Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Comparação do controle do inversor trifásico conectado à rede com filtro LCL considerando o amortecimento passivo e ativo

Distributed generation systems must fulfill standards specifications of current harmonics injected to the grid. In order to satisfy these grid requirements, passive filters are connected between inverter and grid. This work compares the characteristic response of the traditional inductive (L) filter with the inductive-capacitive-inductive (LCL) filter. It is shown that increasing the inductance L leads to a good ripple current suppression around the inverter switching frequency. The LCL filter provides better harmonic attenuation and reduces the filter size. The main drawback is the LCL filter impedance, which is characterized by a typical resonance peak, which must be damped to avoid instability. Passive or active techniques can be used to damp the LCL resonance. To address this issue, this dissertation presents a comparison of current control for PV grid-tied inverters with L filter and LCL filter and also discuss the use of active and passive damping for different regions of resonance frequency. From the mathematical models, a design methodology of the controllers was developed and the dynamic behavior of the system operating in closed loop was investigated. To validate the studies developed during this work, experimental results are presented using a three-phase 5kW experimental platform. The main components and their functions are discussed in this work. Experimental results are given to support the theoretical analysis and to illustrate the performance of grid-connected PV inverter system. It is shown that the resonant frequency of the system, and sampling frequency can be associated in order to calculate a critical frequency, below which is essential to perform the damping of the LCL filter. Also, the experimental results show that the active buffer per virtual resistor, although with a simple development, is effective to damp the resonance of the LCL filter and allow the system to operate stable within predetermined parameters.

Utilização de resíduo lignocelulósico na obtenção de chapa de madeira aglomerada homogênea e compósito reforçado com fibra de vidro-E

This research is about the use of the coconut´s endocarp (nucifera linn) and the waste of derivatives of wood and furniture as raw material to technological use. In that sense, the lignocellulosic waste is used for manufacture of homogeneous wood sheet agglomerate (LHWS) and lignocellulosic load which take part of a polymeric composite with fiber glass E (GFRP-WC). In the manufacturing of the homogeneous wood sheet agglomerate (LHWS), it was used mamona´s resin as waste s agglutinating element. The plates were taken up in a hydraulic press engine, heated, with temperature control, where they were manufactured for different percentage of waste wood and coconuts nucífera linn. Physical tests were conducted to determine the absorption of water, density, damp grade (in two hours and twenty-four hours), swelling thickness (in two hours and twenty-four hours), and mechanical tests to evaluate the parallel tensile strength (internal stick) and bending and the static (steady) flexural. The physical test´s results indicate that the LHWS can be classified as bonded wood plate of high-density and with highly water resistant. In the mechanical tests it was possible to establish that LHWS presents different characteristics when submitted to uniaxial tensile and to the static (steady) flexural, since brittle and elasticity module had a variation according to the amount of dry endocarp used to manufacture each trace of LHWS. The GFRP-WC was industrially manufactured by a hand-lay-up process where the fiber glass E was used as reinforcement the lignocellulósic´s waste as load. The matrix was made with ortofitalic unsaturated polyester resin. Physical and mechanical tests were performed in presence of saturated humidity and dry. The results indicated good performance of the GFRP-WC, as traction as in flexion in three points. The presence of water influenced the modules obtained in the flexural and tensile but there were no significant alteration in the properties analyzed. As for the fracture, the analysis showed that the effects are more harmful in the presence of damp, under the action of loading tested, but despite this, the fracture was well defined starting in the external parts and spreading to the internal regions when one when it reaches the hybrid load

Hidrocarbonetos alifáticos em sedimentos de fundo do estuário do Rio Potengi, grande Natal (RN): implicações ambientais

The Potengi river estuary is located in the region of Natal (RN, Brazil), comprising a population of approximately 1,000,000 inhabitants. Besides the dominant urban presence, the estuary has fragments of mangrove forest. The objective of this study is to determine the aliphatic hydrocarbons found in the bottom sediments of this estuary, identifying their levels, distribution and their possible origins through the diagnostic rates, indexes and results comparisons with the local anthropic and natural characteristics. The samples were obtained according to a plan that allowed sampling of the estuary up to 12 km upstream from it as mounth. 36 stations were selected, grouped into 12 cross sections through the course of the river and spaced on average by 1 km. Each section consisted of three stations: the right margin, the deepest point and the left margin. The hydrocarbon n-alkanes from C10 to C36, the isoprenoids pristane and phytane, the unresolved complex mixture (UCM) and the total resolved hydrocarbons were analyzed by gas chromatography. N-alkanes, pristane, phytane and UCM were detected only at some stations. In the other, the concentration was below the detection limit defined by the analytical method (0.1 mg / kg), preventing them from being analyzed to determine the origin of the material found. By using different parameters, the results show that the estuary receives both the input of petrogenic hydrocarbons, but also of biogenic hydrocarbons, featuring a mixture of sources and relatively impacted portions. Based on the characteristics and activities found in the region, it is possible to affirm that petrogenic sources related to oil products enter the estuary via urban runoff or boats traffic, boat washing and fueling. Turning to the biogenic source, the predominant origin was terrestrial, characterized by vascular plants, indicating contribution of mangrove vegetation. It was evident the presence of, at specific points in the estuary, hydrocarbon pollution, and, therefore is recommended the adoption of actions aimed at interrupting or, at least, mitigating the sources potentially capable of damp petrogenic hydrocarbons in the estuary studied.

Progesterone attenuates microglial-driven retinal degeneration and stimulates protective Fractalkine-CX3CR1 signaling

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.

Limited influence of stream networks on the terrestrial movements of three wetland-dependent frog species

Quantifying functional connectivity is essential for understanding factors that limit or promote animal dispersal in fragmented landscapes. Topography is a major factor influencing the movement behavior of many animal species, and therefore the extent of functional connectivity between habitat patches. For pond-breeding frogs, areas of low topographic relief (such as streams or drainage lines) offer damp microhabitats that can facilitate movement through otherwise dry landscapes. However, the extent of topographic bias of frog movements has rarely been quantified. We used a replicated study to compare captures in high- and low-relief transects, for three species from a pond-breeding frog community in southeastern Australia. We captured frogs significantly more often on low-relief transects. However, capture rates decreased with increasing distance from water at similar rates on both high-relief and low-relief transects, and we observed few differences between adult and juvenile movements. Our results suggest that although low-relief drainage lines are important for the pond-breeding frogs in question, ecologists and landscape managers should not discount the role of high-relief locations. Because low-relief drainage lines represent a low proportion of the pond margin, >90% of movements are likely to occur across high-relief locations. Therefore, for the species that we studied, buffer zones designed to conserve only hydrological networks would provide insufficient protection of frequently used pond margins, while drainage lines are unlikely to act as vital networks facilitating connectivity between breeding ponds. Our study suggests that movement across slopes may be most important for facilitating functional connectivity.