Em 1978, o trabalho realizado por Stephenson e Zamecnik demonstrou a capacidade de um oligonucleotídeo de impedir a expressão de uma proteína específica. Atualmente, duas tecnologias são mais utilizadas para este propósito: os oligodeoxiribonucleotídeos antisense e o RNA de interferência (siRNA), que se aproveitam da capacidade de anelação entre as fitas complementares. A maior diferença entre as duas técnicas é a maquinaria proteica recrutada, isso é, o complexo RISC atua no funcionamento do siRNA, e a protease RNase H atua na clivagem da fita de RNA quando hibridizada com DNA. Apesar da grande aplicabilidade destas tecnologias, tanto para doenças metabólicas quanto para canceres, o veículo de entrega e proteção dessas sequências é de fundamental importância, visto que a aplicação desses oligonucleotídeos livres está sujeita à rápida degradação e ineficiência. A modificação das bases é uma das estratégias para conferir maior estabilidade às sequências, porém estas tem sido relacionadas a um aumento da toxicidade. Nessa dissertação, a quitosana, um polissacarídeo catiônico é utilizado para síntese de nanopartículas e encapsulamento dos oligodeoxiribonucleotídeos antisense (ASO). Para isso, foram realizadas modificações na quitosana comercial como despolimerização, trimetilação ou conjugação com PEG, seguida da síntese das nanopartículas com a adição de tripolifosfato de sódio (TPP) pelo método de gelatinização ionotrópica. A estabilidade das nanopartículas foi medida em função do tempo, da variação de temperatura e da diferença de pH. Além disso, a toxicidade dessas nanopartículas foi analisada através da viabilidade celular em diferentes linhagens, NB-4, HepaRG, HTC e BHK-570. A expressão da proteína verde fluorescente (GFP) na célula NB-4 foi utilizada para avaliar a entrega do ASO desenhado, sendo sua fluorescência monitorada por microscopia confocal. Os resultados demonstram que as nanopartículas se mantiveram estáveis durante o período de tempo analisado, assim como com a temperatura variando de 22 a 45°C e em pH ácido. Cada linhagem celular respondeu de forma diferente ao tratamento com as nanopartículas sem ASO, sendo a linhagem saudável BHK-570 com a maior resistência. Ademais, todas as células apresentaram viabilidade reduzida quando tratadas com concentrações na ordem de 1011 nanopartículas/mL a base de quitosana trimetilada. A fluorescência das células NB-4 quando tratada com as nanopartículas com ASO diminuiu consideravelmente nas 18 primeiras horas, seguida de um aumento após 42 horas. Dessa forma, pode-se concluir que as nanopartículas de quitosana propostas nessa dissertação apresentaram uma excelente alternativa para a entrega de material genético, principalmente para o trato gastro-intestinal, devido à sua estabilidade em pH ácido. / The property of an oligonucleotide to interfere in the expression of a protein was observed in 1978 by Stephenson and Zamecnik. To perform such interference, there are today, two main techniques being explored: antisense oligodeoxyribonucleotides and interference RNA. In both cases, the particularity of their chemical structure is taken into account as soon as they can bind in a complementary manner to the messenger RNA and inhibit its translation. The great difference between these techniques is related to the proteases involved in the process, while for interference RNA the RISC machinery acts, for antisense oligodeoxyribonucleotides RNase H cleaves the RNA in the duplex DNA-RNA. Although these tools to edit the translation process are relevant to the treatment and even cure of metabolic disorders and cancers, it is still not effective when employed without a coating to protect the sequences before it reaches the destiny in vivo. Efforts have been made in developing modified bases to be more stable, but they show some toxicity. In this dissertation, chitosan, a natural cationic polyssacharide, is used to produce nanoparticles to protect the antisense oligodeoxyribonucleotide (ASO). For this reason, the commercial chitosan was modified, depolymerized, trimetilated or PEGlated and the nanoparticles were synthesized with sodium tripolyphosphate (TPP) by ionotropic gelation method. The stability along time, in different pHs and temperatures was assessed. The toxicity of nanoparticles without ASO was quantified by MTT tests in NB-4, HepaRG, HTC and BHK-570 cell lines. A green fluorescent protein (GFP) expressed by NB-4 cells was the target to evaluate the delivery efficiency of the ASO, and its fluorescence was measured by confocal microscopy. Results showed that nanoparticles were stable over time as well as in temperatures ranging from 22 to 45°C and in acidic pH. Each cell line responded in a different manner to the treatment, with the health cell BHK-570 showing higher resistance. Furthermore, all of them presented lower viability when treated with trimetilated chitosan nanoparticles in the highest concentrations (ca 1011 nanoparticles/mL). NB-4 cells presented a decrease in fluorescence in 18 hours of treatment followed by an increase after 42 hours. We conclude that chitosan nanoparticles are a good alternative to the delivery of genetic material even more in the gastro intestinal tract due to its great stability in acid pH values.

Solute carriers (SLCs) constitute the largest family of membrane-bound transporter proteins in humans, and they convey transport of nutrients, ions, drugs and waste over cellular membranes via facilitative diffusion, co-transport or exchange. Several SLCs are associated with diseases and their location in membranes and specific substrate transport makes them excellent as drug targets. However, as 30 % of the 430 identified SLCs are still orphans, there are yet numerous opportunities to explain diseases and discover potential drug targets. Among the novel proteins are 29 atypical SLCs of major facilitator superfamily (MFS) type. These share evolutionary history with the remaining SLCs, but are orphans regarding expression, structure and/or function. They are not classified into any of the existing 52 SLC families. The overall aim in this thesis was to study the atypical SLCs with a focus on their phylogenetic clustering, evolutionary conservation, structure, protein expression in mouse brains and if and how their gene expressions were affected upon changed food intake. In Papers I-III, the focus was on specific proteins, MFSD5 and MFSD11 (Paper I), MFSD1 and MFSD3 (Paper II), and MFSD4A and MFSD9 (Paper III). They all shared neuronal expression, and their transcription levels were altered in several brain areas after subjecting mice to food deprivation or a high-fat diet. In Paper IV, the 29 atypical SLCs of MFS type were examined. They were divided into 15 families, based on phylogenetic analyses and sequence identities, to facilitate functional studies. Their sequence relationships with other SLCs were also established. Some of the proteins were found to be well conserved with orthologues down to nematodes and insects, whereas others emerged at first in vertebrates. The atypical SLCs of MFS type were predicted to have the common MFS structure, composed of 12 transmembrane segments. With single-cell RNA sequencing and in situ proximity ligation assay, co-expression of atypical SLCs was analysed to get a comprehensive understanding of how membrane-bound transporters interact.   In conclusion, the atypical SLCs of MFS type are suggested to be novel SLC transporters, involved in maintaining nutrient homeostasis through substrate transport.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-07-17T21:50:50Z No. of bitstreams: 1 Solange Almeida dos Santos.pdf: 759875 bytes, checksum: f16dbd72a00c1e89bdc4543f27aacd3f (MD5) / Made available in DSpace on 2018-07-17T21:50:50Z (GMT). No. of bitstreams: 1 Solange Almeida dos Santos.pdf: 759875 bytes, checksum: f16dbd72a00c1e89bdc4543f27aacd3f (MD5) Previous issue date: 2016-12-19 / Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by chronic and systemic inflammation, which leads to destruction of the cartilage and bone, and affects tissues in multiple joints. Oxidative stress has been implicated in involvement in various disease conditions, such as rheumatoid arthritis (RA). In vivo experimental studies using photobiomodulation therapy (FBM) have shown positive effects in reducing lipid peroxidation and increasing antioxidant activity. The regular practice of physical exercise has also been reported as a beneficial treatment capable of reducing oxidative damage. The aim of this research was to analyze the effect of photobiomodulation therapy at 2 joules and 4 joules doses associated with physical exercise on oxidative stress in an experimental model of rheumatoid arthritis in protein expression: superoxide dismutase (SOD); Glutathione Peroxidase (GPX) and Catalase (CAT) on thiobarbituric acid reactive substances (TBARS). Twenty-four male Wistar rats divided into 4 groups were submitted to an AR model (CIA). First immunization were performed at the base of the tail on the days 0 and 07, and after 28 days the third dose was administered intra-articular in both knees of the animals. After the last induction, FBM therapy was started immediately, transcutaneously at two points: medial and lateral, with a total of 15 applications. Treadmill exercise started the day after the last induction and lasted 5 weeks. As results we obtained the decrease of the lipid peroxidation and the increase of antioxidant activities of SOD, GPX and CAT with physical exercise associated to FBM in doses of 2 joules and 4 joules. Conclusion: Physical exercise associated with FBM therapy decreases lipid peroxidation and increases antioxidant activity. / A artrite reumatóide (AR) é uma doença inflamatória crônica redicivante caracterizada por uma inflamação crônica e sistêmica. O estresse oxidativo tem sido referido no envolvimento em várias condições de doenças, como artrite reumatóide (AR). Estudos experimentais in vivo, utilizando a terapia de fotobiomodulação têm demonstrado efeitos positivos na diminuição da peroxidação lipídica, e no aumento das atividades antioxidantes. A prática regular de exercício físico também vem sendo relatada como um tratamento benéfico capaz de diminuir os danos oxidativos. Sendo assim, esta pesquisa tem por objetivo analisar os efeitos da terapia de fotobiomodualçao nas doses 2 joules e 4 joules associado ao exercício físico sobre marcadores de estresse oxidativo em modelo experimental de artrite reumatóide. Foram analisadas expressão proteica: superóxido dismutase (SOD); e Glutationa Peroxidase (GPX) e Catalase (CAT), sobre as substâncias reativas ao ácido tiobarbitúrico (TBARS). 24 ratos machos wistar divididos em 4 grupos foram submetidos a um modelo de AR (CIA), 1ª imunização realizada na base da cauda nos dias 0, 07, e após 28 dias foi administrada 3ª dose intra-articular em ambos joelhos dos animais. Após última indução a terapia de fotobiomodulação foi iniciada imediatamente, por via transcutânea em dois pontos: medial e lateral, as aplicações seguintes aconteceram em dias alternados, totalizando 15 aplicações. O exercício na esteira começou no dia subsequente a última indução e teve duração de 5 semanas. Como resultados obtivemos a diminuição da peroxidação lipídica e aumento das atividades antioxidantes da SOD, GPX e CAT com exercício físico associado a terapia de fptpbiomodulação nas doses de 2 joules e 4 joules. Conclusão: O exercício físico associado a terapia de fotobiomodulação diminui peroxidação lipídica e aumenta atividades antioxidantes.

abstract: Flaviviruses (FVs) are among the most medically important arboviruses of the world with the Dengue virus (DENV) accounting for a large percentage of infections observed in tropical and subtropical regions of the world. Globalization, travel, and the expanding range of mosquito vectors, such as Aedes aegypti, have increased the potential of infection rates and illnesses associated with FVs. The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV. A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...

NMDA receptor is necessary for excitatory transmission in the central nervous system. Altered funtion of the NMDA receptors is associated with many neurodegenerative and neuropsychiatric diseases. All available crystal structures of the NMDAR meant great shift towards our understanding of details of the receptor and its function. Unfortunately, these up- to-date available structures present only certain functional states of receptors and also a few structural data are still missing. For complete comprehension of the process of activation and deactivation of NMDA receptors, we need to supplement the current information with more data. The aim of this thesis was to employ a combination of different approaches (computational modelling, cloning, biochemistry, protein expression and purification and mass spectrometry) to obtain new structural data, by which we would be able to fill in the gaps in current receptor models, especially at various functional states of the receptor. Key words: NMDA receptor, glutamate receptor, computational modelling, structure, cloning, protein expression

The aryl hydrocarbon receptor (AHR) is a transcription factor which heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) to regulate downstream gene transcription. For the purpose of studying the crystal structure of human aryl hydrocarbon receptor (hAHR), it is essential to obtain abundant amount of pure recombinant protein.Basing on the benefits of using P. pastoris system to produce recombinant protein, including appropriate folding, secretion of interest proteins to the external environment of the cell, and easier purification process of protein due to the its limited production of endogenous secretory proteins [1], our lab chose P. pastoris yeast as the host to overexpress human AHR. My lab has successfully used the protease-deficient P. pastoris (ySMD1163) strain to express AHR [2], but unfortunately the yield is modest, presumably due to low copy number. My work addressed whether increasing the copy number of hAHR in the yeast genome would increase the expression level of hAHR in Pichia pastoris. Results from my experiments showed that although the copy number correlated with the expression levels of hAHR, the increased expression of the hAHR largely in the pellet, suggesting that the soluble expression of hAHR can’t be enhanced merely by increasing its production.

<div>Cyclic peptides are important sources of medicines. </div><div>They are advantageous compared to linear peptides because they possess lower flexibility, which allows for high-affinity target binding and enhanced proteolytic stability. Unfortunately, achieving head-to-tail cyclization of peptides is quite challenging, as it is hard to control efficiency and regiospecificity of peptide macrocyclization. Many have attempted to improve peptide cyclization, including the use of different synthetic reagents as well as synthetic techniques to allow amide-bond formation and promote cyclization. While these strategies have offered great potential solutions, the aim of this study is to explore an alternative strategy that utilizes biocatalysis as a method of achieving successful peptide cyclization. Biocatalysis is the use of enzymes as natural process catalysts under artificial in vitro conditions. Biocatalysis is often more environmentally friendly and safer compared to traditional organic synthesis methods. Non-ribosomal peptide synthetases (NRPSs) are one of the major sources of cyclic peptides in nature. These are systems of large multifunctional proteins are organized into functional domains that act as an assembly line to generate peptide natural products. Normally, the thioesterase domain is responsible for hydrolysis and cyclization of the peptide. Recently, a novel cyclase (SurE) that is physically discrete from the NRPS was discovered. Based on this unique quality, we hypothesized that SurE would be easier to express compared to thioesterase domains and, for this reason, SurE could be a fantastic biocatalyst for the cyclization of peptides. To test this, we designed and generated an expression vector for SurE. We then expressed and purified the SurE protein. We also synthesized three linear peptides of varying lengths. To test for SurE activity, we attempted to add N-acetylcysteamine (SNAC) to mimic its native substrate. Unfortunately, we were unable to successfully attach the SNAC to our linear peptide. To combat this issue, a new synthesis strategy is currently being developed. This work is currently ongoing in the Parkinson lab, with the aim being to test the SurE protein, as well as other PBP-like cyclases, on other modified linear peptides and demonstrate whether the protein has the ability to cyclase a wide scope of peptides.</div><div><br></div>