Synthesis of Short, Self-Complementary DNA Oligomers and Solution Studies of their Duplex Structure and Formation

A phosphotriester synthesis for short deoxy oligomers was developed based on the RNA synthesis developed in Neilson's laboratory. This synthesis parallels that of Catlin and Cramer (1973) but differs significantly in a number of procedures and reagents used and represents an overall improvement in every aspect of the previous method. This synthetic procedure allows the synthesis of sufficient quantities of DNA for 1H-NMR analysis. The following sequences were successfully synthesized using this procedure: d(GCA) d(AGCT) d(ACGT) d(ACGTp) d (ACGTACGTp). It was found by variable temperature 1H-NMR that the trimer and the tetramer duplexes had a very low Tm (0-10°C) as compared to their RNA counter-parts which had Tms between 29-34°C. This demonstrates quantitatively that the short DNA duplex is significantly less stable than the short RNA duplex. Consequently, sequences of at least five or six bases in length will be required for model studies of DNA duplex stabilities using variable temperature NMR methods. A CD study of d(ACGTACGTp) in conditions of low salt (1M NaCl) and high salt (5M NaCl) demonstrated that a high salt B to Z-helix transition did not occur. Instead, the duplex remained in the right handed B form in both low and high salt.ThesisMaster of Science (MSc

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca(2+)-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca(2+) entry (ROCE). Although store-operated Ca(2+) entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca(2+) imaging. After depleting internal Ca(2+) stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca(2+)]i whereas lower concentrations (≤100 μM) also induced Ca(2+) oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca(2+) oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca(2+) signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca(2+) oscillations (P2X4) and sustained [Ca(2+)]i increases. We demonstrate specific drug effects on purinergic Ca(2+) pathways and provide new pharmacological insights into Ca(2+) oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca(2+)-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro

Hollow resorbable fiber for combined light and drug delivery: fiber development and analysis of release kinetics

A hollow bioresorbable phosphate glass fiber was developed and used for drug and light delivery. The interaction between organic molecules and the fiber’s internal surface was studied. Promising results for the release of Rose Bengal were obtained

L-carnitine co-administration prevents colistin-induced mitochondrial permeability transition and reduces the risk of acute kidney injury in mice

Colistin is a polymyxin antibiotic currently experiencing renewed clinical interest due to its efficacy in the treatment of multidrug resistant (MDR) bacterial infections. The frequent onset of acute dose-dependent kidney injury, with the potential of leading to long-term renal damage, has limited its use and hampered adequate dosing regimens, increasing the risk of suboptimal plasma concentrations during treatment. The mechanism of colistin-induced renal toxicity has been postulated to stem from mitochondrial damage, yet there is no direct evidence of colistin acting as a mitochondrial toxin. The aim of this study was to evaluate whether colistin can directly induce mitochondrial toxicity and, if so, uncover the underlying molecular mechanism. We found that colistin leads to a rapid permeability transition of mitochondria isolated from mouse kidney that was fully prevented by co-incubation of the mitochondria with desensitizers of the mitochondrial transition pore cyclosporin A or L-carnitine. The protective effect of L-carnitine was confirmed in experiments in primary cultured mouse tubular cells. Consistently, the relative risk of colistin-induced kidney damage, calculated based on histological analysis as well as by the early marker of tubular kidney injury, Kim-1, was halved under co-administration with L-carnitine in vivo. Notably, L-carnitine neither affected the pharmacokinetics of colistin nor its antimicrobial activity against relevant bacterial strains. In conclusion, colistin targets the mitochondria and induces permeability transition thereof. L-carnitine prevents colistin-induced permeability transition in vitro. Moreover, L-carnitine co-administration confers partial nephroprotection in mice treated with colistin, without interfering with its pharmacokinetics and antibacterial activity

Effects of acute administration of trimethylamine N-oxide on endothelial function: a translational study

Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery (Roux-en-Y gastric bypass, RYGB), stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg$^{−1}$) 1.5-h before sacrifice and ex-vivo 30-min incubation of thoracic aortas with 10$^{−6}$ M TMAO did not impair vasodilation in response to acetylcholine (Ach), glucagon-like peptide 1, or insulin. Similarly, in lean WT mice (n = 5–6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10$^{−6}$ M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO

Os impactos da evolução do projeto sped em escritórios de contabilidade de Luziânia – GO

Resumo: Com o intuito de modernizar a transmissão de informações fiscais e contábeis dos contribuintes para o fisco, surgiu o projeto Sistema Público de Escrituração Digital (SPED), buscando padronizar as informações contábil e fiscais das empresas. Em constante atualização, torna-se possível o monitoramento dos dados dos contribuintes de forma cada vez mais completa e eficiente, diante disso, percebe-se o papel fundamental da classe contábil para o manuseio dessa ferramenta. O artigo tem por objetivo evidenciar os impactos na rotina dos profissionais contábeis de escritórios de contabilidade da cidade de Luziânia-GO sobre a evolução do projeto SPED. Para alcançar o objetivo proposto, a metodologia utilizada foi de abordagem quantitativa de caráter exploratório, cujo a coleta de dados foi feita por meio de questionário. Aplicando os métodos citados, foram coletadas informações de 10 (dez) escritórios de contabilidade da cidade de Luziânia. Observa-se que a evolução do projeto SPED referente ao que diz respeito aos módulos EFD-Reinf e o E-social tiveram maior impacto na rotina dos escritórios contábeis, que utilizaram ferramentas como os sites de consultoria para adequarem-se às mudanças. Palavras-Chave: Impacto; SPED; evolução; sonegação; escritório

The European Union and United Kingdom's deforestation-free supply chains regulations : implications for Brazil

This paper analyses the potential implications of the proposed European Union Deforestation Regulation (EUDR) and the recently adopted United Kingdom (UK) legislation on deforestation-free supply chains (henceforth ‘the legislation’) for different stakeholders in Brazil. These regulations intend to address global commodity-driven deforestation and forest degradation by ensuring that targeted commodities and products placed on (or exported from) markets are of minimal risk of being associated with - in the EU - deforestation and forest degradation or - in the UK - illegal deforestation. The paper examines potential compliance readiness in cattle, cocoa, coffee, palm oil, soybean and tropical timber supply chains in Brazil, indicating specific challenges that may arise. Through the construction of a “Compliance Likelihood Index”, our research provides comparable indications to policymakers on sectors and stakeholders that may need stronger support to meet the requirements, in order to maintain Brazil's access to EU and UK markets. The paper indicates that coffee is the sector with the highest level of incentivization and smallest hurdles for compliance, while the cattle sector may face stronger challenges to rapidly adjust its production system towards a deforestation-free value chain and prove compliance. Results of our analysis also highlight the need for collaboration between the EU/UK and Brazil in order to promote alignment between domestic and demand-side legislations so that they are mutually reinforcing. Results of this exercise, which has a focus on the producer-country view of demand-side legislation, will contribute to discussions on the merits of different approaches to strengthen the governance of deforestation-risk commodity trade