271734264345; Lanza, Daniel Carlos Ferreira; https://orcid.org/0000-0002-1341-4814; http://lattes.cnpq.br/6851351991421755; Araújo, Josélio Maria Galvão de; Franco, Gloria Regina; Acácio, Stela Mirla da Silva FelipeThe development of high-throughput sequencing technologies in the last decade resulted in large repositories of raw sequencing data and metagenomic projects. Alongside, computational biology and bioinformatics became important allies and many taxonomic classification tools were created. The pandemic of COVID-19 (Coronavirus Disease 2019) has shown the importance of better investigating coronaviruses, specially human coronaviruses that impact health and public services. Among them, HCoV-NL63 is a seasonal human coronavirus that spreads worldwide, causes mild to moderate respiratory disease, and shares the same receptor for host entry as SARS-CoV and SARS-CoV-2. This study uses public databases of sequences to 1) compares two bioinformatic tools, Kaiju and Burrows-Wheeler Aligner (BWA), in identifying from Orthocoronavirinae viral sequences in human metagenomes and 2) conduct a comprehensive phylogenetic analysis of HCoV-NL63. 1169 human samples were randomly selected (from 3670), and taxonomic classification was performed with Kaiju (double-step analysis with different reference databases). 150 samples were found positive for CoVs using Kaiju, however, it was not possible to assembly any genomes. In addition, all 3670 samples were analyzed using BWA, with negative results for CoVs, except for one sample containing sequences from HCoV NL63. Our findings suggest that the investigated samples did not have coronaviruses, only conserved and similar sequences between organisms, which was overestimated by the Kaiju tool. On the other hand, BWA can be used to identify viromes in various sequencing products. In this study, we also used 173 publicly available spike genes sequences from HCoV-NL63 to perform phylogenetic analysis. Maximum likelihood analysis resulted in eight subgenotypes (A1, A2, A3, B1, B2, C1, C2, and C3), consolidating the lineage B division into B1 and B2. Subgenotypes B2 (20.1%), B1 (19.5%), and C3 (16.1%) were the most prevalent. Positive selection was found in S1 (V57, G96, N431) and S2 (V1177, E1206) residues. Seven non-synonymous substitutions were detected in the RBD region. Compared to other genotypes, genotype B has a remarkable number of amino acid substitutions in the S1 region. Our analysis also detected a high prevalence of recombination events within the S1 region (12–307 amino acids). Genotype A, which has the highest number of recombination events, shows a constricted period of emergence in both time and place, displaying a pattern of purifying selection. Our results highlight the importance of continuous epidemiologic and phylogenetic surveillance, which is strongly recommended in order to predict the evolution of future variants.Doctoral Thesis Hidroxiapatita modificada: estudos biológicos para avaliação da biossegurança e das propriedades osteogênicas(Universidade Federal do Rio Grande do Norte, 2023-09-29) Souza, Augusto Monteiro de; Medeiros, Silvia Regina Batistuzzo de; https://orcid.org/0000-0003-2431-0479; http://lattes.cnpq.br/5882662534904226; https://orcid.org/0000-0002-6269-5536; http://lattes.cnpq.br/7633811857140815; Uchoa, Adriana Ferreira; https://orcid.org/0000-0002-3822-6502; http://lattes.cnpq.br/6644671747055211; Menezes, Fabiano Peres; Granjeiro, José Mauro; Moreira, Susana Margarida GomesHydroxyapatite (HA) is a natural mineral compound found in bones and teeth and can be adapted as a widely used biomaterial in clinical and pharmaceutical applications. Studies show that ionic substitutes can be incorporated into HA, making it more similar to natural bone tissue and promising for bone regeneration. However, these modifications can alter the physicochemical characteristics of biomaterials, necessitating a comprehensive assessment of biosafety before large-scale production. This study aimed to investigate the safety and efficacy of HA-functionalized biomaterials for bone regeneration applications. Divided into five independent chapters, it addressed crucial aspects of biosafety and the regenerative potential of these materials. A systematic literature review was conducted to investigate whether nanostructured HA-based compounds are free of genotoxicity. In this study, it was found that genotoxicity of HA-based biomaterials concerning human health is underestimated in scientific literature. In general, needle-shaped HA nanoparticles tend to cause more DNA damage and chromosomal alterations compared to other forms of HA. Additionally, the results suggest that primary cells are more sensitive than cell lines in assessing the genotoxicity of these biomaterials. The biomaterials used in this study were characterized and produced at the Brazilian Center for Physical Research in Rio de Janeiro. In the second chapter, the cytotoxicity and mutagenicity of a biodegradable bone graft based on nHA, consisting of microspheres of HA nanoparticles encapsulated in alginate, with and without the incorporation of bioactive ions (CO3 2- , Zn2+, and Sr2+), were evaluated. The main results of these analyses were that cytotoxicity tests did not reveal significant adverse effects of HA microspheres in cell culture, and no relevant changes in chromosomal levels were observed in CHO-k1 cells after exposure to the different biomaterials. Moreover, there was no increase in the rate of reverse bacterial mutation, using Salmonella Typhimurium strains, after exposure to these biomaterials. In the third chapter, embryotoxicity tests were conducted in zebrafish to assess the safety of hydroxyapatite-based microspheres, including HA with substituted ions. There was no significant mortality (less than 20%), and no neurotoxic effects were detected. Furthermore, the biomaterials did not alter oxidative stress or negatively affect development. The fourth chapter focused on evaluating the biosafety and bone regeneration potential of HA nanoparticles. Preliminary results of the biosafety of HA nanoparticles at concentrations of 25, 50, and 100 µg/mL were investigated. We used mesenchymal stem cells obtained from umbilical cord tissue as a cellular model for this evaluation. Cytotoxicity was assessed through the MTT test, and osteogenic potential was analyzed by measuring alkaline phosphatase levels and alizarin red staining. In the final chapter, we assessed the biosafety of functionalized HA nanoparticles in zebrafish embryos and larvae at concentrations of 50, 100, and 500 µg/m. Additionally, we investigated the impact of these biomaterials on bone mineralization using techniques such as staining and fluorescence microscopy. Overall, the results obtained in the different chapters of this study highlight the biological safety of HA-based biomaterials, as well as their potential in osteogenesis at various concentrations.Doctoral Thesis Desenvolvimento e teste de um dispositivo para estimulação torácica externa com função de marca-passo e avaliação da eficácia da estimulação cardíaca mecânica(Universidade Federal do Rio Grande do Norte, 2023-07-28) Oliveira Neto, Nestor Rodrigues de; Fregonezi, Guilherme Augusto de Freitas; Fregonezi, Guilherme Augusto de Freitas; https://orcid.org/0000-0003-4938-7018; http://lattes.cnpq.br/2201375154363914; http://lattes.cnpq.br/3901786270968653; Soares, Bruno Lobão; https://orcid.org/0000-0003-4564-2288; http://lattes.cnpq.br/3124118595692286; Dias, Fernando Augusto Lavezzo; Pinheiro, Francisco Irochima; Belchior, Hindiael AerafThe transcutaneous pacemaker has been used as therapy in cases of bradycardia with hemodynamic impairment, but it has important limitations, due to the high energy required to promote myocardial activation, causing discomfort and muscle stimulation. Thus, alternative forms of artificial cardiac stimulation have been the object of studies. Mechanical stimulation is capable of causing cardiac activation and functioning as a pacemaker, similarly to activation by an electrical stimulus. A mechanical stimulus of a certain intensity can generate a mechanical deformation in the myocardium capable of initiating myocardial excitation, resulting in ventricular contraction. This work comprises two parts: the development and testing of a prototype electropneumatic device for transthoracic cardiac pacing in humans, and the performance of an experimental study aimed to assess the efficacy the of mechanical cardiac stimulation, comparing this form of stimulation with standard electrical stimulation, regarding cardiac activation, maintenance of ventricular command and induction of ventricular arrhythmia. Hearts from 10 adult toads (402 ± 101 g) of both sexes were partially exposed outside the body and randomly submitted to mechanical or electrical stimulation. Two pairs of copper electrodes were implanted to monitor and stimulate the heart. Mechanical stimulation was performed by means of a back-and-forth movement and impact of a 2 mm diameter metal rod on the external surface of the left ventricle (aiming at the lateral wall and apex). The electrical ventricular stimulation was provided by programmed temporary pacemaker generator in demand mode connected to two pacing wire leads. After performing the threshold measurements, each form of stimulation was maintained for five minutes, with an HR of 10 bpm above the baseline HR of each animal, maintaining the same energy and HR during each phase of the experiment. The electropneumatic device tested showed a linear correlation between the mechanical energy released and the pressure of the compressed air, providing increasing energy values within the predicted range for transthoracic cardiac pacing (considering a threshold of 0.04 to 1.5 J), reaching up to about 2.8J, with the maximum pressures provided by the hospital medical air network (average pressure of 5 bar). Data of the experimental study showed that direct mechanical stimulation (similarly to electric stimulation) can be maintained continuously by five minutes in toad hearts at low heart rate without loss of capture. Nevertheless, the maximum HR during mechanical stimulation before the loss of capture was close to baseline values and significantly lower than during electric stimulation in most toads, possibly because of different types of ion channels activated in mechanical and electrical stimulation. The occurrence of ventricular arrhythmias was similar in both forms of stimulation.Doctoral Thesis Interações agonísticas em pares de espécies inter e cogenéricos e diversificação citogenética na família Osphronemidae(Universidade Federal do Rio Grande do Norte, 2023-09-29) Faria, Camila Fernanda Almeida de; Molina, Wagner Franco; https://orcid.org/0000-0002-6695-0952; http://lattes.cnpq.br/8437464961129518; http://lattes.cnpq.br/5000402126927493; Motta Neto, Clóvis Coutinho da; Hoyos, Daniela Chemim de Melo; Padilha, Deborah de Melo Magalhães; Gomes, Veruska Dilyanne SilvaDoctoral Thesis Características clínicas associadas ao agravamento da Covid-19 e na predisposição ao desenvolvimento da Condição Pós-Covid(Universidade Federal do Rio Grande do Norte, 2023-10-17) Silva, Nayara Sousa da; Luchessi, André Ducati; https://orcid.org/0000-0002-2758-8102; http://lattes.cnpq.br/4420863418928278; https://orcid.org/0000-0002-3864-4106; http://lattes.cnpq.br/2002678643031631; Fregonezi, Guilherme Augusto de Freitas; https://orcid.org/0000-0003-4938-7018; http://lattes.cnpq.br/2201375154363914; Marques, Otavio Cabral; Braga Neto, Pedro; Cobucci, Ricardo Ney OliveiraBrazil has recorded approximately 37 million cases of COVID-19 as of January 2023. Beyond the acute manifestations, COVID-19 might progress to a chronic disease, known as Post-COVID Condition (PCC). This study describes the clinical profile of a cohort of patients infected with SARS-CoV-2 in 2020 and investigates health implications and quality of life two years after infection. In 2020, 704 patients diagnosed with COVID-19 were enlisted from hospitals and through social media postings. Male sex and diabetes during the acute infection displayed higher risks for disease severity (RR=1.76; 95%CI: 1.43 – 2.17 and RR=1.60; 95%CI: 1.28 – 2.00). The use of "COVID Kit" medications— antimalarials, azithromycin, and ivermectin—was significantly higher in severe and critical severity groups (p=0.010, p<0.001, p=0.010, respectively), but no significant impact on patient survival was observed. A total of 105 patients died due to COVID-19 and its complications. Between May and June 2022, 137 surviving individuals were surveyed concerning PCC symptoms. Persistent symptoms were reported by 56.9% of patients, with 70% still experiencing these symptoms two years post-infection. Women and individuals hospitalized during the acute infection phase faced a higher risk of experiencing symptoms after COVID-19 (RR=2.07; 95%CI: 1.47 – 2.94 and RR=1.53; 95%CI: 1.07 – 2.18). Respiratory disturbances, neurological effects, and body pains were the most frequently reported manifestations. At least 20% of individuals reported some degree of difficulty in performing daily activities. We identified that risk factors such as male gender and the presence of comorbidities were associated with COVID-19 severity, while the risk of developing PCC was linked to female gender, hospitalization during the acute phase, and the first SARS-CoV-2 infection. Vaccination against COVID-19 did not demonstrate an effect on the progression of PCC symptoms. In this context, our study is crucial in providing an overview of the Brazilian situation and guiding further studies, as well as the development of public policies aimed at addressing PCC.Doctoral Thesis Identificação e caracterização funcional de variantes genéticas regulatórias da expressão gênica na hipercolesterolemia familiar(Universidade Federal do Rio Grande do Norte, 2023-03-29) Araújo, Jéssica Nayara Góes de; Silbiger, Vivian Nogueira; https://orcid.org/0000-0002-9252-0278; http://lattes.cnpq.br/6121935907512568; http://lattes.cnpq.br/3356845980263437; Simabuco, Fernando Moreira; Navarrete, Luis Antônio Salazar; Martínez, María José Brión; Moreira, Susana Margarida Gomes; https://orcid.org/0000-0002-8333-3016; http://lattes.cnpq.br/7741233949116498Familial hypercholesterolemia (FH) is an inherited disorder of cholesterol metabolism, characterized by prolonged exposure to high serum concentrations of low-density lipoprotein (LDL) cholesterol, which leads to a significantly increased risk of early cardiovascular morbidity and death. The main causes of FH are variants found in genes involved in the LDL clearance pathway (LDLR, APOB and PCSK9), however, causal variants are not identified in a significant portion of clinically diagnosed individuals. Therefore, variants in regions that are not usually included in diagnostic panels, could be influencing the expression of these genes, and contributing to the phenotype. The present study aimed to identify and functionally characterize genetic variants located upstream of FH-related genes. 25 volunteers clinically diagnosed with FH but without confirmatory molecular diagnosis were selected for targeted sequencing of 3 kb upstream regions (Chr19:11086362-11089361; Chr2:21044074- 21047073; Chr1:55036476-55039475 - GRCh38) using the AmpliSeq for Illumina method and the Miseq Reagent Nano Kit v2. Computational analysis of sequencing results included pre-processing, mapping of the generated reads, variant calling, and visual inspection of alignments to confirm the quality of variant calling. Potentially regulatory variants were prioritized according to regulatory probability prediction algorithm, functional annotation with data from public databases, pathogenicity prediction algorithms, variant-related literature, and prediction of altered transcription factor binding. The effect of two of these potentially regulatory variants on the functioning of the promoters of their target genes was evaluated by luciferase reporter gene assays in HepG2 cells. 34 single nucleotide variants (SNV) identified in sequencing, passed the quality filters: 6 were upstream from LDLR, 15 from APOB and 13 from PCSK9. 5 variants were considered potentially regulatory and, consequently, good candidates for functional validation assays: rs36218923-T and rs538300761-T of LDLR; rs934197-T and rs9282606-A of APOB; and g.55038486A>G of PCSK9. In vitro assays revealed that rs36218923-T variant caused a significant mean reduction in luciferase activity (75.4% ± 2.9% SEM) compared to the construct containing the wild-type LDLR promoter, whereas the rs934197-T variant did not cause a significant difference in mean luciferase activity compared to the construct containing the wild-type APOB promoter. These results suggest that the rs36218923-T variant may be contributing to the decrease in LDLR gene expression and causing the FH phenotype. More studies corroborating these findings are needed to confirm the biological relevance of this variant.Doctoral Thesis Desenvolvimento de aditivos bifuncionais derivados do cardanol hidrogenado para aplicação em mistura diesel/biodiesel(Universidade Federal do Rio Grande do Norte, 2023-03-13) Nascimento, Tássio Lessa do; Maciel, Maria Aparecida Medeiros; Rios, Maria Alexsandra de Sousa; http://lattes.cnpq.br/5360188002708095; https://orcid.org/0000-0002-5250-314X; http://lattes.cnpq.br/2860667150743768; Santos, Everaldo Silvino dos; https://orcid.org/0000-0001-7384-1140; http://lattes.cnpq.br/4330639792072559; Lopes, Ada Amélia Sanders; Rodrigues, Francisco Eduardo Arruda; Santos, Hélcio Silva dosBiodiesel is used in the vehicular fleet through its insertion together with diesel, which has high oxidative stability when compared to this biofuel. However, in the diesel/biodiesel blend, instability to oxidation predominates, which in the long term can cause wear and/or corrosion in the mechanical devices of vehicles fueled with this combination. In this scenario, agents with antioxidant and lubricity properties stand out, in which a byproduct of the cashew nut processing industry, cardanol, is present in the Cashew Nut Liquid (CNSL). In the present study we aimed to evaluate the potential of hydrogenated cardanol, 3-pentadecylphenol (compound 1), and some of its derivatives as fuel additives. Tert-butyl chloride (2) was produced in the laboratory to be the alkylating agent to obtain 2-tert-butyl5-pentadecylphenol (3). The other additives synthesized were: 2-nitro-5-pentadecylphenol (4), diethyl-3-pentadecylphenyl phosphate (5), 3-pentadecylphenyl diphenyl (6), tri (3- pentadecylphenyl) phosphate (7), 2-tert-butyl-5-pentadecylphenyl diethyl phosphate (8), and 2-tert-butyl-5-pentadecylphenyl diphenyl phosphate (9). During the syntheses of these compounds, reproduced with modification, principles of green chemistry were employed. The reproduced compounds were characterized by Gas Chromatography Coupled to Mass Spectrometer (GC/MS) and by Infrared (IR) and Nuclear Magnetic Resonance (1H and 13C NMR) spectroscopies. The soy biodiesel used in the diesel/biodiesel blend in the ratio 8:2, was synthesized through the methyl route using the potassium methoxide catalyst, and was characterized according to the ANP Resolution No . 45 of 08/25/2014. The antioxidant activity of hydrogenated cardanol and its derivative compounds (2000 mg/kg), was evaluated using the DPPH• and Rancimat methods, and lubricity by the Four Ball technique. The results of the antioxidant analysis showed that all compounds stabilized the DPPH• radical, especially 3 (81.1), 1 (46.5), and 7 (43%) for the highest percentages of free radical reduction. In the Rancimat test, the compounds that showed the greatest increases in induction time were 7 (32.8%), 3 (31%), and 9 (31%). For lubricity, the 3- pentadecylphenyl diphenyl compound (6) provided the highest percentage (70.4%) of lubricity, observed as a result of its higher torque-reducing effectiveness when compared to the additive-free soy biodiesel. In a comprehensive way, the phosphorus additives (5, 7, 8 and 9) were the most efficient and represent a class of semi-synthetic eco-compatible compounds, therefore, they are inserted in the energy sector that seeks to develop efficient and low-polluting fuels, thus, if commercialized, they will add biotechnological value to the fuel industries.Doctoral Thesis Caracterização de marcadores moleculares do tipo SNPs e Microssatélites em Penaeus vannamei e proposição de painéis para estudos populacionais e de associação(Universidade Federal do Rio Grande do Norte, 2022-12-20) Vieira, Iasmim Santos Mangabeira e Silva; Lanza, Daniel Carlos Ferreira; https://orcid.org/0000-0002-1341-4814; http://lattes.cnpq.br/6851351991421755; Ribeiro, Karina; https://orcid.org/0000-0001-7992-9717; http://lattes.cnpq.br/0589962629835994; Theodoro, Raquel Cordeiro; https://orcid.org/0000-0001-5016-1046; http://lattes.cnpq.br/0977453259767928; Cesar, Aline Silva Mello; https://orcid.org/0000-0001-9921-214X; http://lattes.cnpq.br/6423577693155656; Lima, Juliana Gabriela Silva de; https://orcid.org/0000-0002-8009-6721; http://lattes.cnpq.br/1463822009424883Shrimp farming worldwide, and especially in Brazil, suffers profound impacts due to the lack of standardization in production processes and the occurrence of infectious diseases. In this sense, the study of the genome of the shrimp Penaeus vannamei and the development of new tools that allow the selection of animals with the vigor necessary for production is crucial for the success of the activity. Among these tools, the use of molecular markers and their association with a trait of interest has shown to be a promising strategy, especially considering that vaccines and treatments are not available for shrimp farming. However, information about molecular markers for P. vannamei is still quite fragmented and many markers are not properly characterized. Thus, this work aimed to identify and characterize in detail single nucleotide polymorphism (SNP) and microsatellite (STR) markers already described in the literature for P. vannamei, and based on this characterization, propose panels of markers with potential application in population and association studies. In Chapter 1, 512 SNPs associated with genes involved in disease resistance were identified and classified in detail. It was observed that most SNPs were present in Toll-like receptor proteins 1 and 3, large and small subunits of hemocyanin and anti-lipopolysaccharide factors 1 and 2, allowing to propose their use as targets in studies of association and genetic improvement of populations of P. vannamei In chapter 2, 306 polymorphic STRs were characterized in detail, in order to identify their location in the genome. Among these, 38.8% were considered perfect, with the highest prevalence of dinucleotides (45.3%). The number of uninterrupted repetitions ranged from 3 to 62, with the majority consisting of short repetitions. Putative sequences of 135 STRs were obtained and, after functional alignment, 62 of them were associated with coding genes (mRNA). The identified proteins are mainly related to binding activity to different molecules (53.5%) and catalytic activity (32.14%). Furthermore, 7 perfect STRs located in DNA coding regions were validated in vitro in a shrimp population. The average number of alleles per locus was approximately five, indicating that there may be different protein isoforms in the population. In conclusion, this work summarizes all the markers already described and characterized for P. vannamei and details panels of SNPs and microsatellites in coding regions. These markers have potential for use in population studies and, above all, for studies of direct phenotypegenotype association, increasing the study of genetic plasticity and genotype phenotype correlation in P. vannamei.Doctoral Thesis Avaliação do efeito da losartana e do probiótico Limosilactobacillus mucosae CNPC007 no modelo experimental de mucosite intestinal induzido por 5-fluorouracil em camundongos(Universidade Federal do Rio Grande do Norte, 2022-10-21) Oliveira, Maisie Mitchele Barbosa; Medeiros, Caroline Addison Carvalho Xavier de; Silva Júnior, Arnóbio Antônio da; https://orcid.org/0000-0002-7516-1787; http://lattes.cnpq.br/2593509584288129; https://orcid.org/0000-0001-9224-2434; http://lattes.cnpq.br/2982271986555450; https://orcid.org/0000-0001-7141-3708; http://lattes.cnpq.br/2771654404049896; Maciel, Maria Aparecida Medeiros; Araújo, Aurigena Antunes de; Wong, Deysi Viviana Tenazoa; Raimundo, Juliana MontaniIntestinal mucositis (IM) is a side effect associated with cancer treatment resulting from chemotherapy. IM cytotoxicity resulting from the use of 5-fluorouracil (5-FU) promotes damage to the mucosal epithelial structure and associated gastrointestinal symptoms that induce the patient to discontinue treatment. Currently, there is no efficient therapeutic protocol that improves the symptoms of intestinal mucositis, in this sense, to propose alternatives that aim to attenuate the symptoms presented by patients affected by IM, the objective of the present study was to evaluate the effect of losartan (LOS) and the protective effect of the probiotic (Pb) Limosilactobacillus mucosae CNPC 007 on 5-FU-induced intestinal mucositis in Swiss mice. 5-FU (450 mg/kg, i.p.) was used to induce intestinal mucositis on day 1. Experimental groups included: Saline Group, 5-FU Group, LOS Group (5, 25 or 50 mg/Kg) and Probiotic Group. Losartan was administered 30 min before 5-FU and the probiotic was administered 14 days prior to IM induction. These were euthanized with thiopental (90 mg/kg, i.p.) on the 4th day after induction and blood samples and intestinal segments were collected for further analysis. Blood samples were used for leukocyte count and jejunal segments were separated for enzyme immunosorbent assays for cytokine (TNF-α and IL-1β), oxidative stress (MDA and GSH) and quantitative polymerase chain reactions in time. real (qPCR) for TWEAK, Fn-14 and NF-κB p65 and MUC-2 genes. The animals with untreated IM showed greater intestinal damage in the jejunal portion, observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate, evaluated by histopathological analysis with scores 3 (range 2-3) and morphometry (p < 0.001 vs saline group). LOS 50 mg/Kg presented the best results for the initial analyses, being this the dose adopted for the evaluation of the study. Leukopenia and greater weight loss were observed in the animals with IM (Group 5-FU), compared to the Saline Group. The treatment of the LOS 50 and Probiotic groups significantly improved the appearance of the villi and crypts (p < 0,01), reversing the histopathological changes (scores 1, range 1-2) and morphometry, leukopenia, and weight loss (p < 0,05 vs 5-FU group), induced by 5-FU. There was a reduction in the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β cytokines for the LOS 50 and Probiotic groups. LOS 50 reduced oxidative stress markers and NF-κB p65, TWEAK and Fn-14 gene expression compared to the 5-FU group (p < 0.001). The probiotic reduced p65 NF-κB gene expression and increased MUC-2 expression compared to the untreated group (saline, p < 0,001). These results showed a beneficial effect of LOS 50 and Limosilactobacillus mucosae CNPC 007 on induced MI in mice. In conclusion, LOS 50 mg/Kg and the probiotic L. mucosae CNPC 007 showed promise in attenuating IM symptoms, considering that both treatments improved 5-FU-induced IM injury, reducing the expression of pro-inflammatory mediators. in evaluated jejunal segments.Doctoral Thesis Avaliação da atividade antioxidante de quitosana extraída de fungos e de seu derivado conjugado com ácido gálico através de testes in vitro e in vivo utilizando modelo zebrafish (Danio rerio)(Universidade Federal do Rio Grande do Norte, 2022-07-22) Paiva, Weslley de Souza; Rocha, Hugo Alexandre de Oliveira; https://orcid.org/0000-0003-2252-1221; http://lattes.cnpq.br/4651814546820796; https://orcid.org/0000-0002-2022-5659; http://lattes.cnpq.br/6853669083018775; Scortecci, Katia Castanho; https://orcid.org/0000-0002-4690-2785; http://lattes.cnpq.br/4808910380593455; Theodoro, Raquel Cordeiro; Tosin, Fernanda Fogagnoli Simas; Sabry, Diego de AraújoOxidative stress is a cause of numerous diseases in humans, this causes a constant search for new molecules that may have antioxidant capacity and prevent the emergence of diseases. The objective of this work was to extract chitosan from fungi (Chit-F) and synthesize a new molecule, conjugating it with gallic acid, which was called Chit-FGal, in order to potentiate its antioxidant action. After collection, isolation and identification of the fungus as being of the species Rhizopus arrhizus, it was possible to extract chitosan and after physical-chemical tests such as high performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) ), it was confirmed that the sample was chitosan, with a degree of deacetylation of 86% and low molecular weight, in addition, without the presence of proteins and phenolic compounds. After that, the synthesis of fungal chitosan + AG (Chit-FGal) was successfully performed, confirmed by the 4x increase in the amount of phenolic compounds in relation to Chit-F, as well as the appearance of a peak in the region of the aromatic rings in the NMR analysis. When the 2 samples were extracted/synthesized and characterized, the in vitro antioxidant tests were performed (Copper Chelation, Iron Chelation, Total Antioxidant Capacity (CAT), reducing power and superoxide radical scavenging), where the samples had activities in all the tests, however Chit-FGal obtained results at least 50% (p <0.05) higher, demonstrating the potentiation of activities when modifying the native molecule. When testing Chit-F and Chit-FGal in fibroblast cells (NIH/3T3), there was no decrease in cell viability measured by MTT ((3-(4,5- Dimethylthiazolyl- 2)-2,5-diphenyltetrazolium bromide) in any sample. , in addition, a protection test against damage caused by hydrogen peroxide was performed, where ChitFGal at a concentration of 0.05 mg/mL showed better protection. A protective effect of embryos against damage caused by 0.06 mM hydrogen peroxide (100% live embryos). Subsequently, 3 days after fertilization, regarding the production of reactive oxigen species (ROS), lipid peroxidation and cell death apoptotic rate of larvae treated with Chi-F and Chit-FGal post. In both protection rates against the 3 surveys. These results combine the excellent potential antioxidant of Chit-FGal, as well as this new molecule as a potential to treat various oxidative stress problems.Doctoral Thesis Infertilidade e reprodução assistida: mercado global, avaliação de tecnologias para seleção espermática e prospecção da microbiota seminal(Universidade Federal do Rio Grande do Norte, 2022-07-29) Aderaldo, Janaina Ferreira; Lanza, Daniel Carlos Ferreira; https://orcid.org/0000-0002-1341-4814; http://lattes.cnpq.br/6851351991421755; http://lattes.cnpq.br/4449032685703603; Morais, Danielle Barbosa; http://lattes.cnpq.br/1768180848750009; Cobucci, Ricardo Ney Oliveira; Oliveira, Rodrigo Vasconcelos de; Tilburg, Mauricio Fraga VanInfertility is defined as failure to obtain a pregnancy after 12 months of regular unprotected sexual intercourse, or as conditions that make the meeting of gametes incompatible. Infertility is considered a global health problem, the global market in assisted reproductive technologies (ART) moved around US$ 26 billion dollars in 2019 and is expected to reach US$ 45 billion by 2025. The main factors associated with this market, as well as the elements that can influence the efficiency of fertility-related procedures, are still little discussed, and in many cases unknown. In this context, this thesis aimed to characterize the main topics related to the global market in assisted human reproduction (AHR) and to deepen the research on two topics still little studied: the efficiency of the microfluidics procedure and the occurrence of pathogens in semen. The first chapter presents a scope review that aimed to characterize the RHA market. The topic modeling methodology was applied, which made it possible to identify the most discussed terms in peer-reviewed texts and gray literature related to the topic. Based on this review, it was possible to observe that the vast majority of the works address economic, regulatory and geographic aspects and that topics related to research, development and technical improvement, especially regarding in vitro fertilization (IVF) are not addressed. In this way, we observe the need to deepen the analysis of market elements that may be related to the increase in the efficiency of IVF in the technical field. This first chapter motivated the deepening of the investigation of technologies that are related to increasing the efficiency of IVF. Among the technologies mentioned above, the use of microfluidics for sperm selection (MFSS) is in vogue. In the second chapter of the thesis, we present the first meta-analysis with the objective of identifying potential benefits in the use of this device on the rates of clinical pregnancy and abortion in ART. The results indicate a slight increase in the clinical pregnancy rate and a decrease in abortion rates in relation to the sperm selection techniques by density gradient and swimup; however, these results did not show statistical significance. The most robust indicators of success in ART remain maternal age, ovarian reserve and quali-quantitative sperm analysis. In this context of deepening the knowledge about the less studied topics within the RHA, the third chapter of the thesis presents the first study that applied the shotgun metagenomics approach in seminal samples. Although still little studied, the occurrence of pathogens in semen can be considered a cause of infertility and the choice of technique is justified by the possibility of non-directed identification of the entire group of organisms in the sample. We used a pool of seminal samples provided by volunteers at the Reproductive Rights Unit at Maternity School Januário Cicco (MEJC). We found 71.3% bacterial domain, 27.6% eukaryotic domain and 1.1% viral community. These findings expand the current view on the functional microbiota and point to future investigations as to the role of non-culturable microorganisms in the reproductive system. In addition, the Herv families identified in seminal samples open a new line for studies with a functional and evolutionary perspective. All the data collected in this thesis contribute to new research fronts at market levels, improvement of methodologies and diagnosis of fertility-related diseases.