Effect of Ethyl Ester L-Lysine Triisocyanate addition to produce reactive PLA/PCL bio-polyester blends for biomedical applications

This paper was accepted for publication in the journal Journal of the Mechanical Behavior of Biomedical Materials and the definitive published version is available at http://dx.doi.org/10.1016/j.jmbbm.2017.02.018We report in this paper the effects of Ethyl Ester L-Lysine Triisocyanate (LTI) on the physical-mechanical properties of Poly(lactide)/Poly(ε-caprolactone) (PLA/PCL) polyesters blends. The PLA/PCL ratios considered were 20/80, 50/50 and 80/20 (wt/wt %) and LTI was added in amounts of 0.0-0.5-1.0 phr. PLA and PCL reacted with LTI during processing in a Brabender twin screw internal mixer to produce block copolymers in-situ. The resulting blends have been characterized by torque measurements, uniaxial tensile tests, Differential Scanning Calorimeter, contact angle measurements with a Phosphate Buffered Saline (PBS) solution, ATR analysis and morphological SEM observations. Experimental results highlighted how LTI enhanced interaction and dispersion of the two components, resulting into a synergic effect in mechanical properties. Mechanical and physical properties can be tailored by changing the blend composition. The most noticeable trend was an increase in ductility of the mixed polymers. Besides, LTI decreased blend’s wet ability in PBS and lowered the starting of crystalline phase formation for both polymers, confirming an interaction among them. These reactive blends could find use as biomedical materials, e.g. absorbable suture threads or scaffolds for cellular growth

Heterogeneous nanocomposite adhesive: Experimental testing and computational multiscale modeling

This study is focused on a multiscale adhesive used for the investigation of bone bonding applications. Additionally hydroxyapatite nanoparticles were added to the adhesive to create a composite in attempts to enhance both the mechanical and biological properties. One of the main objectives was creating an adhesive system that is tailored to the biological environment in which it must operate. A solid adhesive layer as found in most engineering applications would be counterproductive to the bone healing process and thus an alternative solution was sought. A preliminary cell culture demonstrated that a polyurethane based adhesive tested was nontoxic to cells, and had the unique chemistry that would allow it to be processed into a foam. This porous structure is advantageous in a fracture healing scenario since the interconnecting pores aid in cell migration and ingrowth. This heterogeneous nanocomposite foam that is able to provide optimum conditions for the biological environment also presents additional issues that are of interest from a fundamental viewpoint. The material is composed of multiscale features with hydroxyapatite particles at the nano-scale level, and pores at the micro-scale level. This porosity and spatial heterogeneity introduces new challenges and opportunities for characterization and modeling. The experimental testing of this composite adhesive with unique characteristics then also provides support for the development of open issues in multiscale heterogeneous adhesive models

Molecular and genetic characterization of the Drosophila morgue gene

The Drosophila morgue gene was identified as a regulator of programmed cell death and encodes a novel ubiquitination (Ub) protein. Morgue protein contains a distinct combination of functional domains including: a zinc finger, F box, and variant Ub E2 conjugase domain. This unique combination suggests that Morgue may influence protein ubiquitination and targeting to the 26S proteasome for degradation. Morgue has been shown to promote turnover of a conserved anti-apoptotic protein, DIAP1. In the first part of this study, I present a published paper that describes the isolation and initial analysis of morgue. In the second part of this thesis, I present a published review that discusses different models for Morgue function and describes the identification of a morgue orthologue in mosquito, Anopheles gambiae. This study compares the architecture of the two Morgue proteins, as the F box and conjugase domain within the two proteins are located in similar positions, and the F box and conjugase domains of AgMorgue exhibit 54% and 66% amino acid sequence identity to Morgue. Interestingly, the glycine substitution for the active site cysteine in AgMorgue is conserved. A zinc finger motif located at the NH2-terminus was also identified in both Morgue proteins. In the third part of this study, I performed additional P-element excision screens to generate specific loss-of-function morgue alleles. Several viable alleles were obtained; however, the mutants appeared weak and were shown to exhibit decreased locomotor capabilities in adult climbing assays. Analysis of mutant embryos using a number of cell-type specific markers revealed partially penetrant disruptions in the number, position, and morphology of specific neurons and glia in both the central and peripheral nervous system. Lastly, I describe strong genetic interactions between morgue mutants and mutations in the effete gene, which encodes a conserved Ub E2 conjugase that also influences DIAP1 levels and programmed cell death. Thus, in contrast to either morgue or effete mutants alone, animals homozygous for mutations in both these genes exhibit major disruptions in life cycle progression as they arrest in the third instar larval stage. These mutant larvae come to exhibit several morphological and anatomical abnormalities

Análisis sectorial de la industria vitivinicola chilena y expectativas futuras de exportaciones al resto del mundo en el corto y mediano plazo

Tesis (Ingeniero Comercial)RESUMEN EJECUTIVO Durante los últimos 10 años la industria vitivinícola ha presentado un desarrollo notable en el cual las exportaciones se han multiplicado por 5 veces. • No obstante parece estar sufriendo los efectos de la fuerte competencia internacional , que ha estrechado márgenes y rentabilidades y por un mercado interno que en términos de consumo per. capita no logra volver a mostrar los niveles alcanzados anteriormente. Algunos factores que han favorecido a Chile son: • Gran rendimiento del viñedo ,conservando calidad de la uva. • Buenas condiciones climáticas y de suelo para el cultivo de la uva. • Filosofía empresarial moderna y competitiva. • Producción enfocada a la exportación hacia mercados importantes. • Empresas bien organizadas y con tecnología de punta • Enólogos titulados como requisito por ley .. • Buenas estrategias de marketing, comercialización y distribución. • Excelente presentación de los vinos. • Excelente relación precio-calidad. • Elaboración de vinos de alta calidad conforme a los gustos actuales del mercado. • Gran afluencia de turistas norteamericanos y británicos incluyendo interesantes rutas del vino en paquetes turísticos

Drosophila morgue associates with SkpA and polyubiquitin in vivo.

Morgue is a unique ubiquitination protein that influences programmed cell death and circadian rhythms in Drosophila. We have found that over-expression of wild-type Morgue results in organismal lethality. This over-expression phenotype was used as the basis for an in vivo functional assay to investigate the importance of the Morgue zinc finger, F box, Ubiquitin E2 Conjugase Variant (UEV) domain, and active site Glycine residue. Removal of the zinc finger or UEV domain reduced Morgue's ability to induce lethality and enhance cell death. In contrast, lack of the F box as well as several different substitutions of the active site Glycine did not alter Morgue-induced lethality or cell death enhancement. To further characterize Morgue functions, a Flag:Morgue protein was used to isolate Morgue-associated proteins from whole adult Drosophila. Mass spectrometry analysis of the Morgue-associated proteins identified SkpA as well as a ubiquitin multimer. The identification of SkpA is consistent with previous in vitro studies and further suggests Morgue acts in an SCF-type ubiquitin E3 ligase complex. The identification of poly-ubiquitin was unexpected and this interaction had not been previously identified. The associated poly-ubiquitin was found to exhibit a Lys-48 topology, consistent with distinct functions of Morgue in proteasome-mediated protein turnover. Multiple regions of Morgue were subsequently shown to be required for poly-ubiquitin binding. Overall, Morgue is a novel multi-functional ubiquitin-binding protein

Purification of three Morgue-associated proteins.

<p><b>A.</b> Silver staining of an analytical SDS polyacrylamide gel separating proteins from whole fly extracts that associate with Morgue. Protein bands corresponding to Morgue-3xFLAG (∼60 kD) (black arrowhead) as well as the anti-FLAG Ig heavy chain (∼50 kD) are indicated (gray arrowhead). Lanes 1–4 correspond to material purified via the anti-FLAG resin from the following flies: Lane 1: P[<i>da</i>-Gal4]; Lane 2: P[UAS-Morgue3xFlag]; Lane 3: P[<i>da</i>-Gal4], P[UAS-3xFlag:Morgue]; Lane 4: P[<i>da</i>-Gal4], P[UAS-Morgue3xFlag]; Lane 5: Protein molecular weight markers. The three major bands corresponding to Morgue-associated proteins purified are indicated (arrows) and correspond to polypeptides migrating between 28 kD and 20 kD. <b>B.</b> Coomassie Brilliant Blue staining of a preparatory SDS polyacrylamide gel separating Morgue-associated proteins from extracts of P[<i>da</i>-Gal4], P[UAS-Morgue:3xFlag] adult flies purified via anti-FLAG resin. Three major bands (arrows) were excised from the gel and the corresponding polypeptides analyzed via mass spectrometry. Lane on right corresponds to protein molecular weight markers.</p

Overexpression of Morgue results in lethality.

<p>Effects of widespread Morgue expression on fly viability. Counts of heterozygote (balancer) and homozygote non-balancer (+) progeny derived from genetic crosses where P[<i>da</i>-Gal4] was used to drive expression of P[UAS-GFP] or P[UAS-Morgue]. Homozygous P[<i>da</i>-Gal4] or P[<i>da</i>-Gal4],P[UAS-GFP] flies are viable while P[<i>da</i>-Gal4],P[UAS-Morgue] homozygotes are completely lethal. Flies containing two copies of P[<i>da</i>-Gal4] and one copy of P[UAS-Morgue] or vice versa exhibit either complete or significant lethality. UAS-morgue1 and UAS-morgue2 represent independent insertions of P[UAS-Morgue].</p

Substitutions of the Morgue Gly421 residue do not affect Morgue-induced lethality or cell death.

<p><b>A.</b> Schematic representation of Morgue point mutant proteins with Alanine, Cysteine, or Serine substitutions of the active site Glycine (residue 421). <b>B.</b> The progeny derived from P[<i>da</i>-Gal4], P[UAS-MorgueG421X]/TM3 parent flies were examined and the percentage of homozygotes/heterozygotes was determined. As for native Morgue, expression of each Morgue point mutant essentially resulted in completely lethality. Note that the expected percentage of homozygotes/heterozygotes is 50% if the homozygotes are fully viable. <b>C.</b> Enhancement of eye cell death in P[GMR-Gal4], P[UAS-R/Grim] flies by Morgue point mutants. Compared to GFP co-expression of native Morgue with R/Grim results in enhanced levels of eye cell death (evidenced by additional loss of pigment cells). Similar enhancement is observed for co-expression of the MorgueG421A, MorgueG421C, and MorgueG421S point mutants. <b>D.</b> Enhancement of Reaper-induced CNS midline cell death by Morgue Gly421 mutant proteins. The P[52a-Gal4] line was used to drive expression of P[UAS-LacZ] and P[UAS-Reaper] in embryonic CNS midline cells. Expression of Reaper alone does not induce significant amounts of cell death. Co-expression of native Morgue induces increased levels of CNS midline cell death. This enhanced death is somewhat enhanced by Morgue421A but is not significantly altered by either the MorgueG421C or MorgueG421S point mutant proteins. All views are sagittal with anterior to left. Stage 12 (P[UAS-Reaper] and PUAS-Morgue421A]), stage 16 (P[UAS-Morgue and P[UAS-MorgueG421C]), and stage15 (P[UAS-Reaper] and P[UAS-MorgueG421S]) embryos are shown.</p