Human immunodeficiency virus (HIV) type 2-mediated inhibition of HIV type 1: a new approach to gene therapy of HIV-infection.

Human immunodeficiency virus (HIV) type 2, the second AIDS-associated human retrovirus, differs from HIV-1 in its natural history, infectivity, and pathogenicity, as well as in details of its genomic structure and molecular behavior. We report here that HIV-2 inhibits the replication of HIV-1 at the molecular level. This inhibition was selective, dose-dependent, and nonreciprocal. The closely related simian immunodeficiency provirus also inhibited HIV-1. The selectivity of inhibition was shown by the observation that HIV-2 did not significantly downmodulate the expression of the unrelated murine leukemia virus; neither did the murine leukemia virus markedly affect HIV-1 or HIV-2 expression. Moreover, while HIV-2 potently inhibited HIV-1, the reverse did not happen, thus identifying yet another and remarkable difference between HIV-1 and HIV-2. Mutational analysis of the HIV-2 genome suggested that the inhibition follows a complex pathway, possibly involving multiple genes and redundant mechanisms. Introduction of inactivating mutations into the structural and regulatory/accessory genes did not render the HIV-2 provirus ineffective. Some of the HIV-2 gene defects, such as that of tat and rev genes, were phenotypically transcomplemented by HIV-1. The HIV-2 proviruses with deletions in the putative packaging signal and defective for virus replication were effective in inducing the suppressive phenotype. Though the exact mechanism remains to be defined, the inhibition appeared to be mainly due to an intracellular molecular event because it could not be explained solely on the basis of cell surface receptor mediated interference. The results support the notion that the inhibition likely occurred at the level of viral RNA, possibly involving competition between viral RNAs for some transcriptional factor essential for virus replication. Induction of a cytokine is another possibility. These findings might be relevant to the clinical-epidemiological data suggesting that infection with HIV-2 may offer some protection against HIV-1 infection.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

Enhanced pathologic properties of Dutch-type mutant amyloid beta-protein.

Cerebrovascular amyloid beta-protein (Abeta) deposition is a pathological feature of several related disorders including Alzheimer disease and hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). HCHWA-D is caused by a point mutation in the gene that encodes the Abeta precursor and results in a Glu --> Gln substitution at position 22 of Abeta. In comparison to Alzheimer disease, the cerebrovascular Abeta deposition in HCHWA-D is generally more severe, often resulting in intracerebral hemorrhage when patients reach 50 years of age. We recently reported that Abeta(1-42), but not the shorter Abeta(1-40) induces pathologic responses in cultured human leptomeningeal smooth muscle cells including cellular degeneration that is accompanied by a marked increase in the levels of cellular Abeta precursor and soluble Abeta peptide. In the present study, we show that the HCHWA-D mutation converts the normally nonpathologic Abeta(1-40) into a highly pathologic form of the peptide for cultured human leptomeningeal smooth muscle cells. These findings suggest that these altered functional properties of HCHWA-D mutated Abeta may contribute to the early and often severe cerebrovascular pathology that is the hallmark of this disorder.

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Gene disruptions using P transposable elements: an integral component of the Drosophila genome project.

Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.

Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.

Electrical investigation of p-type 4H-SiC heavily doped by ion implantation

In the last decades, an increasing interest in the research field of wide bandgap semiconductors was observed, mostly due to the progressive approaching of silicon-based devices to their theoretical limits. 4H-SiC is an example among these, and is a mature compound for applications. The main advantages offered 4H-SiC in comparison with silicon are an higher breakdown field, an higher thermal conductivity, a higher operating temperature, very high hardness and melting point, biocompatibility, but also low switching losses in high frequencies applications and lower on-resistances in unipolar devices. Then, 4H-SiC power devices offer great performance improvement; moreover, they can work in hostile environments where silicon power devices cannot function. Ion implantation technology is a key process in the fabrication of almost all kinds of SiC devices, owing to the advantage of a spatially selective doping. This work is dedicated to the electrical investigation of several differently-processed 4H-SiC ion- implanted samples, mainly through Hall effect and space charge spectroscopy experiments. It was also developed the automatic control (Labview) of several experiments. In the work, the effectiveness of high temperature post-implant thermal treatments (up to 2000°C) were studied and compared considering: (i) different methods, (ii) different temperatures and (iii) different duration of the annealing process. Preliminary p + /n and Schottky junctions were also investigated as simple test devices. 1) Heavy doping by ion implantation of single off-axis 4H-SiC layers The electrical investigation is one of the most important characterization of ion-implanted samples, which must be submitted to mandatory post-implant thermal treatment in order to both (i) recover the lattice after ion bombardment, and (ii) address the implanted impurities into lattice sites so that they can effectively act as dopants. Electrical investigation can give fundamental information on the efficiency of the electrical impurity activation. To understand the results of the research it should be noted that: (a) To realize good ohmic contacts it is necessary to obtain spatially defined highly doped regions, which must have conductivity as low as possible. (b) It has been shown that the electrical activation efficiency and the electrical conductivity increase with the annealing temperature increasing. (c) To maximize the layer conductivity, temperatures around 1700°C are generally used and implantation density high till to 10 21 cm -3 . In this work, an original approach, different from (c), is explored by the using very high annealing temperature, around 2000°C, on samples of Al + -implant concentration of the order of 10 20 cm -3 . Several Al + -implanted 4H-SiC samples, resulting of p-type conductivity, were investigated, with a nominal density varying in the range of about 1-5∙10 20 cm -3 and subjected to two different high temperature thermal treatments. One annealing method uses a radiofrequency heated furnace till to 1950°C (Conventional Annealing, CA), the other exploits a microwave field, providing a fast heating rate up to 2000°C (Micro-Wave Annealing, MWA). In this contest, mainly ion implanted p-type samples were investigated, both off-axis and on-axis <0001> semi-insulating 4H-SiC. Concerning p-type off-axis samples, a high electrical activation of implanted Al (50-70%) and a compensation ratio below 10% were estimated. In the work, the main sample processing parameters have been varied, as the implant temperature, CA annealing duration, and heating/cooling rates, and the best values assessed. MWA method leads to higher hole density and lower mobility than CA in equivalent ion implanted layers, resulting in lower resistivity, probably related to the 50°C higher annealing temperature. An optimal duration of the CA treatment was estimated in about 12-13 minutes. A RT resistivity on the lowest reported in literature for this kind of samples, has been obtained. 2) Low resistivity data: variable range hopping Notwithstanding the heavy p-type doping levels, the carrier density remained less than the critical one required for a semiconductor to metal transition. However, the high carrier densities obtained was enough to trigger a low temperature impurity band (IB) conduction. In the heaviest doped samples, such a conduction mechanism persists till to RT, without significantly prejudice the mobility values. This feature can have an interesting technological fall, because it guarantee a nearly temperature- independent carrier density, it being not affected by freeze-out effects. The usual transport mechanism occurring in the IB conduction is the nearest neighbor hopping: such a regime is effectively consistent with the resistivity temperature behavior of the lowest doped samples. In the heavier doped samples, however, a trend of the resistivity data compatible with a variable range hopping (VRH) conduction has been pointed out, here highlighted for the first time in p-type 4H-SiC. Even more: in the heaviest doped samples, and in particular, in those annealed by MWA, the temperature dependence of the resistivity data is consistent with a reduced dimensionality (2D) of the VRH conduction. In these samples, TEM investigation pointed out faulted dislocation loops in the basal plane, whose average spacing along the c-axis is comparable with the optimal length of the hops in the VRH transport. This result suggested the assignment of such a peculiar behavior to a kind of spatial confinement into a plane of the carrier hops. 3) Test device the p + -n junction In the last part of the work, the electrical properties of 4H-SiC diodes were also studied. In this case, a heavy Al + ion implantation was realized on n-type epilayers, according to the technological process applied for final devices. Good rectification properties was shown from these preliminary devices in their current-voltage characteristics. Admittance spectroscopy and deep level transient spectroscopy measurements showed the presence of electrically active defects other than the dopants ones, induced in the active region of the diodes by ion implantation. A critical comparison with the literature of these defects was performed. Preliminary to such an investigation, it was assessed the experimental set up for the admittance spectroscopy and current-voltage investigation and the automatic control of these measurements.

The Gaia-ESO Survey: The analysis of high-resolution UVES spectra of FGK-type stars

Context. The ongoing Gaia-ESO Public Spectroscopic Survey is using FLAMES at the VLT to obtain high-quality medium-resolution Giraffe spectra for about 105 stars and high-resolution UVES spectra for about 5000 stars. With UVES, the Survey has already observed 1447 FGK-type stars. Aims. These UVES spectra are analyzed in parallel by several state-of-the-art methodologies. Our aim is to present how these analyses were implemented, to discuss their results, and to describe how a final recommended parameter scale is defined. We also discuss the precision (method-to-method dispersion) and accuracy (biases with respect to the reference values) of the final parameters. These results are part of the Gaia-ESO second internal release and will be part of its first public release of advanced data products. Methods. The final parameter scale is tied to the scale defined by the Gaia benchmark stars, a set of stars with fundamental atmospheric parameters. In addition, a set of open and globular clusters is used to evaluate the physical soundness of the results. Each of the implemented methodologies is judged against the benchmark stars to define weights in three different regions of the parameter space. The final recommended results are the weighted medians of those from the individual methods. Results. The recommended results successfully reproduce the atmospheric parameters of the benchmark stars and the expected Teff-log  g relation of the calibrating clusters. Atmospheric parameters and abundances have been determined for 1301 FGK-type stars observed with UVES. The median of the method-to-method dispersion of the atmospheric parameters is 55 K for Teff, 0.13 dex for log  g and 0.07 dex for [Fe/H]. Systematic biases are estimated to be between 50−100 K for Teff, 0.10−0.25 dex for log  g and 0.05−0.10 dex for [Fe/H]. Abundances for 24 elements were derived: C, N, O, Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Mo, Ba, Nd, and Eu. The typical method-to-method dispersion of the abundances varies between 0.10 and 0.20 dex. Conclusions. The Gaia-ESO sample of high-resolution spectra of FGK-type stars will be among the largest of its kind analyzed in a homogeneous way. The extensive list of elemental abundances derived in these stars will enable significant advances in the areas of stellar evolution and Milky Way formation and evolution.

The place of DPP-4 inhibitors in the treatment algorithm of diabetes type 2 : a systematic review of cost-effectiveness studies

Tese de mestrado, Epidemiologia, Universidade de Lisboa, Faculdade de Medicina, 2015