Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites.

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Scavenger receptor A gene regulatory elements target gene expression to macrophages and to foam cells of atherosclerotic lesions.

Transcription of the macrophage scavenger receptor A gene is markedly upregulated during monocyte to macrophage differentiation. In these studies, we demonstrate that 291 bp of the proximal scavenger receptor promoter, in concert with a 400-bp upstream enhancer element, is sufficient to direct macrophage-specific expression of a human growth hormone reporter in transgenic mice. These regulatory elements, which contain binding sites for PU.1, AP-1, and cooperating ets-domain transcription factors, are also sufficient to mediate regulation of transgene expression during the in vitro differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor. Mutation of the PU.1 binding site within the scavenger receptor promoter severely impairs transgene expression, consistent with a crucial role of PU.1 in regulating the expression of the scavenger receptor gene. The ability of the scavenger receptor promoter and enhancer to target gene expression to macrophages in vivo, including foam cells of atherosclerotic lesions, suggests that these regulatory elements will be of general utility in the study of macrophage differentiation and function by permitting specific modifications of macrophage gene expression.

Calcareous nannofossil stratigraphy and datums of sediments from ODP Leg 198 sites

A relatively complete lower Paleocene to lower Oligocene sequence was recovered from the Southern High of Shatsky Rise at Sites 1209, 1210, and 1211. The sequence consists of nannofossil ooze and clay-rich nannofossil ooze. Samples from these sites have been the target of intensive calcareous nannofossil biostratigraphic investigations. Calcareous nannofossils are moderately preserved in most of the recovered sequence, which extends from nannofossil Zones CP1 to CP16. Most traditional zonal markers are present; however, the rarity and poor preservation of key species in the uppermost Paleocene and lower Eocene inhibits zonal subdivision of part of this sequence.

A simple remote sensing based information system for monitoring sites of conservation importance

Monitoring is essential for conservation of sites, but capacity to undertake it in the field is often limited. Data collected by remote sensing has been identified as a partial solution to this problem, and is becoming a feasible option, since increasing quantities of satellite data in particular are becoming available to conservationists. When suitably classified, satellite imagery can be used to delineate land cover types such as forest, and to identify any changes over time. However, the conservation community lacks (a) a simple tool appropriate to the needs for monitoring change in all types of land cover (e.g. not just forest), and (b) an easily accessible information system which allows for simple land cover change analysis and data sharing to reduce duplication of effort. To meet these needs, we developed a web-based information system which allows users to assess land cover dynamics in and around protected areas (or other sites of conservation importance) from multi-temporal medium resolution satellite imagery. The system is based around an open access toolbox that pre-processes and classifies Landsat-type imagery, and then allows users to interactively verify the classification. These data are then open for others to utilize through the online information system. We first explain imagery processing and data accessibility features, and then demonstrate the toolbox and the value of user verification using a case study on Nakuru National Park, Kenya. Monitoring and detection of disturbances can support implementation of effective protection, assist the work of park managers and conservation scientists, and thus contribute to conservation planning, priority assessment and potentially to meeting monitoring needs for Aichi target 11.

Middle Eocene Fe and Ca counts, Ca/Fe ratios, revised splices and astronomical age models form different Sites of IODP Exp342

Accurate age models are a tool of utmost important in paleoclimatology. Constraining the rate and pace of past climate change are at the core of paleoclimate research, as such knowledge is crucial to our understanding of the climate system. Indeed, it allows for the disentanglement of the various drivers of climate change. The scarcity of highly resolved sedimentary records from the middle Eocene (Bartonian - Lutetian Stages; 47.8 - 37.8 Ma) has led to the existence of the "Eocene astronomical time scale gap" and hindered the establishment of a comprehensive astronomical time scale (ATS) for the entire Cenozoic. Sediments from the Newfoundland Ridge drilled during Integrated Ocean Drilling Program (IODP) Expedition 342 span the Eocene gap at an unprecedented stratigraphic resolution with carbonate bearing sediments. Moreover, these sediments exhibit cyclic lithological changes that allow for an astronomical calibration of geologic time. In this study, we use the dominant obliquity imprint in XRF-derived calcium-iron ratio series (Ca/Fe) from three sites drilled during IODP Expedition 342 (U1408, U1409, U1410) to construct a floating astrochronology. We then anchor this chronology to numerical geological time by tuning 173-kyr cycles in the amplitude modulation pattern of obliquity to an astronomical solution. This study is one of the first to use the 173-kyr obliquity amplitude cycle for astrochronologic purposes, as previous studies primarily use the 405-kyr long eccentricity cycle as a tuning target to calibrate the Paleogene geologic time scale. We demonstrate that the 173-kyr cycles in obliquity's amplitude are stable between 40 and 50 Ma, which means that one can use the 173-kyr cycle for astrochronologic calibration in the Eocene. Our tuning provides new age estimates for magnetochron reversals C18n.1n - C21r and a stratigraphic framework for key sites from Expedition 342 for the Eocene. Some disagreements emerge when we compare our tuning for the interval between C19r and C20r with previous tuning attempts from the South Atlantic. We therefore present a revision of the original astronomical interpretations for the latter records, so that the various astrochronologic age models for the middle Eocene in the North- and South-Atlantic are consistent.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

MiRNA 34a: a therapeutic target for castration-resistant prostate cancer

INTRODUCTION: Development of a therapy for bone metastases is of paramount importance for castration-resistant prostate cancer (CRPC). The osteomimetic properties of CRPC confer a propensity to metastasize to osseous sites. Micro-ribonucleic acid (miRNA) is non-coding RNA that acts as a post-transcriptional regulator of multiple proteins and associated pathways. Therefore identification of miRNAs could reveal a valid third generation therapy for CRPC. Areas covered: miR34a has been found to play an integral role in the progression of prostate cancer, particularly in the regulation of metastatic genes involved in migration, intravasation, extravasation, bone attachment and bone homeostasis. The correlation between miR34a down-regulation and metastatic progression has generated substantial interest in this field. Expert opinion: Examination of the evidence reveals that miR34a is an ideal target for gene therapy for metastatic CRPC. We also conclude that future studies should focus on the effects of miR34a upregulation in CRPC with respect to migration, translocation to bone micro-environment and osteomimetic phenotype development. The success of miR34a as a therapeutic is reliant on the development of appropriate delivery systems and targeting to the bone micro-environment. In tandem with any therapeutic studies, biomarker serum levels should also be ascertained as an indicator of successful miR34a delivery.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Determinantes da utilização de sites de apostas online em Portugal

Com a proliferação da internet assistiu-se desde a década de 90 a um aumento exponencial das casas de jogo online, estando muitas delas traduzidas para português e aceitando euros. Sendo este um tema pouco estudado em Portugal, pretende-se desenvolver e validar um modelo conceptual que reflita os fatores que levam o consumidor a utilizar este tipo de sites. Numa altura em que o governo p g ês p de “ eg iz ” es ivid de, é igualmente interessante perceber qual o perfil do público-alvo, as suas motivações e preferências face à oferta existente. A análise fatorial exploratória, a análise de fiabilidade e os modelos de regressão linear foram as técnicas utilizadas para validar este modelo. Com base num questionário, este estudo mostrou que a expectativa de desempenho, influência social, condições facilitadoras, motivações hedónicas, valor do preço, hábito e o risco psicológico, financeiro e de tempo são fatores determinantes da intenção de utilização de sites de jogo online. Do estudo emergem relevantes implicações académicas e para o mundo empresarial.

Genetic variation in growth and wood-quality traits of Corymbia citriodora subsp. variegata across three sites in south-east Queensland, Australia

Ten growth or wood-quality traits were assessed in three nearby Corymbia citriodora subsp. variegata (CCV) open-pollinated family-within-provenance trials (18 provenances represented by a total of 374 families) to provide information for the development of a breeding program targeting both pulp and solid-wood products. Growth traits (diameter at breast high over bark [DBH], height and conical volume) were assessed at 3 and 7 years of age. Wood-quality traits (density [DEN], Kraft pulp yield [KPY], modulus of elasticity [MoE] and microfibril angle [MfA]) were predicted using near-infrared spectroscopy on wood samples collected from these trials when aged between 10 and 12 years. The high average KPY, DEN and MoE, and low average MfA observed indicates CCV is very suitable for both pulp and timber products. All traits were under moderate to strong genetic control. In across- trials analyses, high (>0.4) heritability estimates were observed for height, DEN, MoE and MfA, while moderate heritability estimates (0.24 to 0.34) were observed for DBH, volume and KPY. Most traits showed very low levels of genotype × site interaction. Estimated age–age genetic correlations for growth traits were strong at both the family (0.97) and provenance (0.99) levels. Relationships among traits (additive genetic correlation estimates) were favourable, with strong and positive estimates between growth traits (0.84 to 0.98), moderate and positive values between growth and wood-quality traits (0.32 to 0.68), moderate and positive between KPY and MoE (0.64), and high and positive between DEN and MoE (0.82). However, negative (but favourable) correlations were detected between MfA and all other evaluated traits (−0.31 to −0.96). The genetic correlation between the same trait expressed on two different sites, at family level, ranged from 0.24 to 0.42 for growth traits, and from 0.29 to 0.53 for wood traits. Therefore simultaneous genetic improvement of growth and wood property traits in CCV for the target environment in south-east Queensland should be possible, given the moderate to high estimates of heritability and favourable correlations amongst all traits studied, unless genotype × site interactions are greater than was evident. © 2016 NISC (Pty) Ltd

MEF2C: expression, regulation and interaction with target genes in health and diseases

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015