Colloidal 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 (self-nanoemulsion 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 is being 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 SNEDDS-EHA-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.

World concerns regarding environmental pollution and global warming have  strengthened the search for alternative solutions to the consumption of oil and its  derivatives. For this reason, biodiesel was introduced in the Brazilian energy matrix  through the National Program for Biodiesel Production and Use (PNPB), whose objective  is to implement its production and use it in a sustainable way, with a focus on social  inclusion and regional development, also aiming to reduce the dependence on imports of  fossil oil derivative, diesel oil. Biodiesel is used in the vehicular fleet through its insertion  together with diesel, which has high oxidative stability when compared to biodiesel.  However, in the diesel/biodiesel blend, the oxidation instability of biodiesel  predominates, causing long term wear and/or corrosion in the mechanical devices of  vehicles fueled with this combination. In this scenario, antioxidant agents stand out, from  which cardanol, a by-product of the cashew nut processing industry and is present in the  Cashew Nut Liquid (CNSL), is included. In this present project, the main objective was  to evaluate the potential of cardanol and some of its derivatives as fuel additives, with  antioxidant and lubricity properties. In that, eight LCC derivative additives (AD-LCC)  were synthesized, such as: 3-pentadecylphenol (compound 1), 2-tert-butyl-5- pentadecylphenol (compound 3), 2-nitro-5-pentadecylphenol (compound 4), diethyl-3- pentadecylphenyl phosphate (compound 5), 3-pentadecylphenyl diphenyl (compound 6),  tri (3-pentadecylphenyl) phosphate (compound 7), 2-tert-butyl-5-pentadecylphenyl  diethyl phosphate (compound 8) and 2- tert-butyl-5-pentadecylphenyl diphenyl  phosphate (compound 9). The AD-LCC/derivative compounds were characterized by Gas  Cromatography Coupled to Mass Spectrometer (GC/MS) and by Infrared (IR) and  Nuclear Magnetic Resonance (H1and C13 NMR) spectroscopies. The antioxidant activity  of AD-LCC/derivative products, at concentration 2000 mg/kg was evaluated by DPPH•  and Rancimat methods, in soy biodiesel synthesized in the laboratory and characterized  according to ANP Resolution No. 45 of 08/25/2014. All AD-LCC/derivative compounds  stabilized the DPPH• radical, from which compounds 1, 3, and 7 showed the highest  percentages of free radical reduction (46.5, 81.1, and 43%, respectively). In the test via  Rancimat, all AD-LCC/derivative compounds increased the induction time of soy  biodiesel, where the largest increases correspond to compounds 3 (31%), 7 (32.8%) and  9 (31%). The lubricity of the additives AD-LCC was evaluated by the Four Ball  technique, in the diesel/biodiesel mixture on the ratio 8:2, with satisfactory results,  exception for compound 4, who showed no influence on the lubricity regime in the  analyzed mixture. Comparatively, the AD-LCC/phosphorus derivatives were the most  efficient and represent a class of eco-friendly synthetic compounds, so if commercialized,  they will add biotechnological value to the fuel industries. 

The 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.

The 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.

Hydroxyapatite (HA) is a widely used biomaterial in clinical and pharmaceutical applications. Many studies have shown that various ionic substitutes can be incorporated into HA. This modification can produce a mineral composition more similar to natural bone tissue with promising biological characteristics for use in bone regeneration. However, these substitutions can alter the physicochemical properties of these biomaterials, making it important to assess biosafety before large-scale production and commercialization. The aim of this study was to investigate the safety and efficacy of functionalized HA-based biomaterials for bone regeneration applications. This investigation addressed important aspects, including the assessment of genotoxicity, cytotoxicity, embryotoxicity, and the ability to promote bone mineralization. To achieve this goal, this work was divided into five independent chapters, each addressing specific aspects of the analysis of biosafety and regenerative potential of these biomaterials. The first chapter of this thesis consisted of a systematic literature review with the purpose of investigating whether nanostructured compounds based on HA are free of genotoxicity. The research protocol was structured to address crucial questions related to the genotoxic profile of HA-containing biomaterials. In this study, it was found that the genotoxicity of HA-based biomaterials concerning human health is underestimated in the scientific literature. Both in vitro and in vivo models were employed to assess the genotoxicity of these biomaterials. Overall, needle-shaped HA nanoparticles tended 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 conventional cell cultures in assessing the genotoxicity of these biomaterials. These findings provided a detailed overview of the current state of knowledge regarding the genotoxicity of HA-based biomaterials, highlighting the need for further research for a comprehensive understanding of the risks associated with their use in biomedical applications. In the second chapter, the focus was on evaluating the cytotoxicity and mutagenicity of HA-based biomaterials, both those functionalized with bioactive ions (CO32-, Zn2+, Sr2+) and those that were not. The main results of this analysis were that cytotoxicity tests did not reveal significant adverse effects of HA microspheres on cell culture, and no substantial changes in chromosomal levels were observed in CHO-k1 cells after exposure to different biomaterials. Moreover, there was no increase in bacterial reverse mutation rate following exposure to these biomaterials. These results provided support for the use of these biomaterials in bone regeneration applications, with confidence in their biosafety. In the third chapter, we conducted embryotoxicity tests on zebrafish to assess the safety of hydroxyapatite (HA)-based biomaterials, including HA with substituted ions. There was no significant mortality (less than 20%) or neurotoxic effects detected. Furthermore, the biomaterials did not alter oxidative stress or negatively affect development. These results support the safety and utility of these biomaterials in medical device applications and bone regeneration. In the fourth chapter, our study focused on evaluating the biosafety and bone regeneration potential of HA nanoparticles. Specifically, we investigated the preliminary biosafety of HA nanoparticles at concentrations of 25, 50, and 100 μg/mL. We used umbilical cord-derived mesenchymal stem cells as a cellular model for this evaluation. Cytotoxicity was assessed through the MTT assay, while osteogenic potential was analyzed through alkaline phosphatase activity and alizarin red staining. These assays allowed us to determine the biological safety of these biomaterials as well as their potential to promote osteogenesis. In the final chapter, we assessed the biosafety of functionalized HA nanoparticles in zebrafish embryos and larvae. We used various concentrations (50, 100, and 500 μg/mL). We monitored larval survival and hatching rates and the presence of developmental abnormalities. 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 different concentrations. These findings have significant implications in the field of biomaterials, contributing to the advancement of bone tissue engineering and regenerative medicine applications.

Health biotechnology and regenerative medicine seek alternatives to circumvent chronic or degenerative diseases, including diseases related to bone metabolism and obesity, for which conventional treatments have yet to provide satisfactory solutions. Thus, scientists have been working on the development of treatments based on biomaterials, cells and biomolecules to respond to these problems. Seaweed is a source of new molecules with potential bioactivity. Within this wide variety of molecules, sulfated polysaccharides (SP), which are highly complex and heterogeneous macromolecules, have shown interesting biological activities. Among the different types of seaweed, green seaweeds are the least studied, although several biological activities have already been described for their molecules. With regard to activities in the modeling of cell differentiation, studies with SP of green algae are particularly scarce. Thinking about that, this work evaluated the osteogenic and antiadipogenic activity of SP-containing SP samples obtained from the green marine algae Caulerpa prolifera and Caulerpa sertularioides. Additionally, as an essential part of the evaluation of the biocompatibility of new molecules, the genotoxicity of samples with biological activity was also determined. The results indicate that the SP-rich samples of C. prolifera and C. sertularioides induce differentiation of human stromal stem cells in non-cytotoxic concentrations (<10 µg / mL), promoting an increase in the alkaline phosphatase activity (ALP) and mineralization of the extracellular matrix, without genotoxicity under the conditions tested. The sample containing SP isolated from C. prolifera was also shown to inhibit adipogenic differentiation of pre-adipocytes, in non-cytotoxic concentrations (<200 µg / mL), and to reduce the accumulation of total lipids and triglycerides in the C. elegans model. Taken together, these results highlight the potential of SP in the development of alternative therapies for problems related to bone regeneration and obesity.

Systemic chronic inflammation (SCI) and chronic wounds have been widely treated, but the adverse effects attributed to medications limit prolonged treatments. In this present study Croton cajucara Benth medicinal species was applied as an alternative therapy, by using in vivo models, aiming at to analyze its analgesia, inflammation and wound healing. In that, were used the non-volatil oil (so called fixed oil, FO) and the diterpine trans-dehydrocrotonin obtained from this herbal stem bark. After chromatographic studies, FO and t-DCTN were loaded into a colloidal self-nanoemulsion drug delivery system (SNEDDS-type) aiming at the carrier biocompatibility with FO and t-DCTN, as well as reductions of therapeutic doses and toxic risks, maintenance of the constant level of the drug in the blood (by controlled release of the SNEDDS systems), and prolonged efficacy of the systems therapeutic effects. Based on the polar colloidal O/A (oil in water) systems the formulation SNEDDS was prepared by using Tween® 80 and a vegetable oil of food use (in low concentrations), dispersed in a neutral aqueous medium (with high concentration). The target SNEDDS system was characterized by physicochemical analyses and showed droplet diameter with reduced scale (11 nm), and showed to be biocompatible with FO and t-DCTN. Since it is a system free of cosurfactant, reinforces the proposal of pharmacological application in prolonged periods, due to the reduction in the number of the carrier ingredients and also by applying minimized concentrations of the assayed bioactives from C. cajucara. The SNEDDS-derivatives were called SNEDDS-OFCC (containing 15 mg of fixed oil, rich in sesquiterpenes) and SNEDDS-DCTN (containing 5 mg of t-DCTN). These nanobioproducts were administered orally in experimental pain models through abdominal contortions and formalin tests. Treatment with SNEDDS-OFCC formulation showed 40.2% inhibition in central pain (neuropathic) and 42.8% in inhibition of peripheral inflammatory pain. Meanwhile, SNEDDS-DCTN in reducing abdominal contortions was effective in 76.7%, and in the formalin test inhibited 47.9% (neuropathic pain) and 52.6% (peripheral pain). In the models of inflammation (paw edema and peritonitis) dexamethasone (0.5 mg/kg, s.c.) was used as positive control. The inhibitor rates of leukocyte migration (by carrageenan-induced inflammation) showed significant reductions for the peritoneal cavity, with inhibitions 29.4% for SNEDDS-OFCC and 38% for SNEDDS-DCTN, in relation to the negative control group (vehicle). In the paw edema inhibition, 69.5% was observed for SNEDDS-OFCC and 89.7% for SNEDDS-DCTN. Based on these data, the SNEDDS-DCTN system was used in the experimental healing model, administered topically in Wistar rat lesions (7, 14 and 21 days). For each experimental group, the retraction index (morphometry) of the lesion and the tissue behavior (histopathological and immunohistochemical analysis) were observed. The rate of lesion retraction on the 14th day was quantitative (98.3%), compared to 43.7% for the 7th day, and also to the negative control group. The histopathological analyses sowed reduction in inflammation, with significant gains in cell proliferation and tissue maturation, compared to the control group. Since, SNEDDS-OF and SNEDDS-DCTN presented associated antinociceptive and anti-inflammatory action, as well as healing (SNEDDS-DCTN), so, they may meet a specific demand from patients who are on prolonged treatments (SCI-type) to combat pain and inflammation caused by skin lesions or chronic inflammations.

Oxidative 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  (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 Chit-FGal 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) and 0.5% alcohol (93% live embryos)  was obtained. acridine orange, where both Chit-F and Chit-FGal demonstrated protective  action against cell death caused by hydrogen peroxide and alcohol. These results demonstrate  the good antioxidant potential of chitosan extracted from soil fungi of the Caatinga biome  (Chit-F), as well as the enhancement of this action by conjugating this polymer with gallic  acid (Chit-FGal), which makes this new synthesized molecule promising for use as  antioxidants with the potential to avoid a series of problems caused by oxidative stress. 

Intestinal mucositis (IM) is a side effect associated with cancer treatment resulting from chemotherapy. The cytotoxicity of IM resulting from the use of 5-fluouroracil (5-FU) promotes  damage to the mucosal epithelial structure and associated gastrointestinal symptoms that induce  the interruption of treatment by the patient. Currently, there is no efficient therapeutic protocol  that improves the symptoms of intestinal mucositis, in this sense, in order to propose  alternatives that aim at the attenuation of probable in patients affected by IM, the aim of this  study was to evaluate the effect of losartan ( LOS) and the protective effect of the probiotic (Pb)  Lactobacillus mucosae CNPC 007 on 5-FU-induced intestinal mucosite in swiss mice. 5-FU  (450 mg/kg, i.p.) was used to induce an intestinal mucositis on the 1st day. 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 within 14  days prior to IM induction. These were euthanized with thiopentals (90 mg/Kg, i.p.) on the 4th  day after induction and blood and intestinal segments were collected for further analysis. Blood  samples were used to count leukocytes and jejunal segments were separated for enzyme  immunoabsorbance assays for cytokine dosage (TNF-α and IL-1β), oxidative stress (MDA and  GSH) and quantitative polymerase chain reactions in time real (qPCR) for the TWEAK, Fn14  and NF-κB p65 genes. Animals with IM without major intestinal damage in the jejunum  portion, observed by shortened villi, loss of crypt architecture and intense inflammatory cell  infiltrate, evaluated by histopathological analysis with scores of 3 (variation 2-3) and  morphometry (p < 0.001 vs saline group). LOS 50 mg / Kg presented the best results for the  initial analyses, this being a dose adopted for the evaluation of the study. Leukopenia and  greater weight loss were observed in animals with MI (Group 5-FU) compared to the saline  group. Treatment of the LOS 50 and Probiotic groups improved the appearance of villi and  crypts (p < 0.01), reversing as histopathological changes (scores 1, variation 1-2) and  morphometry, leukopenia and weight loss (p < 0.05 vs 5-FU group), induced by 5-FU.  Reduction in the levels of tumor necrosis factor (TNF) -α and interleukin (IL) -1β cytokines for  the LOS 50 and Probiotic groups. LOS 50 reduces oxidative stress markers and the expression  of NF-κB genes p65, TWEAK and Fn14 compared to the 5-FU group (p <0.001), showing a  beneficial effect on induced MI in mice. In conclusion, LOS 50 mg / Kg and the probiotic L.  mucosae CNPC 007 enhance the attenuation of IM symptoms, considering that both treatments  improved the IM injury induced by 5-FU, an expression of pro-inflammatory mediators in  segments jejunals marketed. 

Neurodegenerative diseases (NDs) are characterized by a progressive neuronal loss leading to motor or cognitive impairment. Although it is still not completely understood, NDs share many molecular mechanisms such as: oxidative stress, protein aggregation, deficiency of the ubiquitin–proteasome–autophagy system, mitochondrial dysfunction, impaired bioenergetics, dysfunction of neurotrophins and neuroinflammatory processes. The clinical observations that deficiencies in the nucleotide excision repair (NER) and base excision repair (BER) pathways cause human pathologies associated with neurological symptoms suggest that NER and BER might also play a critical role in chronic NDs. Here, we evaluated the deficiency of BER and NER pathways, especially EXO-3 and XPA-1, respectively, on the development of neurodegenerative phenotypes in Caenorhabditis elegans. EXO-3 and XPA-1 inhibition affected redox status by increasing ROS levels and up-regulating the expression of stress resistance related genes in a SKN-1 dependent manner. Similar results were found when APN1, XPC1 and CSB1 proteins were evaluated. EXO-3 and XPA-1 deficiency also activated the endoplasmic reticulum and mitochondrial unfolded protein response (UPR) and interfered in proteostasis as indicated by the reduced proteasome activity and proteasome subunits expression. Thesee alterations were associated with neurodegeneration of pan- and cholinergic-marked neurons. The neurodegeneration induced by EXO-3 and XPA-1 deficiency appears to be triggered by hyperactivation of the DNA damage sensor PARP-1 since this phenotype is rescued by PARP-1 inhibition. Inhibition of the apn-1, xpc-1 and csb-1 genes in transgenic animals for Mal de Alzheimer and Hungtinton's disease accelerated neurodegenerative phenotypes, the most marked affect were in animals treted with csb-1(RNAi).Together, these results support a model where deficiency of NER and BER pathways plays an active role generating a network of stress signals sufficiently strong to trigger neurodegeneration.

In the literature, in the last 40 years some test modalities have been developed to assess the resistance of respiratory muscles, especially inspiratory muscles. The tests and prototypes of devices have always sought to assess two aspects that are relatively distinct, such as fatigue and resistance. There are several gaps in the literature on the best way to assess the resistance of respiratory muscles. Despite the importance of the measure, the previous studies using different models, of assessed resistance of respiratory muscles were based on devices, or prototypes and with a low number of subjects, which makes interpretation of the results very difficult. Also in recent decades, significant progress has been made in understanding sleep-disordered breathing and its consequences. There is evidence that treatment with positive airway pressure, facilitating the activity of the respiratory muscles, improves and controls the disease, but there is no solid evidence that this efficacy is repeated in special populations, such as stroke sequelae. This work aimed to develop a device within the scope of Technological Innovation and carry out two other studies on one on Health Technology Assessment (ATS) and another methodological clinical scientific study. The development of the device is in the final stages for filing a patent. As products of this PhD work we present: a product related to Health Technology Assessment: (i) a systematic review scientific article with meta - analysis (published in the Cochrane Database of Systematic Reviews) and a second clinical article related to the theme (published in. Frontiers in Physiology). The device to be patented is a device capable of evaluating the resistance of the respiratory muscles through different protocols, while the first publication is about evidencing the effects of the use of positive pressure in stroke patients, as a way to facilitate respiratory muscle activity and breathing; the second article deals with the analysis of respiratory muscle resistance techniques, through resistance tests, as a predictor of respiratory function in patients with chronic obstructive pulmonary disease.

Advances in molecular, cellular and genetic levels have provided increasingly new discoveries that allow the gradual understanding of key mechanisms of cardiac activity and related pathophysiologies. In this perspective, several cell-signaling pathways are activated for the process of contractility, electrical conduction and vascularization to occur effectively. However, external or endogenous factors can signal significant changes in natural biological processes, leading to cardiac pathophysiology. Among the various diseases that affect the heart, Atrial Fibrillation, AF, is highlighted as one of the main types of arrhythmia most prevalent worldwide. The diagnosis is made by ECG, however the asymptomatology of many patients and the short duration of fibrillation hamper early detection, leading to late diagnoses. Transcriptomic analyzes show that non-coding RNAs play an important modulatory activity, such as miRNAs, which regulate gene expression of mRNA and play a promising role as potential targets for the diagnosis, prognosis and treatments of AF. The activity of miRNAs, in turn, can be regulated by other non-coding RNAs, including lncRNAs and circRNAs, which perform among other functions the activity of capturing miRNAs so that gene expression can be modulated, featuring an activity called competitive endogenous RNA. The activity of miRNAs in AF and of lncRNAs and circRNAs acting on the biological processes involved in this disease were part of the main objectives of this study, in order to obtain a greater understanding of the mechanisms with which they are associated. Scientific articles based on findings about these activities in AF, including the description of the main circulating miRNAs in AF in addition to the miRNAs associated with pathological processes related to this disease, were carried out through database searches. These articles allowed a greater conception about the importance of miRNAs in the pathophysiology of this disease and other associated conditions. Additionally, in an article related to the objective of this study, the expression of circulating miRNAs in the AF event was experimentally evaluated, characterizing some potential targets differently expressed in this condition. Based on these findings, the functional activity of one of the miRNAs differently expressed in this previous study, miR-133b, in the activity of cardiac cells, was demonstrated using in vitro techniques of inhibition, suggesting that this miRNA modulates the expression of some mRNAs expressed in the heart in cardiomyocyte models and that could be regulated by different lncRNAs and circRNAs. Complementarily, through search in database, the main lncRNAs and circRNAs involved in cardiac processes associated with the pathophysiology of AF were analyzed and described, demonstrating that these molecules play a crucial role in the pathological complexity of this disease, whether with a protective or stimulating activity of progression of AF.

Athletes and physical activity practitioners frequently consume protein supplements to achieve gains in muscle mass (hypertrophy) and strength, thereby improving performance. To do so, they make use of these supplements during and/or after exercise. In the case of practicing resistance exercises, a better post-workout recovery is also sought. Faced with this problem, the study aimed to develop and validate protein bars based on powdered coconut water (vegan and whey) as a supplement for physical activity practitioners by analyzing the efficiency and the acute effects of consumption through hormonal markers and injury enzymes muscular resistance training. Twelve physical activity practitioners aged 20-30 years, with experience in resistance exercises (bodybuilding) for at least two years, were trained three to five times a week, with a minimum duration of 30 minutes per day. The study was cross-over. Participants were divided into three groups: G1 - Control Group (commercial bar; 11 g protein); G2 - Group Treatment 1 (vegan bar based on powdered coconut water, 8.22 g protein); and G3 - Treatment Group 2 (whey bar based on powered coconut water, 10.52 g protein). One protein bar was ingested before training and other after training. The mean age of the 12 participants was 23.6 ± 3.2 years, with a body mass of 81.6 ± 14.3 kg, height of 174.5 ± 5.7 cm and BMI of 26.9 ± 4, 8 kg/m², being classified as overweight. On average, study participants performed strength training in their exercise routine at 5.2 ± 2.2 years. Regarding the hormonal serum levels, there were more relevant changes regarding insulin and free testosterone with 48 h post-training; IGF-1 and total testosterone levels remained constant at all times, regardless of the protein ingested. The LDH and CK muscle injury markers showed more marked changes only at 48 h post-training, with lower serum levels when ACP-Whey intake and similar levels between the commercial bar and the ACP-Moringa vegan bar. It is concluded that the vegan protein bar based on powered coconut water and moringa can be considered equivalent to the commercial protein bar. The ACP-Whey protein bar, based on powdered coconut water and Whey Protein Isolate, promoted a better post-workout recovery for those who practiced resisted physical activity.

The orange juice Citrus sinensis L. Osbeck is considered an excellent food source of several bioactive compounds with properties beneficial to human health due to its high content of flavonoids, carotenoids, sugars, minerals and fibers. Some epidemiological and interventional studies have provided evidence to support the inverse correlation between orange juice consumption and the occurrence of cardiovascular disease and cancer. In this study, we used two naturally enriched oranges, originated through spontaneous mutation, called Bahia and Cara Cara. These oranges are high in vitamin C and flavonoids; however, Cara Cara orange is distinguished by being rich in carotenoids (phytoene, phytofluene, lycopene and β-carotene). Considering that oxidative damage and increased neuroinflammation are critically related to pathogenesis and neuronal loss in neurodegenerative diseases, the neuroprotective effect of some components of orange has been of specific interest in the search for effective treatments for these diseases. However, there is still a lack of in vivo studies to show the therapeutic effects of orange juice in its full and complete form, since its compounds have already been shown to have positive antioxidant and neuroprotective effects. In this context, we used the nematode Caenorhabditis elegans which is a widely established model organism for animal behavior, physiology and genetics studies. The objective of this work was to evaluate and compare the antioxidant, neuroprotective and physiological changes related to the aging of the treatment with the pasteurized orange juices Bahia and Cara Cara in the C. elegans model. To perform the experiments, the animals were synchronized in the L1 larval stage and treated at 20 ºC in petri dishes with solid nematode growth medium (NGM), with different concentrations of Bahia orange or Cara Cara, seeded with Escherichia coli OP50 or E. coli HT115. The treatment with orange juice provided positive antioxidant effects in C. elegans, such as reduction in endogenous levels of reactive oxygen species (ROS), increased survival under standard and oxidative stress conditions, and increased expression of antioxidant reporter genes g- glutamyl cysteine synthetase (gcs-1), glutathione-S-transferase (gst-4) and superoxide dismutase (sod-3) and hsp-16.2 chaperonin. Regarding the neuroprotective effects, it was observed that the orange treatment delays the profile of paralysis induced by the superexpression of the Aβ1-42 peptide in the muscle of the C. elegans model for Alzheimer's disease and decreases the polyglutamine aggregation in the C. elegans model for Huntington's disease. To investigate whether stress response signaling pathways would play an important role in juice effects, ERO quantification, oxidative stress resistance, gst-4::GFP expression, paralysis, and longevity in animals with knockdown were performed. the transcription factors SKN-1, DAF-16 and HSF-1 through interference RNA (RNAi). The results showed that the effects provided by the treatment with Bahia and Cara Cara orange juices were mainly dependent on the SKN-1 and DAF-16 transcription factors, especially SKN-1. In relation to physiological changes related to aging, the treatment increased the life span, the body movements and the rate of pharyngeal beats. We concluded that orange juice treatments provided excellent antioxidant and neuroprotective effects and were able to improve physiological parameters related to aging, which are strongly involved in the quality of life of C. elegans with emphasis on Cara Cara orange juice induced significantly better responses, possibly due to the higher carotenoid content.

The present study aimed to produce the enzyme β-galactosidase (β-gal) using “coalho” cheese whey as biotechnological substrate by yeasts of the genus Kluyveromyces sp. and to evaluate processing strategies that enable its application in the food industry. In the first stage of this study, the co-production of β-gal and ethanol by submerged fermentation in different C:N concentrations was assessed. The yeast Kluyveromyces lactis NRRL Y-8279 presented better efficiency for co-production of β-Gal and ethanol. The maximum production of β-Gal was 21.09 ± 0.69 U/mL and ethanol 7.10 ± 0.09 g/L in 16 hours of cultivation. In the second stage, a 2² experimental design was proposed in order to develop a purification strategy to the enzyme. The parameters pH and ionic strength were evaluated in order to obtain a high purification factor without impairment in the yield. The higher levels of both parameters on study enhanced the purification factor of β-gal to 2.00, with greater influence of ionic strength on the FP response. The purified enzyme was submitted to electrophoresis that presented a band with molecular weight around 120 kDa, the enzyme of interest. In the third and final stage of the study, we analyzed the hydrolysis conditions of lactose in the “coalho” cheese whey with the immobilized form of β-Gal in 1% (w/v) sodium alginate. For the immobilization system, the immobilization efficiency reached 66% and high recovered activity was achieved. In addition, the immobilized form of the enzyme presented higher stability to pH and temperature changes. The immobilized enzyme had a slightly lower rate of lactose conversion (46%) when compared to the crude enzyme extract (53%). For the gastrointestinal simulations, around 40% of the enzymatic activity was preserved after 2 hours of exposure to simulated gastrointestinal environments. Overall, the results described here are promising for the industrial applications of β-galactosidase from K. lactis.

Crude oil extraction, transportation and use provoke the contamination of countless ecosystems. Therefore, bioremediation through hydrocarbon biodegradation or surfactants mobilization is an important subject, both economically and environmentally. Bioremediation research had a great boost with the recent advances in Metagenomics, as it enabled the sequencing of uncultured microorganisms providing new insights on surfactant producing and/or oil-degrading bacteria. Countless research studies are making available genomic data from unknown organisms obtained from metagenomics analysis of oil contaminated environmental samples. These new datasets are presently demanding the development of new tools and data repositories tailored for the biological analysis in a context of bioremediation data analysis. This work presents new bioinformatic tools and protocols for the domain of hydrocarbon biodegradation and surfactant production. The first tool created was BioSurfDB, http://www.biosurfdb.org/, acurated relational information system integrating data from: (i) metagenomes; (ii) organisms; (iii) biodegradation relevant genes; proteins and their metabolic pathways; (iv) bioremediation experiments results; and (v) a biosurfactant-curated list. The main goal of this repository is to gather information on the characterization of biological compounds and mechanisms involved in biosurfactant production and/or hydrocarbon biodegradation and make it available in a curated way and associated with a number of computational tools to support studies of genomic and metagenomic data. Secondly, a biogeographical distribution analysis of hydrocarbon degrading and biosurfactant producing genes was performed. It was of great importance for our reseach as it confirmed the correlation of surfactation and degradation, and therefore, the need for analysis of these events together. The results also suggested that terrestrial biomes have higher abundance of biodegradation genes, when compared to water biomes and that near-equatorial biomes have higher abundance of genes with potential for bioremediation. With more than 70 metagenomes analysed, this work was an extensive test to BioSurfDB and its publication validated the database and tools relevance. Meanwhile, the massive amount of data generated by metagenomics, presents specific computational challenges, demanding the development of new collaborative research platforms. In this thesis the Oil and Gas Virtual Research Environment (OGVRE), available at http://aleph.inesc-id.pt/vre/, was developed. The OGVRE that can support studies using metagenomic data with application to the oil and gas field through a responsive web-based working environment, providing: (ii) workflow sharing, both bioinformatics and bench; (ii) access to algorithms and tools; (iii) access to databases and datasets related with biodegradation and biosurfactants; (iv) access to processing resources; (v) access to storage resources; and (vi) access to social tools. The sharing of tailor-made computational and bench workflows is very useful in metagenomics research due to the novelty, heterogeneity and size of the data that is being generated. The final stage of this thesis corresponds to the selection of consortia enhanced for biodegradation of hydrocarbons ans surfactant production, using oil from the pre-salt layer of Brazil. The best consortium, together with a sample of pure oil were sequenced in a 16S amplicon approach and analysed for a taxonomic classification of this still largely unknown oil type, obtaining a research base for this economical important layer.

The evaluation of human movement is an important parameter for therapeutic decision making and evolution reference after rehabilitation programs. Many devices have been developed, but few of them have satisfactory precision measures associated with ease of transport, handling and remote monitoring. Several chronic and degenerative diseases are characterized by compromised joint movement. Among them, Chronic Venous Insufficiency (CVI), which presents pathophysiological changes in the muscles of the lower limbs, with emphasis on the calf muscles and therefore to reduce the movement of the ankle joint. These changes determine a significant impact on the quality of life of these patients due to functional limitations caused by the disease. Thus, the development of angular assessment tools and targeted exercise programs for recovery of muscle performance and angular motion can assist health professionals in the planning and management of treatment and benefit patients with chronic venous insufficiency in reducing functional complaints and improves. Objectives: 1) To develop and execute a patent application for a device for digital measurement, therapeutic equipment and remote monitoring of joint parameters of the ankle joint and 2) assess the effects of a strengthening supervised exercise program of the calf muscle in physical performance, range of motion and myoelectric activity of the lower limbs muscles, cardiovascular hemodynamics and quality of life related to health in patients with CVI. Methods: The study is composed of two types of integrated research modalities: 1) Technological development and patent deposit of a device for evaluation of joint parameters and 2) randomized controlled clinical trial with blinded evaluator. Results: 1) creation of a device for evaluating human movement applied to the health area, called: "Digital Meter Device, Therapeutic Equipment and Remote Monitoring of Articular Parameters" with patent filing at INPI (BR 10 2018 016524 0); 2) Development of a strengthening exercises protocol of the calf muscles, directed to individuals with CVI and 3) Randomized clinical trial. The results of the randomized clinical trial showed that the patients in the Intervention Group - IG showed improvement in functional performance (p <0.0001) as well as in the Control Group - CG (p = 0.02). There was a decrease in the myoelectric activity in the gastrocnemius muscle during the end of the heel-rise teste (p = 0.01) at 25% and 50% moments in the IG patients, as well as a decrease in dyspnea after the end of the heel-rise test (p= 0.04). Quality of life was positively impacted in the domains: perception of improvement of symptoms in the lower limbs (V3, p = 0.01), performance in daily life activities (V5, p = 0.03), pain (V7, p= 0.04) and psychological impact of disease on the person's life (V8, p = 0.04) in IG patients. Conclusion: The present study develop a prototype, portable and precise, for angular evaluation, therapeutic training and remote monitoring to be used in patients with CVI. Furthermore, the results of the randomized clinical trial demonstrated an improvement in physical performance, myoelectric activity of the lower limbs and quality of life in treated individuals.

The use of food processing residues is a strategy to contribute to reducing waste and adding value to new products. Melon is a fruit consumed worldwide and its high consumption accompanies large amounts of residues, such as peel and seeds. The objective of this thesis was to add value to melon peel and seeds (Cucumis melo var. reticulatus), evidencing its nutritional properties, antioxidant capacity, anti-tumor effect, prebiotic potential and its capacity for enzyme production. Flours of melon peel and seed were elaborated using experimental design 22 to evaluate the factors of drying time and temperature, and the responses moisture, water activity and flour yield. Flour characterization was carried out using centesimal composition (moisture, ash, proteins, lipids, dietary fiber), fibrous fractions of cellulose, hemicellulose, lignin and pectin. Flours were evaluated for their in vitro prebiotic properties using Bifidobacterium lactis in submerged fermentation and bile salt tolerance. The potential of the flours in the production of cellulases in semi-solid fermentation using Aspergillus oryzae fungus was also evaluated. For the accomplishment of the analyzes of antioxidant and antiproliferative power, aqueous extracts, hydrometanolic and hydroethanolic of the flours were prepared, characterized as monosaccharide composition, analysis of total sugars and proteins. For antioxidant evaluation, the total phenolic compounds, total flavonoids, total carotenoids and tannins were determined. Phytochemical evaluation of the extracts was carried out by Thin Layer Chromatography. The antioxidant capacity evaluation was verified by the following in vitro tests: total antioxidant capacity, reducing power, iron and copper metal chelation, sequestration of DPPH, hydroxyl and superoxide radicals, and evaluation of the oxygenated radicals absorption capacity. The antiproliferative activity was evaluated using the tumor cell lines (SiHa, HeLa, 786-0 and HT29) by colorimetric test of cell viability evaluation. The results for the drying experimental design showed that the temperature factor significantly influenced the yield response of the flours, being the test of 80 ºC for 24 h which presented better yield. Data showed that the melon residues had important nutritional characteristics for human consumption, with emphasis on dietary fiber content (51.75% for seed, 40.57% for peel), proteins (22% for seed; 18% for peel) and lipids (24% for seed). The residues can be characterized as cellulosic material, with values of 35% cellulose for melon seed and 19% for peel. Satisfactory percentages of pectin were detected, being 28.96% for seed and 32.65% for melon peel. As for the prebiotic potential of the flour, it was observed that the fructo-oligosaccharide standard showed a growth of bifidobacteria of 12 LogUFCmL-1, after 12 hours of fermentation, and the melon seed flour promoted bifidobacterial growth reaching 9.9 LogUFCmL- 1, after 8 hours of culture, indicating prebiotic properties, with tolerance to the action of bile salts for up to 8 hours of fermentation. The melon peel did not provide growth of bifidobacteria. Melon peel and seed proved to be a good substrate for enzyme production. The best CMCase activity (1.045 U / g) was verified on six days of fermentation and FPase (0.190 U / g) after 96 h of fermentation with medium containing the peel and seed flours. In the evaluation of the extracts obtained from the melon residues, phytochemical analysis indicated the presence of compounds characteristic of phenolics, terpenes and saponins. Total phenolic compounds in aqueous, hydrometanolic, and hydroethanolic extracts of melon residue were quantified and expressed as gallic equivalents, highlighting peel extracts (1.016 mg 100g-1). Total carotenoids, total flavonoids and tannins were also detected in extracts of melon residues, with emphasis on 96 μg of total carotenoids per gram of melon peel, 491 μg of catechin equivalents and 309 μg of catechin equivalents in 100 g extract dried of melon peel. The tests to evaluate the antioxidant activity revealed that all extracts presented antioxidant capacity. In the test of total antioxidant capacity (expressed as equivalent in ascorbic acid), hydroethanolic and methanolic extracts for peel and seed presented values of 89.74 and 83 mg / g, respectively; 50.56% and methanolic 67.7% for melon peel. Reducing power and chelating abilities of copper and iron metals presented relevant data for both melon peel and seed, demonstrating an excellent antioxidant potential. The aqueous extract of the peel showed the ability of in vitro of copper ions chelation (84%) and iron ions chelation (61%). Scavenging activity of the DPPH radical was significant only for extracts of melon peel (29.5%).The cell viability test allowed us to observe that the extracts were able to extract the organic extracts, to significantly decrease the viability of the HeLa, SiHa and 786-0 cancer cells. Aqueous seed extracts at the 1.0 mg / mL concentration inhibited the proliferation of SiHa cells by 80% and the peel hydroethanolic extract (0.25 mg / mL) inhibited by 80% the viability of HeLa cells. For human renal tumor cells (786-0) all extracts at the concentration of 1.0 mg / ml inhibited cell proliferation by more than 50%. The extracts evaluated did not present significant inhibition activity of HT29 cells. All melon peel and seed extracts weakly inhibited the proliferation of normal 3t3 fibroblasts (25% inhibition). It is concluded that melon peels and seeds have antioxidant potential in vitro and antiproliferative effects in tumor cells. Flours of melon peel and seed may be substrates for the production of cellulolytic enzymes, in addition the seed flour showed in vitro prebiotic indication.

The big pelagic fish represents one of the most lucrative subjects of the industrial fisheries in Brazil. The State of Rio Grande do Norte attracts special attention because its geographic position constituting nowadays the region in which the commercial oceanic fisheries tends to enlarge with new policies of governmental incentive. Different from other World’s producer regions that are researching the main exploited species, in Brazil the studies that objectify the population delimitation and to list data for the conservation of the species are scarce. Among the pelagic groups is common to observe a predominance of a minor diversity in relation to the abundance, this is a characteristic in two of the most important families of this oceanic region, Sphyraenidae (barracudas), Carangidae (jacks and pompanos) and Coryphaenidae (dolphinfish). Its representatives are valued and intensely exploited by the fisheries industry. Data about these species are surrounded of logistics difficulties for its size, capture way and habitat which occupies. The first cytogenetical informations for some species of this family were obtained only recently. Here is presented a cytogenetical study with 9 pelagic fish species from the Atlantic, of the families Sphyraenidae, Carangidae, Corypahenidae, Istiophoridae and Megalopidae and 1 Indo-Pacific Carangidae. The results revealed numerical divergences and in the karyotipical macrostructure contrasting with the pattern considered basal for the Teleosts, besides the existence of masculine heterogamety, through a system of multiple sexual chromosomes of the kind X₁X₁X₂X₂/X₁X₂Y. Using techniques such as AgNORs, MM/DAPI it was possible to identify single ribosomal sites and showed cytotaxonomic markers for all. It was also applied more resolutive cytogenetic techniques based on multigenic sequences mapping through FISH (Fluorescence in situ Hybridization) enabled for the first time for some of these families to infer about the active karyoevolutive processes and evolutive aspects of the investigated species. Through the developed techniques it will be also possible to expand the knowledge about this important marine resource,subsidizing future policies of the management of stocks, as well as future support for the technological development of marine aquaculture.

Native to Brazil, Carnauba (Copernicia prunifera) has been used for several purposes, including the wax production from its leaves, in the process that generates a considerable amount of residue.  This residue is characterized as a fiber rich in cellulose and therefore with potential latent for use as a source of carbon for the production of cellulolytic enzymes and bioethanol. However, the chemical structure of this material presents cellulose bound to structurally complex components, such as hemicellulose and lignin, which hinders the production of cellulases by filamentous fungi, as well as its enzymatic hydrolysis, being essential to use of a pre-treatment for the viability of these processes. The present study evaluated the effect of different pre-treatments on carnauba straw for the production of lignocellulolytic enzymes and for the enzymatic hydrolysis with a view to the production of cellulosic ethanol through the concepts of biorefinery and micro-distillery. In the first stage, carnauba straw residue was submitted to hydrothermal (HT), alkaline (AL), alkaline acid (AA) and alkaline hydrogen peroxide (A-HP) pre-treatments. The pretreated and untreated residues were chemically characterized according to the National Renewable Energy Laboratory (NREL) protocol and, physically, by Scanning Electron Microscopy (MEV), X-Ray Diffraction (XRD) and Spectroscopy of Infrared by Fourier Transform (FTIR). A part of each residue was used to produce enzymes by means of Solid State Fermentation (FES), using the fungus Trichoderma reesei CCT-2768. The FPAse, CMCase, β-glycosidase and xylanase activities of the extracts were estimated and the production was subsequently optimized. The other part of the residues was subjected to Saccharification and Simultaneous Fermentation (SSF) using commercial enzymes and Saccharomyces cerevisiae UFLA CA11, Saccharomyces cerevisiae CAT-1 and Kluyveromyces marxianus ATCC-36907. The results of the pretreatments AL, AA and A-HP stood out in terms of the removal of lignin, according to the chemical and physical analysis of the residues. The studies showed that pretreatment of carnauba straw with A-HP has a higher capacity to induce the production of lignocellulolytic enzymes when compared to other residues, such as coconut, cashew apple and sugar cane, pretreated by the same method. The optimization of the production of lignocellulolytic enzymes allowed the production of an enzymatic extract with FPase activity of 2.4 U/g and xylanases of 172 U/g. The application of the enzymatic extract in the hydrolysis of pretreated sugarcane bagasse showed efficiency of 86.96%. The use of AL pretreated carnauba residue in enzymatic hydrolysis, with commercial enzymes, showed a higher conversion of sugars (64.43%) and, when submitted to SSF, produced 7.53 g/L of ethanol, using Kluyveromyces marxianus ATCC-36907 cultured at 45 °C. The results showed, therefore, the biotechnological potential of the carnauba residue for the production of cellulolytic enzymes and the production of bioethanol by means of biorefinery and micro distillery.

The preoperative fasting was instituted many years ago, at the beginning of the anesthetic practice, to ensure the emptying of the stomach contents, avoid bronchial aspiration, vomiting, regurgitation, administration of various drugs to reduce gastric acidity and volume. Preoperative fasting protocols suggest shorter fasting periods especially for liquids, providing more comfort to the patient, and less risk of hypoglycemia and dehydration, without increasing the incidence of perioperative pulmonary aspiration. The aim of the study was to assess whether the bioproduct based on coconut water and maltodextrin (ACP Surgery) supplied the needs of clinical trials of ERAS project (Enhanced Recovery After Surgery) with good tolerance and acceptance. It was conducted a prospective, randomized, controlled, double-blind study in humans with a group of 49 patients at the Santa Casa de Misericordia de Fortaleza, to prove the efficiency and effectiveness of ACP Surgery to achieve the goals and planned effects on ERAS project. The experimental groups were: G1 [control group; Nutri Dextrin (Nutrimed, Fortaleza, Ceará); n = 20) and G2 [intervention; ACP Surgery (ACP Biotecnologia, Fortaleza, Ceará;. N = 29]. In the interval between 10 and 20 hours prior to the surgery, blood sample was taken from each patient and sent to the Hospital's Laboratory of Clinics Analysis for measurement of serum levels of total leukocytes, glucose, sodium, potassium and chlorine. The data were submitted to t test (α = 95%, p> 0.05 and t rejection area > 1.96) and expressed as mean and standard deviation. The G1 (Nutri Dextrin) and G2 (ACP Surgery) showed the following results regarding blood tests: total leukocyte G1 = 11,322.64 + 5,033.94, and G2 = 11,074.14 + 4,480.35; sodium G1 = 140.8 + 4.14 mEq/L, and G2 = 139.3 + 4.38 mEq/L; potassium G1 = 4.16 + 0.65 mEq/L, and G2 = 4.18 + 0.47 mEq/L; chloro G1 = 107.4 + 4.69 mEq/L, and G2 = 107.1 + 6.88 mEq/L; and glucose G1 = 130.3 + 39.96 mg/dL, and G2 = 123.9 + 36.47 mg/dL. In all variables p> 0.05, so it can be considered that Nutri Dextrin and ACP Surgery products are equivalent. The acceptance of the ACP Surgery was 97%, with no reports of cough or nausea (100% tolerance), without loss of palatability. It follows that the produced bioproduct met the needs and specifications of preoperative fasting protocols of short duration, showing acceptance and tolerance compatible with the commercial product at the same time reducing costs and facilitating protocol implement.

O controle da respiração é um processo complexo e multifatorial, responsável pela manutenção da adequada homeostase e suprimento de oxigênio nos tecidos corporais. Os músculos respiratórios são parte primordial deste processo e podem ser treinados para que haja melhora na eficiência na ventilação. São descritas na literatura três modalidades de treinamento, a hiperpneia voluntária isocápnica, o treino resistido a fluxo com válvula e o treino resistido à carga pressórica. Atualmente, merece destaque uma nova modalidade definida como Tixotropia, que envolve contrações inspiratória e expiratória contrauma via aérea ocluída em diferentes volumes pulmonares, como Capacidade Pulmonar Total (CPT) e Volume Residual (VR). O presente estudo teve como objetivo desenvolver e avaliar inovações tecnológicas no âmbito de músculos respiratórios e mecânica respiratória. A pesquisa foi desenvolvida envolvendo o Laboratório de Desempenho Pneumocardiovascular e Músculos Respiratórios da Universidade Federal do Rio Grande do Norte (UFRN) e o Laboratorio Tecnologie Biomedichale - TBM do Politécnico de Milão, Itália. A metodologia adotada foi composta por três itens integrados: (i) depósito de patente de um controlador automático para válvula pneumática para treinamento muscular respiratório na modalidade tixotropia; (ii) revisão sistemática; (iii) artigos científicos relacionados à utilização do instrumento desenvolvido. Os resultados compreenderam depósito de patente no Núcleo de Inovação Tecnológica (NIT) vinculado à UFRN, sob número 23077.077363/2014-59, revisão sistemática sobre testes de endurance com carga constante ou incremental usados em pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC), avaliados pela Pletismografia Optoeletrônica e artigos científicos apresentando resultados dos efeitos agudos da modalidade de treinamento tixotropia e suas repercussões sobre os volumes pulmonares em indivíduos saudáveis utilizando o instrumento desenvolvido.  Portanto, o presente estudo propiciou a criação e validação de instrumento inovador e suas evidências científicas, segundo aspectos da Avaliação de Tecnologias em Saúde (ATS), contribuindo para desenvolvimento de uma nova modalidade e instrumento de treinamento muscular respiratório a ser utilizado em pacientes com doenças que afetam a mecânica respiratória.

O objetivo da presente tese foi utilizar a manipulação de oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) como ferramenta para o resgate e otimização do uso de gametas femininos oriundos de espécies silvestres do bioma Caatinga. A tese foi dividida em quatro experimentos. No primeiro, foi realizada a estimativa e descrição das características histológicas e ultraestruturas dos folículos pré antrais (FOPA) de cutias (Dasyprocta leporina), nos quais foram estimados 4419.8 ± 532.26 e 5397.52 ± 574.91 folículos para os ovários direito e esquerdo, respectivamente, sendo a maioria (86,63%) pertencente a categoria de folículos primordiais (P<0,05). A maior parte da população consiste de folículos morfologicamente normais (70,78%), apresentando núcleo oocitário grande e central, com citoplasma uniforme. Na avaliação ultraestrutural verificou-se a presença de um grande número de mitocôndrias arredondadas e gotas de lipídios. No segundo experimento, foi realizada a estimativa e a descrição das características dos FOPA de preás (Galea spixii), bem como avaliação do efeito da vitrificação em superfície sólida (VSS) sobre a morfologia de FOPA in situ. O total de 416,0 ± 342,8 FOPA foi estimado por par de ovários e a presença de uma grande quantidade de folículos primários foi evidenciada (P<0,05). A maior parte dos FOPA apresentou-se morfologicamente normal (94,6%), possuindo núcleo oocitário contendo grânulos condensados de heterocromatina. Mitocôndrias com formato arredondado ou alongado representaram as organelas mais abundantes. Quanto a VSS, o protocolo utilizando o dimetilsulfóxido (DMSO) 3M possibilitou a preservação de 69,5% de FOPA morfologicamente normais, sendo evidenciado através da análise por microscopia de luz e eletrônica de transmissão. No terceiro experimento, foi realizada a avaliação do efeito da VSS sobre a morfologia e viabilidade de FOPA in situ de catetos (Pecari tajacu). Não foram observadas diferenças entre os tratamentos, onde o uso dos crioprotetores DMSO, etilenoglicol (EG) e dimetilformamida (DMF), independente da concentração utilizada (3 ou 6 M) promoveu a preservação da morfologia de mais de 70% dos FOPA. Quanto a viabilidade, os crioprotetores DMSO e EG, demonstraram melhor manutenção da mesma. O quarto experimento teve por objetivo avaliar o efeito do α MEM+ ou TCM199 associados ou não a 50 ng de FSHr sobre a morfologia, ativação e crescimento de FOPA de catetos, cultivados in vitro (CIV) durante 1 ou 7 dias e o efeito sobre a matriz extracelular (MEC). Após 7 dias de CIV apenas o TCM199/FSH manteve a proporção de FOPA intactos similar ao dia 1 (63,2%), contudo nenhuma diferença foi observada entre os tratamentos (P>0,05). Adicionalmente, um aumento na proporção de FOPA em desenvolvimento foi verificada (P>0.05). Através da análise com Ag-NOR, observou-se que apenas o tratamento com TCM199/FSH manteve a proporção de proliferação celular similar ao dia 1 (P>0.05). A coloração com picrosirius red revelou que a MEC permaneceu intacta em todos os tratamentos (P>0.05). Assim, como conclusão geral, o uso da MOIFOPA nas referidas espécies permitiu o conhecimento de aspectos relacionados a sua morfofisiologia reprodutiva, possibilitando tanto a conservação do material genético destas espécies, com a possibilidade de formação de bancos de germoplasma, como a elucidação de mecanismos relacionados a sobrevivência e desenvolvimento dos FOPA in vitro.

O crescente interesse e aplicações dos produtos biotecnológicos vêm aumentando o desenvolvimento de novos processos de recuperação e purificação de proteínas. A adsorção em leito expandido (ALE) tem se destacado como uma técnica promissora para essa finalidade, pois combina em uma única etapa os passos de clarificação, concentração e purificação da proteína alvo, reduzindo assim o tempo e os custos de operação. Neste contexto, o objetivo desta tese foi avaliar a recuperação e purificação do antígeno 503 de Leishmania i. chagasi expresso em E. coli M15 e a remoção de endotoxina por ALE. Para definir as condições ótimas de adsorção e eluição na resina Streamline chelating, foram inicialmente realizados ensaios em taques agitados sob a forma de dois planejamentos experimentais. Nos ensaios de adsorção usando o leito na forma expandida empregou-se uma coluna de 2,6 cm de diâmetro por 30,0 cm de altura, acoplada a uma bomba peristáltica. Na segunda etapa do trabalho, o tensoativo não-iônico Triton X-114 foi adicionado à etapa de lavagem da ALE visando a remoção de endotoxina durante o processo de purificação. Buscou-se também a elaborar um modelo matemático capaz de prever as curvas de ruptura do antígeno 503 em coluna na forma expandida. Os resultados do planejamento experimental para adsorção do antígeno 503 mostraram o pH 8,0 e a concentração de NaCl 2,4 M como melhores condições adsorção. No segundo planejamento, o único fator significativo para eluição foi a concentração de imidazol, definida em 600 mM. A isoterma de adsorção do antígeno 503 mostrou bom ajuste ao modelo de Langmuir (R=0,98) e os valores de q_max (capacidade máxima de adsorção) e K_d (constante de equilíbrio) estimados foram de 1,95 mg/g de adsorvente e 0,34 mg/mL, respectivamente. Através dos testes de purificação diretamente do homogeneizado não clarificado obteve-se uma recuperação de 59,2% da proteína de interesse e um fator de purificação de 6,0.  A adição do tensoativo não-iônico Triton X-114 à etapa de lavagem da ALE proporcionou altos valores (>99%) de remoção do LPS inicialmente presente nas amostras para todas as condições estudadas. Na etapa de modelagem matemática da adsorção do antígeno 503 em coluna na forma expandida, as curvas de ruptura experimentais permitiram determinar-se as eficiências do processo (15% de saturação) e da coluna (85% de saturação), no entanto o modelo apresentou bom ajuste apenas para eficiência do processo. O modelo validado foi utilizado com uma estratégia híbrida na otimização da eficiência do processo, indicando que os valores de 7,8 cm de altura inicial do leito, 248,8 cm/h de velocidade superficial e 0,338 g/L de concentração inicial do antígeno resultam no valor de 89,19% para eficiência do processo.

Uma cepa produtora de quitosanase foi isolada e identificada como Bacillus cereus C-01. A partir disso a purificação da enzima e sua caracterização bioquímica foram estudadas. Para purificação foi utilizado a resina da linha streamline Dietilaminoetil (DEAE) e coluna de vidro (2,6 cm x 30,0 cm) em sistema de Adsorção em Leito Expandido (ALE).  As condições para a purificação da quitosanase foram otimizadas estatisticamente através de um Planejamento Composto Central. As variáveis que influenciaram significativamente os parâmetros fator de purificação (P) e rendimento da enzima (Y) foram determinadas. As análises estatísticas mostraram que as melhores condições para o máximo P foram 150 cm.h^-1 (velocidade de aplicação da amostra/carga), 6,0 cm de altura do leito fixo e 7,36 cm de altura do distribuidor. Em condições otimizadas, a enzima mostrou ligar-se a resina na mesma medida usando caldo clarificado e não clarificado (0,32 e 0,3 U/g adsorvente, respectivamente). A fração recuperada após a eluição exibiu 31% de rendimento com um aumento de 1,35 vezes na atividade específica em relação ao inicial. Se analisada apenas fração eluída com NaCl 0,3 M é possível encontrar um fator de purificação de 1,75. A enzima purificada apresentou estabilidade entre as temperaturas de 30-55ºC durante 60 minutos, pH de 5-8 por 24 horas, e apresentou máxima atividade em pH 5,5 e temperatura de 55ºC. Os íons de Cu²⁺, Fe²⁺ e Zn²⁺ foram inibitórios para a quitosanase. Em contraste, a enzima foi significativamente ativada por Mn²⁺. Os resultados demonstraram que é possível purificar múltiplas proteínas, a partir de um extrato bruto sem qualquer pré-tratamento, com um processo de purificação econômico e de um único passo.

A meningite bacteriana (MB) é uma doença infecciosa que permanece com altas taxas de mortalidade e morbidade em todo o mundo, principalmente em países subdesenvolvidos, apesar dos avanços na antibioticoterapia. Um dos principais mecanismos associados às sequelas durante a MB é a elevada resposta inflamatória, que promove uma exacerbada quantidade de espécies reativas de oxigênio (ERO) levando às células a apoptose ou necrose. A ativação de enzimas de reparo, durante o estresse oxidativo tem sido demonstrada nas mais diversas desordens, entre elas a endonuclease apurínica/apirimidínica 1/ efetor redox 1 (APE1/ref-1). APE1/ref-1 é uma enzima multifuncional envolvida no reparo de DNA e na redução de fatores envolvidos com a resposta inflamatória, tais como o fator nuclear kappa B (NFκB) e proteína ativadora 1 (AP-1). Portanto, este trabalho teve como objetivo buscar novos alvos terapêuticos para a redução de sequelas durante MB. Para isto, inicialmente foi realizado uma análise no perfil de expressão de citocinas em líquor de pacientes com meningite causada por Streptococcus pneumoniae e Neisseriae meningitidis. Em seguida, foi avaliado o papel da APE1 como um possível alvo terapêutico na modulação das citocinas expressas em monócitos durante resposta celular a lipopolissacarídeo (LPS). Por fim, foi observado a expressão de APE1 no córtex (CX) e hipocampo (HC) de ratos com MB frente a terapia adjuvante com vitamina B6. Nosso estudo mostrou principalmente que interferon gama (IFNγ) é significativamente mais expressa em pacientes com S. pneumoniae do que N. meningitidis. Por outro lado, o uso de inibidores das funções redox e de reparo de APE1 reduzem a expressão de citocinas, principalmente o fator de necrose tumoral-alfa (TNF-α), bem como, o nível de ERO nas células estimuladas com LPS. Ainda, a expressão protéica de APE1, no CX e HC dos ratos, foi modulada após introdução da vitamina B6. Estes dados fornecem um novo olhar para a fisiopatologia da MB, em que citocinas como IFNy podem ser usadas em um diagnóstico diferencial, enquanto que APE1 pode ser usada como alvo para modular a resposta inflamatória e a vitamina B6 pode ser usada para modular APE1. Consequentemente, o conhecimento obtido neste estudo pode ser importante na melhoria do prognóstico da MB, além de contribuir para entender a associação entre o reparo de DNA e inflamação.

Células-tronco mesenquimais humanas (CTMH) são muito úteis na terapia celular. O longo período de cultivo pode resultar em senescência replicativa ou estar relacionado com o aparecimento de alterações cromossômicas responsáveis pela aquisição de um caráter tumorigênico in vitro. Neste estudo, foi comparado o transcriptoma de CTMH jovens e senescentes obtidas de diferentes doadores. Além disso, pela primeira vez, o perfil de expressão de CTMH com uma inversão cromossômica paracêntrica (CTMH/inv) foi comparado ao de CTMH que possuem cariótipo normal (CTMH/n) em passagens jovens e senescentes de cultivo in vitro. As CTMH utilizadas neste estudo foram isoladas da veia do cordão umbilical de três dadores, dois CTMH/n e de um CTMH/inv. Após a criopreservação, elas foram expandidas in vitro até alcançarem a senescência. O RNA total foi extraído utilizando o RNeasy mini kit (Qiagen), marcado, purificado e fragmentado com o ® 3 'GeneChip IVT expresso Kit (Affymetrix, Inc.). Subsequentemente, o RNA fragmentado foi hibridado no microarranjo Affymetrix Human Genome U133 Plus 2.0 (Affymetrix, Inc.). A análise estatística da expressão diferencial foi realizada usando o Partek Suite Software Genomic, versão 6.4 (Partek, Inc.). Foram consideradas estatisticamente significativas as diferenças na expressão com valor de P ˂0.01 corrigido com Bonferroni. Apenas os sinais com fold change ˃3.0 foram incluídos na lista de diferencialmente expressos. Diferenças na expressão gênica observadas no estudo dos microarranjos foram confirmadas por resultados de RT-PCR em tempo real. Para a interpretação biológica dos dados foram utilizados: IPA (Ingenuity Systems) para análise de enriquecimento de funções; STRING 9,0 para a construção de redes de interações; Cytoscape 2,8 para a visualização das redes e análises de bottlenecks com o auxílio do software GraphPad Prism 5.0. O pluggin BiNGO do Cytoscape foi utilizado para avaliar a representação de categorias funcionais no Gene Ontology nas redes biológicas. A comparação entre senescentes e jovens em cada grupo de CTMH mostrou que há uma diferença no perfil de expressão, sendo maior nas senescentes do grupo CTMH/inv. Os resultados também mostraram que há diferença nos perfis de expressão entre as CTMH/inv e CTMH/n, sendo maior a diferença quando as células estão senescentes. Novas redes foram identificadas para genes relacionados com a resposta ao longo do tempo de cultivo nos dois grupos de CTMH. Foram identificados genes que podem coordenar funções importantes mais enriquecidas nas redes, como por exemplo, CXCL12, SFRP1,  EGF, SPP1, MMP1 e THBS1. A interpretação biológica destes dados sugere que a população de células CTMH/inv tem diferentes características constitucionais, relacionadas com o seu potencial de proliferação, diferenciação e resposta a estímulos, responsáveis por um processo de senescência replicativa em CTMH/inv  distinto das CTMH/n. Os genes identificados neste estudo são candidatos a marcadores da senescência celular em CTMH, mas a sua relevância funcional neste processo deve ser testada em experiências adicionais in vitro e/ou in vivo. 

          A busca por genes baseado na construção e análise de bibliotecas metagenômicas a partir de solo gera oportunidades para explorar uma enorme diversidade genética  e metabólica de microrganismos. Os rios são ecossistemas com alta diversidade  biológica mais pouco explorado por meio de metagenomas. Com esse objetivo, uma biblioteca metagenômica foi construída a partir de DNA extraído de substrato do rio em três pontos ao longo do rio Jundiaí-Rio Grande do Norte. Os pontos de amostragem são derivados de área aberta, terreno acidentado e com a incidência direta da luz solar. O solo é úmido, compacto, de cor escura e coberto com finas camadas de sal. O rio é caracterizado por drenagem intermitente e alta salinidade, que pode ser superior a quatro vezes a concentração do mar. Esta biblioteca foi analisada funcionalmente e também com base em sequências. Para a análise funcional, foi utilizado meio de  cultura sólido de Luria-Bertani (LB) com a concentração de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com características halotolerantes. Os DNAs recombinantes foram extraídos e retransformados em DH10B e curvas de sobrevivência foram obtidas para confirmação e quantificação da resistência ao estresse abiótico. As sequências dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam proteínas hipotéticas. Um dos clones apresentou uma  ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na síntese do percurso de glicerol e serve como um soluto compatível para equilibrar a pressão osmótica no interior e no exterior das células. Posteriormente, com o intuito  de identificar genes que codificam osmolitos relacionados com a halotolerância, amostras de DNA ambiental do substrato do rio e da coluna d´água do estuário e oceano foram piro sequenciadas.sequências de osmolitos de diferentes microrganismos foram obtidas a partir do UniProt e usado como RefSeqs para a identificação por homologia (TBLASTN) nos bancos de dados. As sequências foram submetidas a HMMER para a identificação de domínios funcionais e sequências não redundantes para evitar a quantificação repeti da de um mesmo microrganismo e permitir o agrupamento dos dados por metagenoma. Foram identificados trealose, ectoine, sorbitol, glicerol, inositol-3-fosfato desidrogenase, chaperone, a betaína de glicina e prolina oxiredutase. Estes são osmólitos comumente relacionados a ambientes salinos, no entanto, é aceitável que microrganismos presentes no rio possam produzir esses solutos devido a alta concentração de sal no solo e na água durante a estação de seca neste rio. Isso confirma a existência de uma resposta especializada contra o estresse salino por bactérias no ambiente do rio Jundiaí.

Os Perciformes são dominantes no ambiente marinho, caracterizados como a maior e mais diversificada Ordem de peixes dentre os teleósteos. Muitas de suas famílias, como os Gerreidae, conhecidos popularmente como carapicus, carapebas, ou mojarras, têm um alto potencial econômico, no que se diz respeito à piscicultura marinha, extrativismo e pesca esportiva. Informações genéticas destas espécies são de fundamental importância para seu manejo e produção. Mesmo assim, das 13.000 espécies de peixes marinhos descritos apenas 2% foram estudadas sob o ponto de vista citogenético e menos de 1% sobre suas características reprodutivas. A reprodução induzida, a citogenética e a criopreservação de gametas, representam importantes áreas aplicadas de estudo em peixes. No presente projeto análises citogenéticas foram empregadas na caracterização genética de espécies da família Gerreidae, ocorrentes no litoral e regiões estuarinas do Nordeste do Brasil. Diferentes métodos de identificação de regiões cromossômicas foram empregados por meio de técnicas convencionais (Ag-RONs, bandamento C), coloração com fluorocromos base-específicos (DAPI-CMA3), e mapeamento cromossômico de genes ribossomais marcadores DNAr 18S e 5S, através da hibridação in situ com sondas fluorescentes (FISH). As seis espécies analisadas revelaram marcante conservadorismo cromossômico. Os genes ribossomais 18S e 5S quando analisados em perspectiva filogenética demonstram dinâmica evolutiva variada, podendo apresentar estase em alguns grupos e maior dinamismo em outros. As análises por duplo-FISH dos sítios 18S e 5S se revelaram eficientes marcadores citotaxonômicos nos cariótipos homogêneos deste grupo de espécies. Os padrões cariotípicos identificados, além dos aspectos evolutivos do cariótipo identificados, são sugestivos de baixa potencial de barreiras pós-zigóticas, instigando pesquisas futuras de prospecção de hibridação interespecífica de valor comercial destas espécies.

As condições ambientais interferem nos processos de decomposição de corpos e remanescentes humanos. Variáveis associadas ao clima e ao solo como alta temperatura, umidade elevada, incidência da radiação solar, composição do solo e o pH do solo podem degradar o DNA de amostras biológicas coletadas de cenas de crimes e de corpos não identificados. Os dentes representam uma importante fonte de DNA, pois são os tecidos mais rígidos do corpo humano e mais resistente à putrefação. Os dentes são coletados quando o corpo encontra-se esqueletizados ou quando os outros tecidos estão em pequenas quantidades e putrefeitos. A genotipagem forense com STRs (Short Tandem Repeat) é um método padrão que apresenta alto poder de discriminação entre indivíduos e a amplificação de amostras degradadas.  A presente pesquisa objetivou avaliar a quantidade e qualidade do DNA de amostras dentárias expostas às condições ambientais de clima e de solo tropicais por T0, T1 (4 meses), T2 (9 meses) e T3 (24 meses). A amostra foi constituída por 65 dentes molares hígidos de indivíduos com média de idade de 24 anos (dp ± 3). Os dentes foram limpos física-quimicamente e pulverizados criogenicamente. O DNA dentário foi extraído através do protocolo de extração AFDIL, quantificados em PCR em tempo real através do kit Quntifiler^® Human DNA Quantification e os STRs autossômicos (FGA, D16S539, D7S820, D13S317, D3S1358, Penta E, D18S51, D12S391, TH01, D19S433, CSF1PO, TPOX, vWA, D5S818, D2S1338, SE33 e a amelogenina) amplificados em reações de PCR multiplex. As análises de variância (ANOVA) e o pós-teste de Tukey mostraram haver diferenças significativas (p ≤ 0,05) nas médias de quantidades de DNA entre o grupo controle e grupo experimental no tempos de exposição T1, T2 e T3 e no grupo superfície entre T1 e T3. A quantificação foi possível em 92% (n = 60) das amostras. A diferença na médias de amplificação dos loci de STRs foi significativa (p<0,05) nos grupos superfície, argila e areia ao longo dos tempos T1, T2 e T3. Em 5 amostras do tempo T3 não houve amplificação. Os três maiores marcadores CSF1PO, FGA e Penta E amplificaram em 60% das amostras da superfície, 94% da argila e 87% da areia no tempo T1. Os três menores marcadores D5S818, D19S433 e D3S1358 amplificaram em 94% das amostras da superfície, 94% da argila e 100% da areia no tempo T1. A amelogenina amplificou em 86% das 65 amostras e 47% no grupo experimental no tempo T3. Houve variação significativa (p<0,05) para as médias das áreas da amelogenina entre o grupo controle e o grupo experimental em T1 e T3. Para a área do marcador CSF1PO houve variação significativa (p<0,05) entre o grupo controle e o grupo experimental no tempo T1. Face ao exposto, conclui-se que a submissão dos dentes às condições de clima e de solo foi determinante para a redução significativa da quantidade média de DNA no primeiro tempo de exposição T1 (4 meses) e os STRs podem ser indicados eficientemente para genotipagem com até 4 meses de exposição às condições de clima e de solo tropicais.

Células tronco mesenquimais humanas (CTMH) são células multipotentes utilizadas em várias pesquisas de terapia celular regenerativa, pois apresentam a capacidade de se diferenciar em múltiplas e diferentes linhagens, têm grande capacidade de expansão in vitro, excelentes propriedades imunossupressoras e são capazes de secretar moléculas bioativas que exercem efeitos tróficos.  O cordão umbilical é uma fonte de CTMH cuja extração não necessita de um procedimento invasivo, além de não envolver controvérsias éticas, políticas e religiosas. Um dos problemas que pode ocorrer em linhagens de CTMH de diferentes fontes é a possibilidade de ocorrência de alterações cromossômicas e instabilidade genômica, que podem aparecer durante a expansão in vitro. Além disso, as CTMH de um dos cordões apresentaram uma alteração cromossômica constitucional: inversão paracêntrica no braço curto do cromossomo 3, cariótipo: 46,XY,inv(3)(p13p25~26). Em 3p25-26, estão localizados vários genes de grande importância biológica, como genes envolvidos com o reparo de DNA e outros responsáveis pelo desenvolvimento de tumores. O titânio é um dos materiais mais utilizado para fabricação de implantes ortopédicos e dentários, e é considerado um excelente biomaterial, entretanto as partículas derivadas de próteses acumulam-se nos tecidos periprostéticos e na medula óssea local, disseminam-se para linfonodos, fígado e baço. As implicações biológicas a longo prazo da disseminação sistêmica de partículas de metais e seus efeitos cito- e genotóxicos não estão bem caracterizados. Considerando-se que possíveis alterações  em genes em 3p25~26 poderia predispor as CTMH com inv(3) à transformação espontânea in vitro, ainda mais no cultivo na presença de titânio, neste trabalho investigamos a estabilidade genética de CTMH isoladas da veia do cordão umbilical durante a expansão in vitro, após o congelamento, e verificamos as características das CTMH com cariótipo alterado comparadas com CTMH com cariótipo normal em diferentes condições de cultivo in vitro, na presença e na ausência de titânio, antes e após a senescência. A análise da estabilidade genômica das CTMH foi realizada através da análise do cariótipo e da análise do citoma / micronúcleo das CTMH. Ficou estabelecido que as CTMH são capazes de internalizar as partículas de titânio, mas as células mantém sua capacidade de proliferação, diferenciação e preservam os mesmos  marcadores de membrana. Além disso, demonstrou-se que existe um aumento na instabilidade genômica com o decorrer do tempo de expansão in vitro, e esta instabilidade foi maior na presença de grande concentração de partículas de titânio. É necessário sempre avaliar os riscos/ benefícios da utilização do titânio na terapia tecidual envolvendo CTMH, considerando a biossegurança da utilização da regeneração óssea guiada que utiliza CTMH e titânio. Mesmo não se utilizando o titânio, é importante que o uso terapêutico de tais células seja baseado em análises que garantam a qualidade, segurança e estabilidade celular, com a implementação de programas de controle de qualidade adequados. Como conclusão, sugere-se que a análise citogenética  e a análise do citoma sejam realizadas nas CTMH antes de implantar num paciente, sejam elas cultivadas por longo tempo ou não.

Espécies   Atlânticas   da   família   Lutjanidae,   conhecidas   como  snappers,   têm   sido amplamente   estudadas   sobre   diversos   aspectos   biológicos,   contudo   a   estruturação genética   de   suas   populações   é  em   grande   parte   desconhecida.  Lutjanus   analis  (mutton snapper)   com   extensa   distribuição   geográfica   no   Atlântico   têm   sido   historicamente sobrexplorada ao  longo da  costa brasileira  mesmo  na  ausência  de  informações  sobre  o status de divergência genética de suas populações. Análises das sequências hipervariáveis HVR 1 do DNA mitocondrial de amostras de extensa área do litoral brasileiro indicam que L. analis,   nesta   região   do   Atlântico,   pode   ser   considerada   como   uma   única   população panmítica com alta diversidade genética, cujos padrões histórico-demográficos demonstram ter sofrido efeitos de eventos ocorridos no Pleistoceno.

A Leucemia Linfóide Aguda (LLA) é uma doença de caráter maligno do sistema  imune  que  se  origina  a  partir  da  proliferação  neoplásica  de  uma  única  célula  linfocitária  progenitora  que  sofre  alteração  no  seu  processo  de  crescimento  e  reprodução  resultando  na  formação  de  um  clone  neoplásico  (teoria  monoclonal).  As  células  deste  clone  vão  se  expandindo  ao nível  dos  diversos órgãos linfóides e da medula óssea, atingindo a corrente sanguínea, podendo assim  atingir  vários  órgãos  ou  tecidos.  Atualmente  as  LLAs  são  classificadas pelo aspecto morfológico associado às características do perfil imunológico das células  malignas.  O  grupo  French-American-British  (FAB)  classificou  as  LLAs em  três  subtipos  distintos  baseados  somente  em  critérios  morfológicos  de medula  óssea  e  de  sangue  periférico  em  L1,  L2  e  L3.  Métodos  de imunofenotipagens  por  citometria  de  fluxo  tornaram  possível  a  detecção  de populações de células específicas obtidas de suspensões celulares através do uso  dos  anticorpos  monoclonais.  A  proposta  deste  estudo  foi  diagnosticar  e classificar  192  pacientes  com  LLA  com  base  na  imunofenotipagem  por citometria de fluxo com um painel de AcMo específico para leucemias agudas. Foram realizadas análises e correlações entre os dados demográficos, clínicos, laboratoriais  e  imunofenotípicos  desses  pacientes.  A  expressão  de  antígenos linfóides  como  o  CD3  e  CD19,  para  as  LLAs  T  e  B,  respectivamente,  associados na maioria dos casos ao CD34 caracterizou imunofenotipicamente  a  presença  de  precursores  linfóides  muito  imaturos.  A  expressão  dos marcadores linfóides B mais comuns nos pacientes com LLA foram os CD19 e sCD22,  e  dos  marcadores  linfóides  T  foram  os  CD7  e  cCD3.  Foi  notificada  a expressão  de  fenótipos  aberrantes  quando  observamos  a  expressão  dos marcadores linfóides T mais comuns nos pacientes com LLA B como o sCD3, e também  quando  observamos  a  expressão  dos  marcadores  linfóides  B  mais comuns nos pacientes com LLA T como os CD19 e o cCD79a. Concluímos que a imunofenotipagem por citometria de fluxo é uma metodologia necessária para o diagnóstico e a classificação das leucemias agudas.

O seqüenciamento do genoma da espécie Chromobacterium violaceum identificou um cromossomo simples circular de 4.8 Kb, onde aproximadamente 40% das ORFs encontradas são classificadas como hipotéticas conservadas ou hipotéticas. Algumas regiões gênicas de interesse biotecnológico vêm sendo caracterizadas, como por exemplo, genes de reparo de DNA. Diante disso, objetivou-se identificar genes de  C. violaceum envolvidos com mecanismos de reparo de DNA e manutenção da integridade genômica. Foram realizados ensaios de complementação funcional com uma biblioteca genômica de  C. violaceum transformada na cepa DH10B de Escherichia coli deficiente em RecA. Cinco clones foram selecionados após ensaios com luz UVC. No clone PLH6A foram identificadas quatro ORFs (CV_3721 a 3724). Duas ORFs, CV_3722 e  CV_3724, foram subclonadas e uma atividade sinérgica de complementação foi observada. O ensaio de mutagênese com rifampicina após tratamento de luz UVC mostrou alta freqüência de mutagenicidade para a ORF CV_3722 (Pol III δ’ subunit). Dessa forma, propomos que a subunidade  δ’ de C. violaceum pode agir em DH10B na síntese translesão utilizando Pol IV em uma via  RecA independente. A ocorrência de um operon foi
confirmada usando cDNA de  C. violaceum em um ensaio de RT-PCR. Adicionalmente, foi observada a indução do operon após tratamento com UVC, dessa forma, esse operon composto pelas quatro ORFs foi identificado estar associado a resposta ao estresse em  C. violaceum. Em ensaios de curva de crescimento os outros quatro clones foram capazes de complementar a função na dose de 5 J/m2. E em ensaios de mutagenicidade três deles apresentaram freqüências de mutação com diferenças significativas em relação ao controle (DH10B), demonstrando que de alguma forma eles estão envolvidos com a resposta ao estresse em C. violaceum e manutenção da integridade genômica. Os resultados obtidos contribuem para um melhor conhecimento da genética desta espécie. 

A quitosana é um polímero natural, biodegradável, não tóxico e de alta massa molecular
obtido  a  partir  de  animais  marinhos,  insetos  e  microrganismos.  Os  oligômeros  de
glicosamina  (GlcN)  e  N-acetilglicosamina  (GlcNAc)  têm  atividades  biológicas
interessantes,  incluindo  efeitos  antitumorais,  atividade  antimicrobiana,  antioxidante
entre  outras.  A  alternativa  proposta  por  este  trabalho  foi  estudar  a  viabilidade  de
produção de quitooligossacarídeos utilizando um  extrato bruto de  enzimas produzidas
pelo fungo Metarhizium anisopliae. A hidrólise da quitosana foi realizada em diferentes
tempos,  de  10  a  60 minutos  para  a  produção  de  quitooligossacarídeos  e  a  detecção  e
quantificação foi realizado pela Cromatografia Liquída de Alta Eficiência. A avaliação
da  citotoxicidade  dos  oligômeros  de  quitosana  foi  realizada  em  células  tumorais
(HepG2 e HeLa) e não  tumoral (3T3). As células foram  tratadas durante 72 horas com
os oligômeros a viabilidade celular foi feita usando o método do MTT. A produção de
oligômeros de quitosana  teve maiores rendimentos durante 10 minutos de hidrólise, os
pentâmeros  apresentaram  concentração  0,15  mg/mL,  porém  os  hexâmeros,  que
apresentam maior interesse pelas suas propriedades biológicas, só foram detectados com
30 minutos de hidrólise  apresentando uma  concentração de 0,004 mg/mL. Um estudo comparando  os  oligômeros  puros  de  quitosana  com  a  mistura  dos  oligômeros
produzidos  pelo  extrato  bruto  enzimático  na  toxicidade  das  células  tumorais  revelou
maior  eficiência  da mistura  dos  quitooligossacarídeos. Além  disso,  o  composto  com
maior citotoxicidade entre os oligômeros puros foi a glicosamina, apresentando valores
de IC50 0.30; 0.49; 0.44 mg/mL para células HepG2, HeLa e 3T3, respectivamente. Os
oligômeros  produzidos  por  hidrólise  durante  20  minutos  foram  analisados  quanto  à
capacidade  de  inibir  células  tumorais mostrando  inibição  da  proliferação  apenas  nas células  HeLa,  não  apresentando  nenhum  efeito  em  células  HepG2  e  células  de
fibroblastos (3T3).