Self-immolative linkers in polymeric delivery systems

There has been significant interest in the methodologies of controlled release for a diverse range of applications spanning drug delivery, biological and chemical sensors, and diagnostics. The advancement in novel substrate-polymer coupling moieties has led to the discovery of self-immolative linkers. This new class of linker has gained popularity in recent years in polymeric release technology as a result of stable bond formation between protecting and leaving groups, which becomes labile upon activation, leading to the rapid disassembly of the parent polymer. This ability has prompted numerous studies into the design and development of self-immolative linkers and the kinetics surrounding their disassembly. This review details the main concepts that underpin self-immolative linker technologies that feature in polymeric or dendritic conjugate systems and outlines the chemistries of amplified self-immolative elimination.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352 +/- 21 and 272 +/- 25 mu M, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1 degrees C and pH 8.6. Above 37 degrees C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly. (C) 2010 Elsevier Inc. All rights reserved.

O projeto de arquitetura de espaços temporários com o uso de sistema construtivo remontável: um estudo exploratório

The purpose of this research is to study the portable or reassemblable architectures, which, different from conventional architecture (whose designs are of permanent buildings), corresponds to the designing of spaces with temporary purposes. The focus of the study is the architectural design of spaces that are produced from building systems that can to be moved to different places (process of assembly / disassembly / reassembly) in order to identify the types of spaces generated and the processes used in their design / projecting. The aim is to investigate relationships between the initial project conceived based on a Reassemblable Construction System (RCS) and its application in the architectural design of professionals and students in order to contribute to the understanding of the specificities of this type of design activity. To this end it was developed the exploratory research based on multimedia methods, which includes: documentary analysis, technical visits, interviews, surveys, academic exercise and documentation by images. Although the study is not conclusive, the results indicate significant differences between the point of view of the RCS´s designers and its users (architects and architecture students) since the users demonstrated to have some difficulty to access the features provided for the first group, in particular the students. It is also demonstrated that the use of RCSs seems to change the appreciation / hierarchization of the conditions of project design, since, unlike what happens in traditional architectural design, the designers who use them seem to be more concerned with constructive issues, especially the structural elements (support and covering), instead of functionality, aesthetics and even physical characteristics of the site

Lixiviação de potássio em função da textura e da disponibilidade do nutriente no solo

O manejo correto da adubação potássica pode minimizar perdas assim como evitar o esgotamento de K do solo. Objetivou-se estudar a dinâmica do K no perfil do solo em função do teor de argila e do teor do nutriente resultantes da adubação da soja. Foram coletados solos de texturas média e argilosa, que vinham sendo adubados com 0, 60, 120, e 180 kg ha-1 de K2O na soja por 6 anos. O experimento foi conduzido em casa de vegetação, em tubos de PVC estratificados nas profundidades de 0-5, 5-10, 10-20, e 20-40 cm. Na superfície das colunas, aplicaram-se mais 80 kg ha-1 de K2O. Durante 16 semanas, foi aplicada, semanalmente, água em quantidade equivalente a 100 mm de chuva. em cada aplicação, o volume de solução percolada foi determinado, assim como as quantidades de K contidas nessa solução. Após a desmontagem das colunas de solo, foram determinados os teores de K trocável e não-trocável nas diferentes profundidades. A percolação de K foi maior no solo de textura argilosa, que tinha mais K disponível devido ao maior efeito residual da adubação potássica anterior. A intensidade de lixiviação foi proporcional ao teor de K disponível, mas inicialmente a lixiviação foi mais intensa no solo mais arenoso, decrescendo com o tempo. No argiloso, as perdas foram mais constantes. Houve proporcionalidade entre teor inicial e lixiviação no perfil do solo, nas duas formas de K e nos dois tipos de solo. Conclui-se que o efeito residual da adubação potássica aumentou as quantidades de K percolado. A movimentação de K no perfil do solo teve boa relação com o teor inicial de K no solo, resultante da adubação potássica anterior nos dois tipos de solo. A passagem de K considerado não-trocável para trocável foi rápida e influenciou na lixiviação.

Adolescentes e jovens e suas bases de apoio :relações de amizade como suporte social no enfrentamento à violência

The purpose of this work is to map the family and community social supports for adolescents and young students from Bom Pastor Distric, West Zone of Natal/RN, as well as to describe how such resources are used by these individuaIs in that community. Social support refers not only to formal activities or organizations, but also to spontaneous or informal forms of support - friendship and solidarity nets available in the community, affective relations that are meaningful in the lives of children and young people. Our discussion is based on a research performed with 382 adolescents and young students from Jean Mermoz Public School (students from 5th to 11th grades, aged 13 to 14). We emphasized the situations of violence derived from family or community spheres faced by these students. In relation to this specific aspect, we observed the participants more frequently look for help from the informal social supports, mostly from their friends, which indicates that the formal ones are not considered to be effective instruments for social assistance. The search for informal social supports shows the relations informally established in the streets (for instance when they look for help from friends, rei atives or neighbors) have more effect and play an important role in which there are values and affections exchange. Thinking the strengthening of these social links is of extreme importance and leads to the weakening of the hegemonic logics focused on the production of subjects as private identities, and to the amplification of an ethics committed to the disassembly of a sociability anchored to fear, impotence, intolerance, discrimination, and reduction of spaces for circulating and confronting mechanisms of social exclusion. It is crucial that we concentrate our attention to building friendship as a system of reciprocity and affective exchanges, as a space for political actions and production of forms of lives that are potent against social anesthesia

Nucleolar behaviour during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identiied by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia per spicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.

Synaptonemal polycomplexes in spermatids: a characteristic trait of Orthoptera?

Spermatogenesis was analysed in a cricket, Eneoptera surinamensis (Gryllidae, Orthoptera), using ultrathin serial sections and transmission electron microscopy. Special attention was placed on documentation of the development and structure of synaptonemal polycomplexes (PCs) within spermatid nuclei. Pachytene spermatocytes showed the usual tripartite synaptonemal complexes in the nuclear lumen. PCs were situated close to chromosomes at the periphery of spindles in prometaphase I spermatocytes, where microtubule density was low. The PCs are probably incorporated into the daughter nuclei of both meiotic divisions by adhesion to chromosomes. Finally, PCs end up within spermatid nuclei. Analysis of serial sections through three nuclei of young spermatids revealed at least one PC within each. The PCs were intimately attached to an electrondense spherical nuclear body. This topographical correlation was confirmed through inspection of random sections. The PCs may have an affinity to the spherical bodies. In more developed spermatids, PCs and nuclear bodies were missing. Disassembly products of the PCs may play a role in spermatid maturation. In a series of other Orthoptera species, PCs have been reported to occur in the cytoplasm or the nuclei of spermatids. In most other systematic groups, PCs do not form at all or disassemble earlier. The presence of PCs in young spermatids, therefore, seems to be typical of Orthoptera.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Influência de parâmetros construtivos e operacionais na vida em fadiga de uniões aparafusadas em tração

Pós-graduação em Engenharia Mecânica - FEG

Predominant Rab-GTPase amplicons contributing to oral squamous cell carcinoma progression to metastasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EPTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the fast multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

Characterization of anti-silencing factor 1 in Leishmania major

Anti-silencing factor 1 (ASF1) is a histone chaperone that contributes to the histone deposition during nucleosome assembly in newly replicated DNA. It is involved in chromatin disassembly, transcription activation and in the cellular response to DNA damage. In Leishmania major the ASF1 gene (LmASF1) is located in chromosome 20 and codes for a protein showing 67% of identity with the Trypanosoma brucei TbASF1a. Compared to orthologous proteins, LmASF1 conserves the main residues relevant for its various biological functions. To study ASF1 in Leishmania we generated a mutant overexpressing LmASF1 in L. major. We observed that the excess of LmASF1 impaired promastigotes growth rates and had no impact on cell cycle progress. Differently from yeast, ASF1 overproduction in Leishmania did not affect expression levels of genes located on telomeres, but led to an upregulation of proteins involved in chromatin remodelling and physiological stress, such as heat shock proteins, oxidoreductase activity and proteolysis. In addition, we observed that LmASF1 mutant is more susceptible to the DNA damaging agent, methyl methane sulphonate, than the control line. Therefore, our study suggests that ASF1 from Leishmania pertains to the chromatin remodelling machinery of the parasite and acts on its response to DNA damage.

Valutazione di impatto ambientale di un gruppo elettrogeno mediante Life Cycle Assessment: il caso COGEM.

Il presente lavoro ha come oggetto l’analisi di impatto ambientale, svolta mediante la metodologia Life Cycle Assessment (LCA) di un gruppo elettrogeno prodotto da COGEM s.r.l., azienda italiana situata a Castel d’Argile, nel bolognese, con l’obiettivo di supportare eventuali scelte di riprogettazione del prodotto anche in termini di Design for Disassembly. Dopo una prima analisi del contesto attuale in cui si colloca, la metodologia LCA è stata studiata nel dettaglio per poterla poi applicare al prodotto in oggetto. Esso è stato individuato mediante un’analisi delle vendite di COGEM, in seguito si è svolta una fase di raccolta dati e si sviluppata l’analisi LCA usando il software SimaPro 7.1. I risultati ottenuti hanno consentito di individuare le possibili aree di miglioramento dell’impatto ambientale dell’intero ciclo di vita del gruppo elettrogeno. In particolare si sono valutate due soluzioni innovative: un gruppo elettrogeno alimentato a olio vegetale e uno progettato in ottica DFD per consentire un corretto smaltimento dei rifiuti.