Elementos de regulação e microRNAs envolvidos na modulação dos níveis de hemoglobina fetal em indivíduos portadores de beta-hemoglobinopatias

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analisi idrogeologica dell'area carsica dei Monti Alburni (Salerno, Campania)

The Alburni Massif is the most important karstic area in southern Italy and It contains about 250 caves. Most of these caves are located on the plateau, between 1500 m a.s.l. and 700 m a.s.l., and only a few reach the underground streams that feed the springs and the deep aquifer. The main springs are Grotta di Pertosa-Auletta (CP1) and Auso spring (CP31), both located at 280 m a.s.l., the first on the south-eastern margin whereas the second on south-west margin, and the springs present in Castelcivita area, the Castelcivita-Ausino system (CP2) and Mulino di Castelcivita spring (CP865), located at 60 m a.s.l.. Some other secondary springs are present too. We have monitored Pertosa-Auletta’s spring with a multiparameter logger. This logger has registered data from November 2014 to December 2015 regarding water level, electric conductivity and temperature. The hydrodynamic monitoring has been supported by a sampling campaign in order to obtain chemical water analyses. The work was done from August 2014 to December 2015, not only at Pertosa but also at all the other main springs, and in some caves. It was possible to clarify the behavior of Pertosa-Auletta’s spring, almost exclusively fed by full charge conduits, only marginally affected by seasonal rains. Pertosa-Auletta showed a characteristic Mg/Ca ratio and Mg2+ enrichment, as demonstrated by its saturation index that always showed a dolomite saturation. All other spring have characteristic waters from a chemical point of view. In particular, it highlights the great balance between the components dissolved in the waters of Mulino’ spring opposed to the variability of the nearby Castelcivita-Ausino spring. Regarding the Auso spring the variable behavior in terms of discharge and chemistry is confirmed, greatly influenced by rainfall and, during drought periods, by full charge conduits. Rare element concentrations were also analyzed and allowed to characterize further the different waters. Based on all these data an updated hydrogeological map of the Alburni massif has been drawn, that defines in greater detail the hydrogeological complexes on the basis of lithologies, and therefore of their chemical characteristics.

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

Neogene and Paleocene-Maestrichtian calcareous nannofossils in sediments of the upper continental rise off New Jersey

Maestrichtian to Holocene calcareous nannofossils from two closely spaced sites on the upper continental rise some 100 miles (161 km) southeast of Atlantic City, New Jersey, were zoned in order to help date a major canyon-cutting event in the late Miocene and to delineate and correlate other hiatuses with seismic stratigraphy. Mid-middle Eocene through middle Miocene sediments (Zones CP14 to CN6) were not recovered in these holes, but nearly all other zones are accounted for. The Eocene section is described in a companion chapter (Applegate and Wise, 1987, doi:10.2973/dsdp.proc.93.118.1987). Nannofossils are generally sparse and moderately preserved in the clastic sediments of Site 604. Sedimentation rates are extremely high for the upper Pleistocene (201 m/m.y. minimum) above a hiatus calculated to span 0.44 to 1.1 Ma. The associated disconformity is correlated with local seismic reflection Horizon Pr . Sedimentation rates continue to be high (93 m/m.y.) down to a second hiatus in the upper Pliocene dated from about 2.4 to 2.9 (or possibly 3.3) Ma. The disconformity associated with this hiatus is correlated with local seismic reflection Horizon P2 and regional Reflector Blue, which can be interpreted to mark either the onset of Northern Hemisphere continental glaciation or circulation changes associated with the closure of the Central American Seaway. Sedimentation rates in the pre-glacial lower Pliocene are only about a third those in the glacial upper Pliocene. A prominent disconformity in the upper Miocene marks a major lithologic boundary that separates Messinian(?) glauconitic claystones above from lower Tortonian conglomeratic debris flows and turbidites below. The debris flows recovered are assigned to nannofossil Zones CN8a and CN7, but drilling difficulties prevented penetration of the bottom of this sequence some 100 m below the terminal depth of the hole. Correlation of the lower bounding seismic reflector (M2/Merlin?) to a drift sequence drilled on the lower rise at DSDP Site 603, however, predicts that the debris flows began close to the beginning of the late Miocene (upper Zone CN6 time) at about 10.5 Ma. The debris flows represent a major canyon-cutting event that we correlate with the beginning of the particularly severe late Miocene glaciations believed to be associated with the formation of the West Antarctic Ice Sheet. The existence of these spectacular debris flows strongly suggest that the late Miocene glacio-eustatic low stand occurred during Vail Cycle TM3.1 (lower Tortonian) rather than during Vail Cycle TM3.2 (Messinian) as originally published. Beneath a set of coalesced regional disconformities centered upon seismic reflection Horizon Au, coccoliths are abundant and in general are moderately preserved at Site 605 in a 619-m carbonate section extending from the middle Eocene Zone CP13b to the upper Maestrichtian Lithraphidites quadratus Zone. Sedimentation rates are 37 m/m.y. in the Eocene down to a condensed interval near the base (Zone CP9). A disconformity is suspected near the Eocene/Paleocene boundary. Sedimentation rates for the upper Paleocene Zone CP8 are similar to those of the Eocene, but Zones CP7 and CP6 lie within another condensed interval. The highest Paleocene rates are 67 m/m.y. down through Zones CP5 and CP4 to a major disconformity that separates the upper Paleocene from the Danian. This hiatus spans about 2.6 m.y. (upper Zone CP3 to lower Zone CP2) and corresponds to the major sea-level drop at the base of Vail Cycle TE2.1. As the most prominent break in this Paleogene section, it may correspond to seismic reflection Horizon A* of the North American Basin. Sedimentation rates from this point to the Cretaceous/Tertiary boundary drop to 11 m/m.y., still high for a Paleocene DSDP section. No major break in deposition could be detected at the Cretaceous/Tertiary boundary.