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Dissertations |
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1
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Filipe Barbosa Marques dos Santos
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"Characterization of halophilic Archaea present in table salts ".
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Advisor : CYNTHIA MARIA KYAW
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COMMITTEE MEMBERS :
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ALINE BELMOK DE ARAUJO DIAS IOCCA
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CYNTHIA MARIA KYAW
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ILDINETE SILVA PEREIRA
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RICARDO HENRIQUE KRUGER
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Data: Apr 27, 2023
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Show Abstract
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Halophile organisms grow in environments with high concentrations of salts and are distributed in the three domains of life: Bacteria, Eukarya and Archaea. Such environments include salt waters, soils, fermented salt foods and food salts. The presence of halophilic prokaryotes in food salts is common and has been described in several scientific works, however, there is no information about their occurrence in salts sold in Brazil. Thus, this work had as main objective the characterization of extreme halophilic prokaryotes present in food salts, initially through culture-dependent approaches. Aliquots of four commercial salts were inoculated into two culture media for halophiles (ATCC and MGM), with the addition of 3 or 4 M NaCl, in search of extreme halophile organisms. The results obtained suggest the presence of both bacteria and archaea in the analyzed salts, since microbial growth was observed in all analyzed media. Most of the colonies obtained had a reddish coloration, typical of halophilic archaea, while microscopic analysis revealed the presence of both cocci and bacilli, predominantly Gram negative. All cultures were subjected to DNA extraction, followed by PCR, with specific primers for Archaea and Bacteria. Amplicons obtained from some of the salts were then ligated into cloning vectors and transformed into Escherichia coli XL1-Blue cells. Recombinant clones were analyzed by DNA sequencing and the results reveal the presence of archaea whose 16S rRNA genes exhibit a high degree of identity with the respective genes of archaea of the genera Halobacterium and Halolamina.
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2
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Felipe de Araujo Mesquita
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In silico analysis of the genomic potential for terpene production in aerobic endosporeforming bacteria
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Advisor : MARLENE TEIXEIRA DE SOUZA
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COMMITTEE MEMBERS :
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Thaís Amaral e Sousa
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GABRIEL SERGIO COSTA ALVES
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MARCELO DE MACEDO BRIGIDO
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MARLENE TEIXEIRA DE SOUZA
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Data: Jun 28, 2023
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Show Abstract
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Secondary or specialized metabolites are non-essential molecules for cell growth. However, they play an important adaptive role for the producing organisms, such as bacteria, fungi, and plants. Bacteria isolated from soils are known for synthesizing a wide range of specialized metabolites with different physicochemical properties. Bacillus species and from related genera, collectively quoted as aerobic endospore-forming bacteria (AEFB), are promising for searching these compounds. Nevertheless, reports on the synthesis of terpenes by AEFB are very scarce in the scientific literature. This study aimed to analyse the genomic potential for terpene production in ten AEFB strains isolated from soil in the Federal District (SDF strains). The software antiSMASH 6.0 was used to track the genetic information involved in specialized metabolites syntheses, known as biosynthetic gene clusters (BGC). Metabolic pathway reconstruction to detect the enzyme methylerythrotol phosphate (MEP), engaged in the terpene synthesis in prokaryotes, was also investigated using Pathway Tools 26.5. The results indicate that part of the probed SDF strains has the complete genetic apparatus to produce the terpenes squalene, hopanoid, and lycopene. Phylogenetic analyses based on amino acid sequences of the detected terpene synthases revealed that these catalysts are well conserved, indicating that the potential for terpene production is widely distributed among AEFB. In addition to terpenes, the results showed that AEFB are promising sources of polyketides and non-ribosomal peptides. Therefore, these bacteria are good candidates for prospecting new bioproducts, notably antimicrobials.
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3
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RAFAELLA CHRISTINA ROCHA MOREIRA DA SILVA
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Carbohydrate metabolism influence virulence determinants in Uropathogenic Escherichia coli
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Advisor : TATIANA AMABILE DE CAMPOS
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COMMITTEE MEMBERS :
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TATIANA AMABILE DE CAMPOS
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ANA FLAVIA ALVES PARENTE
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VICENTE DE PAULO MARTINS
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LIVIA PIMENTEL DE SANT ANA DOURADO
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Data: Jun 30, 2023
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Show Abstract
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Urinary tract infections (UTIs) are the most frequent cause of bacterial infections in humans. It is estimated that each year, approximately 150 million people are affected by this pathology worldwide. Responsible for 80%-90% of cases, the Gram-negative enterobacteria known as Uropathogenic Escherichia coli (UPEC), is the most common causative agent of community-acquired UTIs. The ability of UPEC to survive in the urinary tract depends as much on its physiology and metabolism as on its virulence determinants. Recently, it has been demonstrated that there is a link between the transport and metabolism of various carbohydrates and virulence in enterobacteria. However, as the precise role of carbohydrate utilization in the expression and regulation of bacterial virulence has not yet been determined, we seek to understand the impact that the metabolism of different carbon sources has on the expression of fitness and virulence factors in the pathogenesis of UPECs resistant to the multiple drugs (MDR), with emphasis on the expression of type 1 fimbriae. The objective of this work is to understand the effect that the use of different carbon sources has on the expression of genes and mechanisms of virulence of UPECs MDR strains. For this purpose, four different strains of MDR UPECs (representatives of the group A, B1, B2 and D) were submitted to the determination of the profile of resistance to antibiotics, the analysis of the capacity of survival and reproduction (bacterial fitness), of expressing type fimbriae 1 functional, to form biofilm using different carbon sources and to survive in blood and serum. In anaerobic conditions similar to those found along the urinary tract, all UPECs analyzed used D-(-)-Sorbitol as a high-yield carbon source. Three of the four analyzed strains do not use D-(-)-Fructose as a carbon source for their growth. Data obtained from the Yeast Agglutination Assay suggest that D-(-)-Fructose metabolism is related to the expression of functional type 1 fimbriae on the cell surface of three of the four strains analyzed. UPEC 76 did not use D-(-)-Fructose as a carbon source for its growth or to express functional type 1 fimbriae. N-acetyl-D-glucosamine metabolism promotes biofilm formation in UPECs MDR. When grown in the presence of N-acetyl-D-glucosamine as the sole carbon source, three of the four UPECs showed an increased ability to survive in blood and serum. Taken together, our data suggest that the metabolism of different carbon sources (sugars) modulate the expression of virulence factors such as exacerbated growth, expression of functional type 1 fimbriae, biofilm production and serum resistance in different strains of Uropathogenic Escherichia coli resistant to multiple drugs of clinical origin.
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4
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RAFAEL ÍCARO MATOS VIEIRA
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PRODUCTION AND CHARACTERIZATION OF PECTINOLYTIC ENZYMES BY Paecilomyces formosus IN SOLID WASTE FROM COFFEE PROCESSING
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Advisor : EDIVALDO XIMENES FERREIRA FILHO
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COMMITTEE MEMBERS :
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EDIVALDO XIMENES FERREIRA FILHO
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LUIS HENRIQUE FERREIRA DO VALE
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Gilvan Caetano Duarte
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HELDER ANDREY ROCHA GOMES
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Data: Jul 4, 2023
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Show Abstract
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Brazil stands out as the world's largest coffee producer, more than 50 million bags of coffee are produced annually, after coffee processing there is waste generation (husk, pulp, mucilage) in the same proportion, reaching up to 50% of total production. In this work, the potential for pectinase enzyme production by Paecilomyces formosus in Robusta coffee husk residues and the characterization of this enzyme were investigated. In a previous study, it was found that the best cultivation conditions are at 20ºC, 87 rpm, pH 4.0, without supplementation. An enzymatic screening was carried out, revealing that the peak of pectinase production occurs on the seventh day. The sample was concentrated (1.54UI/mL) and a comparison was made between the concentrated portion and the crude extract (0.43UI/mL). The best biochemical conditions for pectinase for pH occurred in acid (pH 4-5) and basic (pH 9-10) ranges, the best temperature was 60ºC, it was activated by the phenolic compounds transferulic acid and tannic acid, Mn2+ and Ca2+ ions and EDTA. Pectinase was stable in terms of thermostability for up to 72 hours at temperatures of 30, 60 and 80ºC in the crude extract and also in the concentrated fraction at 30 and 60ºC. The hydrolysis test demonstrated the ability to degrade the lignocellulosic substrate by pectinase, which was maintained until the final 24-hour period of the test, and there was no pectinolytic activity when using pre-treated coffee husks. The results of the present work demonstrated the potential of pectinase production by Paecilomyces formosus, being characterized, contributing with new information within the biotechnological scenario, making further research necessary.
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5
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Giovanna Alves de Sousa Dutra
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INTRACELLULAR AND EXTRACELLULAR PROTEOME CHARACTERIZATION OF Corynebacterium glutamicum (ATCC 13032) IN THE PRESENCE OF TWEEN 40
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Advisor : LUIS HENRIQUE FERREIRA DO VALE
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COMMITTEE MEMBERS :
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CARLOS ANDRE ORNELAS RICART
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CONSUELO MEDEIROS RODRIGUES DE LIMA
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Gilvan Caetano Duarte
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LUIS HENRIQUE FERREIRA DO VALE
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Data: Jul 27, 2023
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Show Abstract
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Corynebacterium glutamicum is a non-pathogenic bacterium widely used in the industrial production of amino acids, which ranks third worldwide among the most produced molecules through fermentation, reaching millions of tons with increasing numbers in the global market. C. glutamicum has been extensively studied as traditional methods used for amino acid production are cost-intensive, and alternatives are being sought to reduce production costs and increase productivity. In this context, proteomics has played a crucial role in understanding and improving the efficiency of amino acid production, as the understanding of biosynthetic pathways relies on the interpretation of proteomic analyses. Proteomics offers the possibility of describing and characterizing the functioning of metabolic pathways, including the identification of post-translational modifications (PTMs) and protein degradation. Thus, the present study aimed to identify and characterize proteins and protein complexes involved in the amino acid biosynthetic pathways of this bacterium, particularly in glutamate production with the influence of Tween 40, using a bottom-up proteomic approach with mass spectrometry, analyzing the intracellular and extracellular proteomic profiles. Analysis of amino acids secreted by the microorganism resulted in a glutamate concentration of 0.70 ± 0.14 mM after 18 hours of cultivation in the Tween 40 condition, while in the control condition, it was 0.04 ± 0.02 mM (t-test p<0.05). Protein complexes were separated via BN-PAGE electrophoresis, revealing intracellular protein complexes ranging from 20 kDa to 720 kDa. Main proteins involved in C. glutamicum metabolism were identified, such as enolase and glyceraldehyde-3-phosphate dehydrogenase, belonging to the glycolytic pathway, and glutamine synthetase, involved in glutamate biosynthesis. Analysis of the extracellular proteomic profile by SDS-PAGE showed proteins ranging from 10 to 80 kDa, suggesting differences in the abundance of some proteins between the conditions. Bottom-up analysis of the secretome by LC-MS/MS identified some more abundant regulated proteins in the Tween 40 condition, such as 2-oxoglutarate dehydrogenase, which is a precursor for L-glutamate synthesis. These results contribute to a better understanding of the biochemical machinery involved in the production and secretion of amino acids by Corynebacterium glutamicum can aid in the improvement of its scientific manipulation towards biotechnological interests.
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6
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João Marcos Teixeira Martins
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Taxonomic relocation of some pucciniales from the cerrado based on morphophylogenetic analysis
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Advisor : JOSE CARMINE DIANESE
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COMMITTEE MEMBERS :
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CECILIA BEATRIZ FIUZA FAVALI
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LEONARDO SILVA BOITEUX
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ADALBERTO CORREA CAFE FILHO
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DIRCEU MACAGNAN
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Data: Aug 25, 2023
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Show Abstract
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The studies of Pucciniales from the Cerrado carried out at the Coleção Micológica do Herbarário UB (CMHUB) led to the description of 25 new species and, more importantly, five new genera (Batistopsora, Crossopsorella, Kimuromyces, Pseudocerradoa, Raveneliopsis), in addition to the reestablishment of the genus Mimema. Furthermore, the set of Pucciniales at CMHUB exceeds 10% of its total inventory, with around 2,500 accessions distributed in 167 different species. However, virtually all material was identified based on morphotaxonomic criteria. Thus, the objective of the research reported here was to systematically initiate the application of the phylogenetic species concept to the Pucciniales collection, leading to the essential analyzes of molecular phylogeny based on sequences of fragments of nuc ITS, nuc rLSU (28S), nuc rSSU (18S) rDNA and Cytochrome-c-oxidase subunit 3 (COX3) of mitochondrial origin. In the present case, the results of molecular taxonomical and phylogenetic analyzes involving the following materials are reported: 1. The species Aplopsora henneni and Phakopsora rossmaniae; 2. Discovery of the second species of Austropuccinia which is a parasite of Licania tomentosa (Oití); 3. Morphological analysis and molecular phylogeny of a new rust infecting an endangered species of the genus Coracoralina (= Paepalanthus); 4. Morphological analysis and molecular phylogeny of the Pouteria (Sapotaceae) rust currently allocated in the genus Catenulopsora; 5. Molecular phylogeny of Esalque, genus with currently undefined allocation. 6. Molecular phylogeny of Dietelia duguetiae. The results of the analyzes showed that: A. henneni and P. rossmaniae are congeneric, but belonging to a new genus to be designated Cerradopsora (in press) (Appendix 1). On the other hand, the phylogenetic analysis of Phakopsora tomentosae showed that it is a second species of the important genus Austropuccinia, until now monospecific, containing only Austropuccinia psidii (Appendix 2). The rust agent of two species of Coracoralina was identified as a new species of Puccinia solidly established both morphologically as well as based on molecular phylogenetic analyses. Catenulopsora hennenae was morphologically reassessed and shown to be phylogenetically close to Cerradopsora species (Appendix 1), within the suborder Raveneliineae. The same occurred with the monospecific genus Esalque, type species Esalque holwayi, previously morphologically well studied, and Dietelia duguetiae needing more morphological details, however, both need phylogenetic analyses. The result indicated that Esalque was not a member of the Raveleniaceae family, as indicated by Hennen et al. (2000), but was placed in Family incertae sedis within Raveneliineae, alongside Neopuccinia and Allodus. The species D. duguetiae, parasite of Duguetia furfuracea (Annonaceae) was morphologically restudied and phylogenetically inserted in the Sphaerophragmiaceae family, confirming the finding of Zhao et al. (2020) and by Aime and McTaggart (2021). Most of the rust-causing fungi in Annonaceae are placed in the Spaherophragmiaceae family. So, it was for D. duguetiae and for the rust agent of Cardiopetalum calophyllum (Annonaceae), yet to be classified.
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7
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MARIANA NOGUEIRA DE MOURA FREITAS
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"Genetic engineering of Komagataella phaffii to increase tolerance to inhibitors present in lignocellulosic hydrolyzate".
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Advisor : JOÃO RICARDO MOREIRA DE ALMEIDA
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COMMITTEE MEMBERS :
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JOÃO RICARDO MOREIRA DE ALMEIDA
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JANICE LISBOA DE MARCO
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SÍLVIA BELÉM GONÇALVES
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VIVIANE CASTELO BRANCO REIS
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Data: Oct 20, 2023
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Show Abstract
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The global energy demand and the concern with the development of a sustainable economy have encouraged the search for processes to obtain chemical compounds with the potential to replace, at least in part, those derived from non-renewable fossil sources. In this context, lignocellulosic biomass presents great potential as an alternative and renewable raw material for the production of high value-added compounds through bioprocesses. One of the main challenges in these processes refers to the presence of inhibitors in lignocellulosic hydrolysate, which are formed or released during the stages of pretreatment and hydrolysis of biomass. These are compounds that can inhibit microbial metabolism and, consequently, the yield and productivity of bioprocesses. The inhibitors are gathered in 3 main groups: organic acids (e.g., acetic, formic and levulinic acids), furaldehydes (furfural and 5-hydroxymethyl-2-furaldehyde) and phenolic compounds. Previous studies demonstrate the potential of the yeast Komagataella phaffii to remain active in the presence of these inhibitors. Thus, physiological and transcriptomic analyses of K. phaffii revealed genes with potential to increase the yeast tolerance to furaldehyde, acetic acid and sugarcane bagasse hydrolysate. This work aims to validate the expression of five putative genes - YJR096W, YDL124W, SAP30, FLR1 and 2661 - in the response of K. phaffii M12 in the presence of different inhibitors present in different fermentative culture conditions. Initially, each of the genes was amplified by PCR and cloned into the pGEM®-T Easy cloning vector. For cloning into the pKLD expression vector, under control of the PGK1 promoter, the BamHI/NotI and BcuI cloning sites were employed. Until then, K. phaffii strains expressing the YJR096W gene have been successfully constructed. Microplate and Erlenmeyer flask assays were performed to evaluate the responses of the recombinant strain to the inhibitors furfural, HMF and lignocellulosic hydrolysate. The control and recombinant strains showed the same growth profiles, substrate consumption and product formation in the presence of HMF 2 g/L, furfural 3 g/L and lignocellulosic hydrolysate 30%. Thus, under the culture conditions evaluated so far, overexpression of the YJR096W gene in K. phaffii M12 did not result in increased tolerance to the presence of the inhibitors furfural, HMF and lignocellulosic hydrolysate. The next steps involve overexpression of the remaining genes and validation of the recombinant strains.
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8
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LARISSA VERGINIA DO NASCIMENTO MIRANDA
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"LARGE-SCALE PRODUCTION OF RECOMBINANT LACASE 1 FROM Cryptococcus neoformans IN THE HETEROLOGOUS EXPRESSION SYSTEM Pichia Pastoris".
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Advisor : PATRICIA ALBUQUERQUE DE ANDRADE NICOLA
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COMMITTEE MEMBERS :
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HERDSON RENNEY DE SOUSA
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LARISSA FERNANDES MATOS
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PATRICIA ALBUQUERQUE DE ANDRADE NICOLA
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VIVIANE CASTELO BRANCO REIS
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Data: Dec 7, 2023
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Show Abstract
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Meningoencephalitis caused by the fungus Cryptococcus neoformans in immunocompromised patients, especially in those with HIV, has resulted in approximately 122,000 deaths per year. The mechanisms of infection and its virulence are the subject of intense investigation. Fungal melanization is one of the factors that enhance its virulence and is catalyzed by an enzyme called laccase 1 (Lac1). Furthermore, beyond the described activity, it has been observed that this enzyme might be one of the factors enabling the fungus to survive within the cerebrospinal fluid (CSF), contributing to its persistence in the brain. The yeast Pichia pastoris serves as a suitable model for protein production. The X-33 strain of this yeast was genetically transformed with a plasmid containing the coding sequence of Lac1, aiming to produce recombinant laccase. The expression of the enzyme in the transformed strains was assessed using ABTS, and optimization was achieved through temporal production tests. It was noted that, in flask cultures, varying induction times did not result in significant differences in protein production. Additionally, it was established that Lac1 exhibits sensitivity to pH changes, with optimal activity within the pH range of 5.1 - 6.1. Moreover, as the scale of production increased, it was observed that there was a need for an increased feeding rate with the inducer methanol.
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9
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LETÍCIA OLIVIER SUDBRACK
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Characterization of an outbreak of bacteremia caused by Pan-resistant Serratia marcescens through different approaches: phenotypic, genotypic and genomic methods.
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Advisor : TATIANA AMABILE DE CAMPOS
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COMMITTEE MEMBERS :
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ANA FLAVIA ALVES PARENTE
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LUIS JANSSEN MAIA
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TANISE VENDRUSCOLO DALMOLIN
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TATIANA AMABILE DE CAMPOS
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Data: Dec 15, 2023
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Show Abstract
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A resistência antimicrobiana (RAM) acontece quando bactérias não respondem às terapias ofertadas tornando as infecções mais difíceis de serem tratadas podendo levar à morte do paciente. Serratia marcescens é uma enterobactéria responsável por causar infecções relacionadas à assistência à saúde (IRAS) e, frequentemente, apresentam RAM. Esta espécie é considerada um agente causador de infecções oportunistas de tratamentos desafiadores devido à presença de vários tipos de mecanismos promotores de RAM. Neste contexto, é fundamental que se amplie o estudo dos mecanismos de disseminação de RAM visando o diagnóstico rápido e preciso, além do rastreamento epidemiológico para interromper a cadeia de transmissão de RAM dentro do ambiente. No presente trabalho, Quatro (4) isolados de Serratia marcescens causadores de bacteremia em pacientes internos em uma Unidade de Terapia Intensiva (UTI) foram submetidos à determinação do perfil de susceptibilidade antimicrobiana e de epidemiologia molecular. A determinação do perfil de susceptibilidade antimicrobiana foi determinada por testes fenotípicos (antibiograma por métodos automatizados, disco difusão e imunocromatografia), genotípico para a detecção de beta-lactamases (PCR) e por genômica (sequenciamento WGS). O delineamento da cadeia de transmissão dos isolados foi determinado por epidemiologia molecular através da análise de fingerprinting de sequencias repetitivas intergênicas para enterobactérias (ERIC-PCR) e por pangenoma. As análises fenotípicas e de genômica para não susceptibilidade demonstram que todos os isolados se apresentaram resistentes a todos antimicrobianos disponíveis sendo classificados como pan-resistentes (PDR). A imunocromatografia identificou as carbapenemases KPC e NDM em todos os isolados, contudo a genotipagem por PCR identificou KPC apenas em 2 isolados e a genômica não identificou a enzima em nenhum deles. As análises de similaridade genética por ERIC-PCR e pangenomica determinou que os isolados se apresentaram filogeneticamente idênticos e, portanto, clonais. Em sua totalidade os resultados indicam que análises genômicas por WGS são ferramentas precisas e robustas para o diagnóstico de RAM, contudo a mesma deve ser realizada imediatamente após o isolamento bacteriano para evitar diagnósticos imprecisos devido a perdas de elementos genéticos instáveis, como no caso de blaKPC que codifica a enzima KPC. A análise de pan-genoma, também, demonstrou-se precisa para a identificação de clo
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Thesis |
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1
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Luana Assis Serra
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Thermophilic fungi: enzymatic potential, biochemical characterization of lignocellulolytic enzymes and its biotechnological applications
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Advisor : JOÃO RICARDO MOREIRA DE ALMEIDA
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COMMITTEE MEMBERS :
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CRISTIANE SANCHEZ FARINAS
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ELIANE FERREIRA NORONHA
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JESSICA CARVALHO BERGMANN
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JOÃO RICARDO MOREIRA DE ALMEIDA
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NEI PEREIRA JUNIOR
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Data: Feb 3, 2023
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Show Abstract
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Lignocellulosic biomass is a renewable source that has great potential for producing energy and chemical compounds in a more sustainable way. Enzymatic cocktails containing different classes of lignocellulolytic enzymes (cellulases, hemicellulases and auxiliary proteins) are needed to deconstruct the biomass and release the sugars present in its composition. Thermophilic filamentous fungi have an arsenal of enzymes that can be applied in these cocktails and in other sectors of the industry. In this context, this work aims to evaluate the enzymatic arsenal of Humicola grisea var. thermoidea and characterize thermophilic enzymes from filamentous fungi and its potential applications. Initially, the genome and transcriptome of the fungus H. grisea var. thermoidea were obtained for the first time. The fungus genome was 28.75 Mbp in size, containing 8736 putative genes. The analysis of its transcriptome showed that it expresses more than 200 genes for enzymes capable of degrading carbohydrates when cultivated in an inducing carbon source (sugarcane bagasse), and also the differential expression of genes when the fungus is cultivated in alkaline pH (pH 8) instead of acidic (pH 5). At the same time, the expression and characterization of a putative pectinase and esterase of the thermophilic fungus Thermomyces lanuginosus was sought. The yeast Komagataella phaffii was utilized to express the genes coding for a polygalacturonase (TL-PG1) and a feruloyl esterase (TL-FE1). The genes were cloned into the pPICZA expression vector under the control of the AOX1 promoter. Recombinant clones were successfully obtained, and the expression of the heterologous protein was confirmed by SDS-PAGE and Western-blot. The enzymes TL-PG1 and TL-FE1 were detected by both techniques and purified on a His-tag affinity column, reaching concentrations of 1.45 and 0.76 mg/mL, respectively. The TL-PG1 enzyme has an activity of 462,6 U/mL using polygalacturonic acid (PGA) as substrate under optimal conditions (pH 6 and 55 ˚C). In addition, the TL-PG1 demonstrated synergy in the hydrolysis of lignocellulosic biomass when used in conjunction with Ctec3 and efficiency in bioscouring fabric (denim) for application in the textile industry. Finally, a small-scale method was developed to evaluate the enzymebased liquefaction potential of corn-derived pellets. This method allowed the evaluation of the effectiveness of previously produced heterologous proteins in the enzymatic liquefaction of pelleted corn stover biomass.
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2
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Macária Ferreira Duarte
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Metagenomic analysis of plant virus sequences in sewage and characterization of putative ORFN1 from pepper ring spot virus
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Advisor : TATSUYA NAGATA
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COMMITTEE MEMBERS :
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ELIANE FERREIRA NORONHA
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RENATO DE OLIVEIRA RESENDE
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ROBERTO RAMOS SOBRINHO
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ROSANA BLAWID
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TATSUYA NAGATA
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Data: Apr 27, 2023
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Show Abstract
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The study of viruses is extremely important for society, as it comprises the study of organisms responsible for causing numerous diseases. Regular monitoring of viruses isolated from cultures is very important with regard to plant virology. Fast and accurate identification of potentially dangerous viruses can prevent serious outbreaks in economically important crops. Currently, High Throughput Sequencing (HTS) technologies have become an affordable and powerful tool for this purpose. In this study, we evaluated the use of wastewater samples to monitor plant viruses using HTS analysis and RT-qPCR. Plant viruses belonging to 12 virus families were found, of which Virgaviridae, Solemoviridae, Tymoviridae, Alphaflexiviridae, Betaflexiviridae, Closteroviridae and Secoviridae were the most abundant with more than 20 species. In addition, a quarantine virus and a new species of tobamovirus were also discovered. The relevance of processed foods as a source of these viruses in wastewater was also maintained. Pepper tobamo mild mottle virus (PMMoV) was detected in abundance in samples of processed pepper-based foods and sewage samples, and latent common garlic carlavirus (GarCLV) was detected in smaller amounts in samples of dried and fresh garlic and sewage sample. This showed a high explanation between viral abundance in sewage and in processed food sources. The potential use of wastewater for virus research is discussed in this study. In addition to virus monitoring, it is also of great importance to study known viruses that may also have economic importance not only in the phytosanitary aspect, but also technologically, such as the use of expression vectors. Pepper ringpot virus (PepRSV) is the only tobravirus reported in Brazil so far. It has been used as a viral vector for the production of recombinant proteins. However, as it is a virus restricted to Brazil, it is still a little studied virus when compared to other species also used for this purpose. This work also aims to study a putative new ORF N1 of PepRSV, for which mutant constructions of ORF N1 were agroinfiltrated in leaves of Nicotiana benthamiana and its intracellular location, from the fusion of the protein with flowering proteins to be observed by confocal microscope. With the mutant constructions, we can observe distinct symptoms such as the activation of local lesions, wilting and dropping, previously not seen in the original infectious clones. ORFN1/GFP showed localization associated with the endoplasmic reticulum.
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3
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Clara Vida Galrão Corrêa Carneiro
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" Synthetic biology for the production of ethylene glycol in Komagataella phaffii from xylose".
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Advisor : JOÃO RICARDO MOREIRA DE ALMEIDA
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COMMITTEE MEMBERS :
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FERNANDO ARARIPE GONCALVES TORRES
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JOÃO RICARDO MOREIRA DE ALMEIDA
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MÔNICA CARAMEZ TRICHES DAMASO
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NADIELLE TAMIRES MOREIRA MELO UMPIERRE
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VIVIANE CASTELO BRANCO REIS
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Data: Dec 12, 2023
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Show Abstract
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Ethylene glycol (EG) is a versatile molecule, produced in the petrochemical industry and widely used in the manufacture of plastic polymers (PET), anti-freeze, and automotive fluids. It can also be used as a building block monomer to produce molecules in the chemical and pharmaceutical industries. Currently, ethylene glycol production is chemical or semi-biological, generating a negative impact on the environment. Biotechnological production of ethylene glycol can occur by synthetic routes through the reduction of glycolaldehyde, using xylose as a substrate. In the biorefineries context, which are based on the integration of biomass conversion processes, the production of ethylene glycol is advantageous. Therefore, this work aims to develop a process for ethylene glycol production from xylose, a pentose present in sugarcane biomass hydrolysates using the yeast Komagataella phaffii as a platform. To this end, a synthetic metabolic pathway for ethylene glycol production was designed and evaluated by expression in K. phaffii X-33. The complete synthetic pathway for ethylene glycol production was constructed in silico and evaluated in vivo by flask and bioreactor fermentation experiments. The synthetic pathway for ethylene glycol production from xylose employed in this work consists of four enzymes: xylose dehydrogenase (XDH), xylonate dehydratase (XD), aldolase (ALDO), and aldose reductase (ALDR). In addition, to identify and evaluate the functionality of new sequences encoding for xylonate dehydratase (XD) enzymes, the effect of different combinations of XDH and XD on EG production, in recombinant strains of K. phaffii, was analyzed. All the recombinant strains were able to metabolize xylose, in a defined medium, with maximum EG production of 1.34 g/L, results that show the functionality of the synthetic pathway inserted in the yeast. A new XD identified in this work allowed 30% higher EG production than the strain expressing the control XD. Bottlenecks in the synthetic pathway were identified aiming for a better understanding of K. phaffii’ metabolism. This work results contributed to the development of an EG production process through biotechnological routes with renewable substrates.
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