Made available in DSpace on 2016-04-28T20:15:33Z (GMT). No. of bitstreams: 1 2009 - Aline Rodrigues Bernardo.pdf: 1109499 bytes, checksum: 4b0f2adb9ceeb43406ffa4f7d20978ba (MD5) Previous issue date: 2009-02-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The production of specific antibody (IgY) in chickens (Gallus gallus domesticus) and its purification from the yolk of the egg is attracting the interest of the scientific community due to the numerous advantages of the IgG of mammals. Low cost, high volume production, recognition proteins highly conserved in mammals, in addition to preventing the suffering and death of animals for experimentation, are the main advantages. The aim of our study was to develop, standardize and deploy the technology IgY for aviaries production of antibodies with aim to obtain IGY-specific forms to promastigotes of Leishmania (Leishmania) amazonensis. Initially, matching was performed by two methods of precipitation and the comparison between them, to obtain the pure IgY from the yolk of egg white obtained in trade. The method of purification employing polietienoglicol (PEG-6000) was able to extract IgY with purity comparable to commercial standard IgY (Sigma) and more than obtained by the method with 25% ammonium sulfate. Later, chickens were inoculated with three doses of inactivated antigen (promastigotes forms of L. amazonensis) mixed with incomplete Freund's adjuvant, at 0, 14 and 21 days, IM route. High yield of total and specific IgY was obtained, as determined by the methods of Bradford and ELISA, respectively. The IgY antibodies showed high specificity (indirect immunofluorescence), sensitivity and avidity (ELISA) for promastigotes forms of L. amazonensis, from the second immunization. The level of IgY-specific antibodies remained high until the end of the analysis, around 56 days. Based on the results we can conclude that the "Technology IgY" allows to obtain antibodies with high specificity and sensitivity, in ethics, can be used successfully in various fields of science. / A produ??o de anticorpo espec?fico (IgY) em galinhas (Gallus gallus domesticus) e sua purifica??o a partir da gema do ovo vem atraindo o interesse da comunidade cient?fica devido as in?meras vantagens sobre os anticorpos IgG de mam?feros. Baixo custo, produ??o em quantidades elevada, reconhecimento de prote?nas altamente conservadas de mam?feros, al?m de se evitar o sofrimento e morte de animais de experimenta??o, s?o as principais vantagens. O objetivo geral de nosso estudo foi desenvolver, padronizar e implantar a Tecnologia IgY para produ??o de anticorpos avi?rios, tendo como meta a obten??o de IgY-espec?fica para formas promastigotas de Leishmania (Leishmania) amazonensis. Inicialmente, foi realizada adequa??o de dois m?todos de precipita??o e a compara??o entre eles, visando a obten??o da IgY pura a partir da gema de ovos brancos adquiridos no com?rcio. O m?todo de purifica??o empregando polietienoglicol (PEG-6000) foi capaz de extrair IgY com pureza compar?vel ao padr?o IgY comercial (Sigma) e superior a obtida pelo m?todo com sulfato de am?nio 25%. Posteriormente, galinhas foram inoculadas com tr?s doses de ant?geno inativado (formas promastigotas de L. amazonensis) misturados com adjuvante incompleto de Freund, nos tempos 0, 14 e 21 dias, por via intramuscular. Elevado rendimento da IgY total e espec?fica foi obtido, sendo determinado atrav?s dos m?todos de Bradford e ELISA, respectivamente. Os anticorpos IgY apresentaram elevada especificidade (Imunofluoresc?ncia indireta), sensibilidade e avidez (ELISA) para formas promastigotas de L. amazonensis, a partir da segunda imuniza??o. O n?vel de anticorpos IgY-espec?ficos permaneceu elevado at? o final da an?lise, por volta do 56? dia. Com base nos resultados obtidos podemos concluir que a Tecnologia IgY possibilita a obten??o de anticorpos com alta especificidade e sensibilidade, de forma ?tica, podendo ser utilizada com sucesso em diversos campo da ci?ncia.

Gene therapy can potentially treat a wide range of diseases ranging from inherited diseases to cancer. The successful use of nucleic acids to treat genetic diseases is limited by rapid capture and degradation of the nanoparticle by Kupffer cells in the liver. Scavenger receptors on the cell surface, capture both viral and non-viral nanoparticles leading to reduced efficacy. PEG-peptides were found to inhibit scavenger receptors on the surface of Kupffer cells by forming albumin nanoparticles when intravenously dosed. This work explores the development of potent, low-molecular weight PEG-peptide inhibitors. In order to study the in vivo activity of the nanoparticle, an in vivo assay was developed to directly assess the potency of inhibition. High molecular weight polylysine peptides (33.5 kDa) inhibited liver uptake with an IC50 of 18 μM. Incorporation of four leucine residues, to improve albumin binding, allowed for a decrease in PEG molecular weight and number of lysine residues, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys (7.4 kDa) that inhibited scavenger receptors with an IC50 = 20 μM. Further decrease in the PEG molecular weight resulted in the discovery of PEG2kDa- Cys-Tyr- (Leu-Lys4)3-Leu-Lys (4.4 kDa) with potency of 3 μM. The increase in potency could be attributed to a decrease in the zeta potential of the albumin nanoparticle resulting in more efficient scavenger receptor mediated uptake. Co- administration of PEG2kDa- Cys-Tyr-(Leu-Lys4)3-Leu-Lys with a stable PEGylated polyacridine DNA polyplex resulted in inhibition of rapid polyplex uptake by the liver with an IC50 = 11 μM. Other properties including spatial distribution of leucine, hydrophobicity and peptide length were also explored to determine their effect on liver uptake. Hydrophobic peptides resulted in the formation of micelles which were inactive as scavenger receptor inhibitors and exhibited increased liver uptake upon dose escalation. Reduction in the peptide length resulted in peptides that were not captured by the liver. Inhibition scavenger receptors has the potential to improve the efficacy of viral and non-viral nanoparticles. The findings of this work provide a framework for the development of PEG-peptide inhibitors capable of blocking live uptake of viral and non-viral nanoparticles.

In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.

Many diseases have been associated with oxidative stress (OS) which is caused when the production of reactive oxygen species (ROS), such as superoxide (O2•-) and hydroxyl radical (•OH), overcome the scavenging efficiency of living organisms. It is known that ROS production is worsened during traumas related to ischemic events and subsequent reperfusion in which the treatment with fast and effective antioxidants is critical to prevent cell and tissue damage. PEG-HCCs are carbon nanoparticles that showed O2•- and •OH scavenging properties according to electron paramagnetic resonance (EPR) experiments and peroxyl scavenging properties based on oxygen radical absorbance capacity (ORAC) assays. The O2•- quenching capability was also examined in vivo using a mild traumatic brain injury (mTBI) model complicated with hypotension. As result of the PEG-HCCs treatment, the cerebral blood flow (CBF) was restored while normalizing O2•- and nitric oxide (NO•) levels, primarily in the cerebral vasculature

Pathological changes to tumor extracellular matrix (ECM) composition, mechanics, and architecture promote cancer progression and metastasis. Exploration of tumor-ECM interactions using in vitro matrix-mimetic culture systems has largely been restricted to naturally-derived matrix materials that permit limited experimental control. Such study of a novel lung adenocarcinoma model in Matrigel™ (MG) has suggested key matrix cues that mediate epithelial-mesenchymal transition (EMT) and metastasis. In this thesis work, synthetic hydrogel scaffolds based on poly(ethylene glycol) (PEG) featuring high experimental control and modular bioactivity were used to study matrix influences on the EMT-prone model line 344SQ. Encapsulation of 344SQ cells in PEG hydrogels modified for cell adhesivity and cell-mediated enzymatic degradability induced formation of lumenized, polarized spheres mimicking the epithelial phenotype observed in three-dimensional MG. Tuning matrix stiffness, adhesive ligand concentration, and ligand spatial presentation altered epithelial morphogenesis. Exploration of the EMT phenotype of PEG-encapsulated 344SQ cells revealed TGFβ-initiated changes in morphology, polarity, expression levels of EMT marker genes and their epigenetic controller, and the organization of cell-secreted ECM. Notably, a potent role for adhesive ligand was illuminated as matrices with low PEG-RGDS concentration even in the absence of TGFβ induced formation of spheres with a post-EMT phenotype by several of these measures. A matrix-invasive phenotype was also revealed by altering matrix structural parameters and tuned with incorporation of an alternative protease-cleavable sequence. Finally, the influence of cell-cell contacts was explored by covalent incorporation of cadherin proteins into the matrix. Matrix-tethered E- and -N-cadherin affected 344SQ sphere development in otherwise non-cell-adhesive matrices and modulated polarity and the degree of TGFβ response. Further, in 344SQ with a knockdown of the essential polarity-determining protein Scribble, matrix-tethered cadherin influenced the formation of a phenotype with partially normalized epithelial polarity with corresponding differences in membrane localization of cell-expressed E-cadherin. Overall, this thesis demonstrates the utility of the more experimentally controllable PEG system in studying ECM influences on cancer progression with findings providing greater insight into stromal biomechanical, biochemical, and cell-cell factors that mediate lung adenocarcinoma epithelial morphogenesis and EMT. These contributions help advance the state of the field towards a goal of developing new metastasis-targeting cancer therapeutics.

The groupers are vital fish in the market of over 350 million dollars, while grouper nervous necrosis virus (NNV) has caused mass mortality at about 100% in larvae and juveniles, which impacts on economic of marine cultured fish. The monoclonal antibody is one of the best methods to identify the epitopes on the 3D structure. For evaluation, the Balb/c mice were injected with DGNNV and virus-like particles (VLPs) in this study. The results showed that ascite of mAb-cells produced 1200 times higher than the cell secretion in the medium whereas our best clone hAb_VLP8 can only produced 100 times less antibody than the cell secretion. In the meantime before the monoclonal producer is established, the hAb_VLP8 could be used for ascite production to gain high antibody production.

Both position and angular errors during the insertion process, which cannot be easily predicted because of indeterminate collision situations, may cause failure of the assembly. One of the frequently applied strategies is to use a passive remote center compliance. We break the insertion problem down in to two phases: chamfer-crossing, and inserting (after chamfer-crossing)phase. In this article, the relationship between the position and angular errors during chamfer-crossing with different chamfer size and locality are thoroughly analysis. We also try to design a technological processes of minimizing the angular errors during chamfer-crossing. Besides single round peg insertion, two dimensional dual peg-in-hole insertion problems are briefly analysis.

第23回名古屋大学年代測定総合研究センターシンポジウム平成22(2010)年度報告

Neural electrodes are an important part of brain-machine interface devices that can restore functionality to patients with sensory and movement impairments including spinal cord injury and limb loss. Currently, chronically implanted neural electrodes induce an unfavorable tissue response which includes inflammation, scar formation, and neuronal cell death, eventually causing loss of electrode functionality in the long term. The objective of this research was to develop a coating to improve the tissue response to implanted neural electrodes. The hypothesis was that coating the surface of neural electrodes with a non-fouling, anti-inflammatory coating would cause reduced inflammation and a better tissue response to the implanted electrode. We developed a polymer coating with non-fouling characteristics, incorporated an anti-inflammatory agent, and engineered a stimulus-responsive degradable portion for on-demand release of the anti-inflammatory agent in response to inflammatory stimuli. We characterized the coating using XPS and ellipsometry, and analyzed cell adhesion, cell spreading, and cytokine release in vitro. We analyzed the in vivo tissue response using immunohistochemistry and microarray qRT-PCR. Although no differences were observed among the samples for inflammatory cell markers, lower IgG penetration into the tissue around PEG + IL-1Ra coated electrodes suggests an improvement in BBB integrity. Gene expression analysis showed higher expression of IL-6 and MMP-2 around PEG + IL-1Ra samples, as well as an increase in CNTF expression, an important marker for neuronal survival. An important finding from this research is the increased neuronal survival around coated electrodes compared to uncoated controls, which is a significant finding as neuronal survival near the implant interface is an essential part of maintaining electrode functionality.

Advancements in the biomedical field are driven by the design of novel materials with controlled physical and bio-interactive properties. To develop such materials, researchers rely on the use of highly efficient reactions for the assembly of advanced polymeric scaffolds that meet the demands of a functional biomaterial. In this thesis two main strategies for such materials have been explored; these include the use of off-stoichiometric thiol-ene networks and dendritic polymer scaffolds. In the first case, the highly efficient UV-induced thiol-ene coupling (TEC) reaction was used to create crosslinked polymeric networks with a predetermined and tunable excess of thiol or ene functionality. These materials rely on the use of readily available commercial monomers. By adopting standard molding techniques and simple TEC surface modifications, patterned surfaces with tunable hydrophobicity could be obtained. Moreover, these materials are shown to have great potential for rapid prototyping of microfluidic devices. In the second case, dendritic polymer scaffolds were evaluated for their ability to increase surface interactions and produce functional 3D networks. More specifically, a self-assembled dendritic monolayer approach was explored for producing highly functional dendronized surfaces with specific interactions towards pathogenic E. coli bacteria. Furthermore, a library of heterofunctional dendritic scaffolds, with a controllable and exact number of dual-purpose azide and ene functional groups, has been synthesized. These scaffolds were explored for the production of cell interactive hydrogels and primers for bone adhesive implants. Dendritic hydrogels decorated with a selection of bio-relevant moieties and with Young’s moduli in the same range as several body tissues could be produced by facile UV-induced TEC crosslinking. These gels showed low cytotoxic response and relatively rapid rates of degradation when cultured with normal human dermal fibroblast cells. When used as primers for bone adhesive patches, heterofunctional dendrimers with high azide-group content led to a significant increase in the adhesion between a UV-cured hydrophobic matrix and the wet bone surface (compared to patches without primers). / <p>QC 20140116</p>