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ABSTRACTInstantaneous determinations of photosynthetically active radiation (PAR) over the sea from the Ocean and Land Color Instrument (OLCI) on Sentinel-3 are compared with in-situ measurements at the island of Lampedusa in the central Mediterranean Sea. Radiative transfer calculations show that the PAR measured at the island site is representative for open ocean conditions. Satellite data show a good agreement (5.2% positive bias, R2 = 0.97) with in-situ data, in line with similar analyses for other satellite sensors. Larger satellite-in situ differences are found during summer, and the possible role of aerosols in degrading PAR estimate has been investigated by comparing AOD values measured at Lampedusa and derived by OLCI. The relative difference between OLCI and in-situ PAR appears to be negatively correlated with the relative differences between OLCI and in-situ AOD, suggesting that a more accurate determination of AOD, in particular, for cases with AOD > 0.2, mostly related to Saharan dust, may lead to improved satellite PAR estimates.

Ocean surface radiation measurement best practices have been developed as a first step to support the interoperability of radiation measurements across multiple ocean platforms and between land and ocean networks. This document describes the consensus by a working group of radiation measurement experts from land, ocean, and aircraft communities. The scope was limited to broadband shortwave (solar) and longwave (terrestrial infrared) surface irradiance measurements for quantification of the surface radiation budget. Best practices for spectral measurements for biological purposes like photosynthetically active radiation and ocean color are only mentioned briefly to motivate future interactions between the physical surface flux and biological radiation measurement communities. Topics discussed in these best practices include instrument selection, handling of sensors and installation, data quality monitoring, data processing, and calibration. It is recognized that platform and resource limitations may prohibit incorporating all best practices into all measurements and that spatial coverage is also an important motivator for expanding current networks. Thus, one of the key recommendations is to perform interoperability experiments that can help quantify the uncertainty of different practices and lay the groundwork for a multi-tiered global network with a mix of high-accuracy reference stations and lower-cost platforms and practices that can fill in spatial gaps.

An international team of researchers finds high potential for improving climate projections by a more comprehensive treatment of largely ignored Arctic vegetation types, underscoring the importance of Arctic energy exchange measuring stations.

This research identifies the critical risk factors in the management of construction projects and assesses their relationship on the project implementation constraints of cost, time, and quality, in order to gain a deeper understanding of those factors hindering project success in in Caribbean small island developing states experiencing the dilemma of transitioning to be sustainable in economy and environment. Through three-phase surveys, we identify 26 risk factors from five risk source categories that are more region-specific to influence construction project management, discover strongly positive relationships between a set of risk variables and a set of constraints, and estimate the relative importance of every dimension to the integrated risk-constraint canonical correlation. In risk categories, management has the dominant effect followed by design, contractual, and financial source. For implementation constraints, cost exhibits the most dominance, followed by time and quality. Overall, the directions and magnitudes of interactions gauged in the risk-constraint nexus provide a guideline for improving project planning, thus offering a reference value to other developing countries.

Abstract Background Men who have Sex with Men (MSM) represent the risk group that are disproportionately most affected by the human immunodeficiency virus (HIV) and continue to drop-off from the steps of the continuum of HIV services that have been adopted to overcome poor engagement and retention in care. This realist evaluation aimed at: (1) describing the evaluation carried out in Haiti aiming to ascertain why, how and under which circumstances MSM are linked and retained along the continuum, (2) assessing the outcomes of this approach and (3) exploring the motivators and facilitators for the HIV continuum of services through mechanisms and pathways. Methods Guided by a realist approach, first, an initial program theory (IPT) was developed based on literature and frameworks review, participant observations and discussions with stakeholders. Then, the IPT was tested using a mixed method explanatory study: a quantitative phase to build the continuum from a cross-sectional analysis, and a qualitative phase to explore the motivators and facilitators related to proper linkages along the continuum. Finally, the IPT was refined by eliciting the mechanisms and pathways for outcomes improvement. Results The results showed that the current service delivery model is suboptimal in identifying, engaging, linking and retaining MSM, resulting in loss to follow-up at every step of the continuum and failure to fully realize the health and prevention benefits of antiretroviral. However, the mechanisms through which linkages across the continuum can be improved are: self-acceptance, sense of community support and sense of comprehensive and tailored HIV services. These mechanisms are based on 10 different pathways: self-esteem, awareness and pride, perception of HIV risk, pcceptance and HIV status, addressing community stigma, strengthening of MSM organizations and community networks, societal acceptation and tolerance, stigma reduction training for healthcare providers, engagement of peers as educators and navigators and, adapted services delivery through drug dispensing points and mobile technology and financial assistance. Conclusions The study findings show that engagement, adherence and retention to the continuum of HIV service for MSM are affected by a multi-layer of factors, thus highlighting the importance of taking a comprehensive approach to improve the program.

Mastitis is a most common disease of dairy cows and causes tremendous economic loss to the dairy industry worldwide. Somatic cell counts (SCC) reflect the inflammatory response to infections and is a metric used as key indicator in mastitis screening programs, typically within the framework of national milk recording schemes. Besides the determination of total SCC, the differentiation of cell types has been described to be beneficial for a more definite description of the actual udder health status of dairy cows. Differential somatic cell count (DSCC) represents the combined proportion of polymorphonuclear leukocytes (PMN) and lymphocytes expressed as a percentage of the total. The aim of this study was to investigate the relationship between SCC and differential somatic cell count (DSCC) in individual quarter milk samples collected at different time points: at dry-off, after calving and at the lactation peak. We used individual quarter data from farms representing the specialized production system of Parmigiano Reggiano cheese in Northern Italy. Average DSCC values ranged between 44.9% and 56.3%, with higher values (60.4%-72.1%) in milk samples with ≥ 1 million SCC/ml (where the proportion of samples with DSCC > 70% can be as high as 0.73). Moderate overall correlations between DSCC and log(SCC) were estimated (Pearson = 0.42, Spearman = 0.38), with a clear increasing trend with parity and around the lactation peak (e.g. Pearson = 0.59 at 60 DIM in parity 4). Taking SCC values as indicators of subclinical mastitis, DSCC would diagnose mastitis with 0.75 accuracy. Data editing criteria do have an impact on results, with stricter filtering leading to lower correlations between log(SCC) and DSCC. In conclusion DSCC and SCC provide different descriptions of the udder health status of dairy cows which, at least to some extent, are independent. DSCC alone doesn't provide more accurate information than SCC at quarter level but, used in combination with SCC, can be of potential interest within the framework of milk recording programs, especially in the context of selective dry-cow therapy (SDCT). However, this needs further investigation and updated threshold values need to be selected and validated.

Since May 2022, the Mediterranean Sea has been experiencing an exceptionally long marine heatwave event. Warm anomalies, mainly occurring in the Western basin, have persisted until boreal spring 2023, making this event the longest Mediterranean marine heat wave of the last four decades. In this work, the 2022/2023 anomaly is characterized, using in-situ and satellite measurements, together with state of the art reanalysis products. The role of atmospheric forcing is also investigated; the onset and growth of sea surface temperature anomalies is found to be related to the prevalence of anticyclonic conditions in the atmosphere, which have also caused severe droughts in the Mediterranean region over the same period. Analysis of in-situ observations from the Lampedusa station and of ocean reanalyzes reveals that wind-driven vertical mixing led to the penetration of the warm anomalies below the sea surface, where they have persisted for several months, particularly in the central part of the basin. The evolution of the 2022/23 event is compared with the severe 2003 event, to put recent conditions in the context of climate change.

Anthropogenic aerosol emissions are expected to change rapidly over the coming decades, driving strong, spatially complex trends in temperature, hydroclimate, and extreme events both near and far from emission sources. Under-resourced, highly populated regions often bear the brunt of aerosols’ climate and air quality effects, amplifying risk through heightened exposure and vulnerability. However, many policy-facing evaluations of near-term climate risk, including those in the latest Intergovernmental Panel on Climate Change assessment report, underrepresent aerosols’ complex and regionally diverse climate effects, reducing them to a globally averaged offset to greenhouse gas warming. We argue that this constitutes a major missing element in society’s ability to prepare for future climate change. We outline a pathway towards progress and call for greater interaction between the aerosol research, impact modeling, scenario development, and risk assessment communities.

In this work, the stress relaxation behavior of 3D printed PLA was experimentally investigated and analytically modeled. First, a quasi-static tensile characterization of additively manufactured samples was conducted by considering the effect of printing parameters like the material infill orientation and the outer wall presence. The effect of two thermal conditioning treatments on the material tensile properties was also investigated. Successively, stress relaxation tests were conducted, on both treated and unconditioned specimens, undergoing three different strains levels. Analytical predictive models of the viscous behavior of additive manufactured material were compared, highlighting and discussing the effects of considered printing parameters.

Objective: To detect high-risk human papillomavirus (hrHPV) messenger-RNA (mRNA) in urine samples, compare their concordance with cervical samples including HPV 16 & 18/45 genotyping, and to determine the utility in detecting ≥ CIN 2 lesions. Methods: A cohort of 189 non-pregnant patients (age ≥ 25) was recruited in three groups: Group 1 with abnormal pap-smears and hrHPV positivity, Group 2 with normal pap-smears and hrHPV positivity, and Group 3 with normal pap-smears and hrHPV negativity. Urine samples were tested for hrHPV-mRNA and subsequent hrHPV-mRNA genotype if positive. High-risk HPV detection and genotyping were performed using Aptima assays which are validated for cervical HPV testing. Colposcopy results from groups 1 & 2 were analyzed. Results: The sensitivity of urine hrHPV-mRNA detection was 31.5% while the specificity and PPV were above 95% (96.9% & 95.1% respectively) (p < 0.001). The kappa agreement with cervical samples was fair (0.22, p = 0.04). The sensitivity and specificity of urine hrHPV-mRNA genotyping were 20.0% & 100% respectively (p < 0.001) with 100% genotype-specific concordance. The kappa agreement with cervical samples was fair (0.25, p = 0.16). For urine hrHPV-mRNA detection of ASC-H/HSIL when grouped by age ≥ 30, the sensitivity and specificity were 45.4% & 63.9% respectively (p = 0.009). For urine hrHPV-mRNA detection of ≥ CIN 2 for all ages, the sensitivity and specificity were 45.5% & 75.0% respectively (p = 0.03). Conclusion: Using the Aptima Assay, urine hrHPV-mRNA detection is suboptimal for cervical cancer screening but given the high specificity, it has the potential to identify high-grade lesions (≥ CIN 2). Urine hrHPV-mRNA genotyping via this modality is not beneficial in triage settings of normal or abnormal cytology to determine the need for colposcopy.

Boreal fires have increased during the last years and are projected to become more intense and frequent as a consequence of climate change. Wildfires produce a wide range of effects on the Arctic climate and ecosystem, and understanding these effects is crucial for predicting the future evolution of the Arctic region. This study focuses on the impact of the long-range transport of biomass-burning aerosol into the atmosphere and the corresponding radiative perturbation in the shortwave frequency range. As a case study, we investigate an intense biomass-burning (BB) event which took place in summer 2017 in Canada and subsequent northeastward transport of gases and particles in the plume leading to exceptionally high values (0.86) of Aerosol Optical Depth (AOD) at 500 nm measured in northwestern Greenland on 21 August 2017. This work characterizes the BB plume measured at the Thule High Arctic Atmospheric Observatory (THAAO; 76.53∘N, 68.74∘W) in August 2017 by assessing the associated shortwave aerosol direct radiative impact over the THAAO and extending this evaluation over the broader region (60∘N–80∘N, 110∘W–0∘E). The radiative transfer simulations with MODTRAN6.0 estimated an aerosol heating rate of up to 0.5 K/day in the upper aerosol layer (8–12 km). The direct aerosol radiative effect (ARE) vertical profile shows a maximum negative value of −45.4 Wm−2 for a 78∘ solar zenith angle above THAAO at 3 km altitude. A cumulative surface ARE of −127.5 TW is estimated to have occurred on 21 August 2017 over a portion (∼3.1×106 km2) of the considered domain (60∘N–80∘N, 110∘W–0∘E). ARE regional mean daily values over the same portion of the domain vary between −65 and −25 Wm−2. Although this is a limited temporal event, this effect can have significant influence on the Arctic radiative budget, especially in the anticipated scenario of increasing wildfires.

Air–sea heat fluxes are essential climate variables, required for understanding air–sea interactions, local, regional and global climate, the hydrological cycle and atmospheric and oceanic circulation. In situ measurements of fluxes over the ocean are sparse and model reanalysis and satellite data can provide estimates at different scales. The accuracy of such estimates is therefore essential to obtain a reliable description of the occurring phenomena and changes. In this work, air–sea radiative fluxes derived from the SEVIRI sensor onboard the MSG satellite and from ERA5 reanalysis have been compared to direct high quality measurements performed over a complete annual cycle at the ENEA oceanographic observatory, near the island of Lampedusa in the Central Mediterranean Sea. Our analysis reveals that satellite derived products overestimate in situ direct observations of the downwelling short-wave (bias of 6.1 W/m2) and longwave (bias of 6.6 W/m2) irradiances. ERA5 reanalysis data show a negligible positive bias (+1.0 W/m2) for the shortwave irradiance and a large negative bias (−17 W/m2) for the longwave irradiance with respect to in situ observations. ERA5 meteorological variables, which are needed to calculate the air–sea heat flux using bulk formulae, have been compared with in situ measurements made at the oceanographic observatory. The two meteorological datasets show a very good agreement, with some underestimate of the wind speed by ERA5 for high wind conditions. We investigated the impact of different determinations of heat fluxes on the near surface sea temperature (1 m depth), as determined by calculations with a one-dimensional numerical model, the General Ocean Turbulence Model (GOTM). The sensitivity of the model to the different forcing was measured in terms of differences with respect to in situ temperature measurements made during the period under investigation. All simulations reproduced the true seasonal cycle and all high frequency variabilities. The best results on the overall seasonal cycle were obtained when using meteorological variables in the bulk formulae formulations used by the model itself. The derived overall annual net heat flux values were between +1.6 and 40.4 W/m2, depending on the used dataset. The large variability obtained with different datasets suggests that current determinations of the heat flux components and, in particular, of the longwave irradiance, need to be improved. The ENEA oceanographic observatory provides a complete, long-term, high resolution time series of high quality in situ observations. In the future, more similar sites worldwide will be needed for model and satellite validations and to improve the determination of the air–sea exchange and the understanding of related processes.

Using semi-automated software simplifies quantitative analysis of the visible burden of disease on whole-body MRI diffusion-weighted images. To establish the intra- and inter-observer reproducibility of apparent diffusion coefficient (ADC) measures, we retrospectively analyzed data from 20 patients with bone metastases from breast (BCa; n = 10; aged 62.3 ± 14.8) or prostate cancer (PCa; n = 10; aged 67.4 ± 9.0) who had undergone examinations at two timepoints, before and after hormone-therapy. Four independent observers processed all images twice, first segmenting the entire skeleton on diffusion-weighted images, and then isolating bone metastases via ADC histogram thresholding (ADC: 650–1400 µm2/s). Dice Similarity, Bland-Altman method, and Intraclass Correlation Coefficient were used to assess reproducibility. Inter-observer Dice similarity was moderate (0.71) for women with BCa and poor (0.40) for men with PCa. Nonetheless, the limits of agreement of the mean ADC were just ±6% for women with BCa and ±10% for men with PCa (mean ADCs: 941 and 999 µm2/s, respectively). Inter-observer Intraclass Correlation Coefficients of the ADC histogram parameters were consistently greater in women with BCa than in men with PCa. While scope remains for improving consistency of the volume segmented, the observer-dependent variability measured in this study was appropriate to distinguish the clinically meaningful changes of ADC observed in patients responding to therapy, as changes of at least 25% are of interest.

We describe and implement a data selection algorithm aimed at identifying background atmospheric CO2 observations from in situ continuous measurements. Several selection criteria for detecting the background data have been developed and are currently used: the main objective of this work was to define a common methodology to extract the atmospheric background signal minimizing heterogeneities due to the use of different selection algorithms. The algorithm used in this study, (BaDS, Background Data Selection) was tested and optimized using data (from 2014 to 2018) from four Italian stations characterized by markedly different environmental conditions (i.e., mountain, coastal and marine): Plateau Rosa (PRS), Mt. Cimone (CMN), Capo Granitola (CGR) and Lampedusa (LMP). Their locations extend from the Alps to the central Mediterranean. The adopted algorithm proved to be effective in separating the local/regional from the background signal in the CO2 time series. About 6% of the data at LMP, 11% at PRS, 20–38% at CMN and 65% at CGR were identified as non-background. LMP and PRS can be used as reference sites for the central Mediterranean, while CMN and CGR were more impacted by regional sources and sinks. Finally, we discuss a possible application of BaDS screened data.

Atrial myxoma is one of the most common primary cardiac tumors reported in the literature. In very rare instances, stroke has been the sequelae after a myxomatous tumor resection. We report this unique case of late ischemic cerebral event in a 46-year-old female some days after resection of a left atrial myxoma.

This article lists some tips for reducing gear case noise. With this aim, a static analysis was carried out in order to describe how stresses resulting from meshing gears affect the acoustic emissions. Different parameters were taken into account, such as the friction, material, and lubrication, in order to validate ideas from the literature and to make several comparisons. Furthermore, a coupled Eulerian–Lagrangian (CEL) analysis was performed, which was an innovative way of evaluating the sound pressure level of the aforementioned gears. Different parameters were considered again, such as the friction, lubrication, material, and rotational speed, in order to make different research comparisons. The analytical results agreed with those in the literature, both for the static analysis and CEL analysis—for example, it was shown that changing the material from steel to ductile iron improved the gear noise, while increasing the rotational speed or the friction increased the acoustic emissions. Regarding the CEL analysis, air was considered a perfect gas, but its viscosity or another state equation could have also been taken into account. Therefore, the above allowed us to state that research into these scientific fields will bring about reliable results.

In the context of the Copernicus Program, EUMETSAT prioritizes the creation of an ocean color infrastructure for system vicarious calibration (OC-SVC). This work aims to reply to this need by proposing the European Radiometry Buoy and Infrastructure (EURYBIA). EURYBIA is designed as an autonomous European infrastructure operating within the Marine Optical Network (MarONet) established by University of Miami (Miami, FL, USA) based on the Marine Optical Buoy (MOBY) experience and NASA support. MarONet addresses SVC requirements in different sites, consistently and in a traceable way. The selected EURYBIA installation is close to the Lampedusa Island in the central Mediterranean Sea. This area is widely studied and hosts an Atmospheric and Oceanographic Observatory for long-term climate monitoring. The EURYBIA field segment comprises off-shore and on-shore infrastructures to manage the observation system and perform routine sensors calibrations. The ground segment includes the telemetry center for data communication and the processing center to compute data products and uncertainty budgets. The study shows that the overall uncertainty of EURYBIA SVC gains computed for the Sentinel-3 OLCI mission under EUMETSAT protocols is of about 0.05% in the blue-green wavelengths after a decade of measurements, similar to that of the reference site in Hawaii and in compliance with requirements for climate studies.

The following paper is about the analysis and planning of the cash flows at the “Empresa de Aseguramiento y Servicios” of the Agriculture Ministry of Santiago de Cuba with the goal of knowing how is generated and applied the cash at the enterprise in the period of 2008-2009 and its behavior for the year 2013. This paper makes for the five-year period related the Statements of Cash Flows and its interpretation as an analysis tool for the directors of the company, and also its foreseeing for the next period from the making of the Pro forma Financial Statements applying the method of the rate of sustainable growing of the sales. As a result of the analysis it was able to verify the difficulties of the enterprise to generate cash operative flows in opportune quantities and sufficient that limits the operation with levels of adequate solvency.

The planetary boundary layer structure in the coastal areas, and particularly in complex orography regions such as the Mediterranean, is extremely intricate. In this study, we show the evolution of the planetary boundary layer based on in situ airborne measurements and ground-based remote sensing observations carried out during the MORE (Marine Ozone and Radiation Experiment) campaign in June 2010. The campaign was held in a rural coastal Mediterranean region in Southern Italy. The study focuses on the observations made on 17 June. Vertical profiles of meteorological parameters and aerosol size distribution were measured during two flights: in the morning and in the afternoon. Airborne observations were combined with ground-based LIDAR, SODAR, microwave and visible radiometer measurements, allowing a detailed description of the atmospheric vertical structure. The analysis was complemented with data from a regional atmospheric model run with horizontal resolutions of 12, 4 and 1 km, respectively; back-trajectories were calculated at these spatial resolutions. The observations show the simultaneous occurrence of dust transport, descent of mid-tropospheric air and sea breeze circulation on 17 June. Local pollution effects on the aerosol distribution, and a possible event of new particles formation were also observed. A large variability in the thermodynamical structure and aerosol distribution in the flight region, extending by approximately 30 km along the coast, was found. Within this complex, environment-relevant differences in the back-trajectories calculated at different spatial resolutions are found, suggesting that the description of several dynamical processes, and in particular the sea breeze circulation, requires high-resolution meteorological analyses. The study also shows that the integration of different observational techniques is needed to describe these complex conditions; in particular, the availability of flights and their timing with respect to the occurring phenomena are crucial.

New calculations of the relative optical air mass function are made over the 0°–87° range of apparent solar zenith angle θ, for various vertical profiles of background aerosol, diamond dust and thin cirrus cloud particle extinction coefficient in the Arctic and Antarctic atmospheres. The calculations were carried out by following the Tomasi and Petkov (2014) procedure, in which the above-mentioned vertical profiles derived from lidar observations were used as weighting functions. Different sets of lidar measurements were examined, recorded using: (i) the Koldewey-Aerosol-Raman Lidar (KARL) system (AWI, Germany) at Ny-Ålesund (Spitsbergen, Svalbard) in January, April, July and October 2013; (ii) the Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) satellite-based sensor over Barrow (Alaska), Eureka (Nunavut, Canada) and Sodankylä (northern Finland), and Neumayer III, Mario Zucchelli and Mirny coastal stations in Antarctica in the local summer months of the last two years; (iii) the National Institute of Optics (INO), National Council of Research (CNR) Antarctic lidar at Dome C on the Antarctic Plateau for a typical “diamond dust” case; and (iv) the KARL lidar at Ny-Ålesund and the University of Rome/National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA) lidar at Thule (northwestern Greenland) for some cirrus cloud layers in the middle and upper troposphere. The relative optical air mass calculations are compared with those obtained by Tomasi and Petkov (2014) to define the seasonal changes produced by aerosol particles, diamond dust and cirrus clouds. The results indicate that the corresponding air mass functions generally decrease as angle θ increases with rates that are proportional to the increase in the pure aerosol, diamond dust and cirrus cloud particle optical thickness.

In remote marine areas, biogenic productivity and atmospheric particulate are coupled through dimethylsulfide (DMS) emission by phytoplankton. Once in the atmosphere, the gaseous DMS is oxidized to produce H2SO4 and methanesulfonic acid (MSA); both species can affect the formation of cloud condensation nuclei. This study analyses eight years of biogenic aerosol evolution and variability at two Arctic sites: Thule (76.5° N, 68.8° W) and Ny Ålesund (78.9° N, 11.9° E). Sea ice plays a key role in determining the MSA concentration in polar regions. At the beginning of the melting season, in April, up to June, the biogenic aerosol concentration appears inversely correlated with sea ice extent and area, and positively correlated with the extent of the ice-free area in the marginal ice zone (IF-MIZ). The upper ocean stratification induced by sea ice melting might have a role in these correlations, since the springtime formation of this surface layer regulates the accumulation of phytoplankton and nutrients, allowing the DMS to escape from the sea to the atmosphere. The multiyear analysis reveals a progressive decrease in MSA concentration in May at Thule and an increase in July August at Ny Ålesund. Therefore, while the MSA seasonal evolution is mainly related with the sea ice retreat in April, May, and June, the IF-MIZ extent appears as the main factor affecting the longer-term behavior of MSA.

Ettore Grimaldi (1934-2015) was one of the brightest and active ichthyologists in Italy. He was a man of great enthusiasm, boundless energy and great curiosity. He was a person with an open mind. He originally visited the Institute of Pallanza in June 1957 as a master student at the Faculty of Veterinary Medicine, University of Milan. He was formally called back by the Director Vittorio Tonolli, as a research assistant in 1962....

Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX 302 and TMX 306, characterized by TLR7 partial agonistic activity, therefore expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared to equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma and silicosis following LPS, allergen (ovalbumin) and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibro-granulomatous response, the number of silica particles in lung interstitial space and improved respiratory function in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.

In a simple procedure, the intramolecular hydroarylation of N-propargyl-pyrrole-2-carboxamides was accomplished with the aid of gold(III) catalysis. The reaction led to differently substituted pyrrolo[2,3-c]pyridine and pyrrolo[3,2-c]pyridine derivatives arising either from direct cyclization or from a formal rearrangement of the carboxamide group. Terminal alkynes are essential to achieve bicyclic pyrrolo-fused pyridinones by a 6-exo-dig process, while the presence of a phenyl group at the C–C triple bond promotes the 7-endo-dig cyclization giving pyrrolo-azepines.

Lung deep lymphatic drainage (LDLD) plays an important role in the removal of foreign materials from lungs being alveolar macrophages the first line of phagocytic defence with high affinity for pathogenic microorganisms. Bacillus subtilis is a well-known genome-decoded saprophyte of the human respiratory tract used in research and in the biotechnology industry. Lung deep lymphatic chains (LDLC) constitute one of the first sites of lung tumours’ dissemination. In this work we intended to develop and validate a non-invasive method for assessing LDLC by nanoradioliposomes aerosolised modulated on the Bacillus subtilis spore wall. The final goal was to produce a nanoradioliposome formulation that can mimics the dynamics of preferential removal of spores by LDLD and present the ideal properties as a tracer for molecular imaging studies. Seven different liposomal formulations were tested, and the formulation-F demonstrated physicochemical and radiopharmaceutical properties that make it an ideal candidate as an in vivo probe for molecular imaging studies of the LDLC. Nanoradioliposomes of the formulation-F after labelling with 99mTc-HMPAO were administered as aerosols to 20 Sus scrofa. Hilar and interpulmonary communications were visualized in first 5 minutes post-inhalation, infradiaphragmatic chains between 10 and 20 minutes, the ganglia of the aortic chain at 20 minutes and those of the renal hilar region at 30 minutes. Conclusion: the proposed method enables visualization of deep lymphatic lung network and lymph nodes. Besides, this technique involving the modulation of nanoradioliposomes targeting specific organs or tissues may be an important tool for diagnostic or even for therapeutic purposes.A drenagem linfática pulmonar profunda (DLPP) desempenha um papel importante na remoção de materiais estranhos, constituindo os macrófagos alveolares a primeira linha de defesa fagocitária, dada a grande afinidade para microrganismos patogénicos. Os Bacillus subtilis são saprófitas do tracto respiratório humano com ampla utilização em investigação e em biotecnologia. As cadeias linfáticas pulmonares profundas (CLPP) constituem um dos primeiros locais de disseminação de tumores pulmonares. Neste trabalho pretendeu-se desenvolver e validar um método não invasivo para avaliar as CLPP através de nanorradiolipossomas aerosolisados e modulados pela parede do esporo do Bacillus subtilis. O objectivo final foi produzir uma formulação de nanorradiolipossomas capaz de imitar a dinâmica da remoção de esporos pelas CLPP e simultaneamente ter propriedades ideais como traçador para imagiologia molecular. Testámos sete diferentes formulações lipossómicas, tendo a formulação F demonstrado possuir propriedades fisicoquímicas e radiofarmacêuticas que a tornam o traçador ideal para imagiologia molecular in vivo das CLPP. Os nanorradiolipossomas da formulação F após marcação com 99mTc-HMPAO foram administrados sob a forma de aerossóis a 20 Sus scrofa. Visualizaram-se comunicações hilares e interpulmonares nos primeiros 5 minutos após a inalação, as cadeias infradiafragmáticas entre os 10 e os 20 minutos, os gânglios da cadeia aórtica aos 20 minutos e os da região hilar renal aos 30 minutos. Em conclusão, o método proposto visualiza os gânglios linfáticos e a rede linfática pulmonar profunda. A modulação dos nanorradiolipossomas permite que eles atinjam órgãos ou tecidos específicos, conferindo-lhes importantes potencialidades no âmbito do diagnóstico e/ou da terapêutica.

Resumo: A drenagem linfática pulmonar profunda (DLPP) desempenha um papel importante na remoção de materiais estranhos, constituindo os macrófagos alveolares a primeira linha de defesa fagocitária, dada a grande afinidade para microrganismos patogénicos. Os Bacillus subtilis são saprófitas do tracto respiratório humano com ampla utilização em investigação e em biotecnologia.As cadeias linfáticas pulmonares profundas (CLPP) constituem um dos primeiros locais de disseminação de tumores pulmonares.Neste trabalho pretendeu-se desenvolver e validar um método não invasivo para avaliar as CLPP através de nanorradiolipossomas aerosolisados e modulados pela parede do esporo do Bacillus subtilis. O objectivo final foi produzir uma formulação de nanorradiolipossomas capaz de imitar a dinâmica da remoção de esporos pelas CLPP e simultaneamente ter propriedades ideais como traçador para imagiologia molecular.Testámos sete diferentes formulações lipossómicas, tendo a formulação F demonstrado possuir propriedades fisicoquímicas e radiofarmacêuticas que a tornam o traçador ideal para imagiologia molecular in vivo das CLPP.Os nanorradiolipossomas da formulação F após marcação com 99mTc-HMPAO foram administrados sob a forma de aerossóis a 20 Sus scrofa. Visualizaram-se comunicações hilares e interpulmonares nos primeiros 5 minutos após a inalação, as cadeias infradiafragmáticas entre os 10 e os 20 minutos, os gânglios da cadeia aórtica aos 20 minutos e os da região hilar renal aos 30 minutos.Em conclusão, o método proposto visualiza os gânglios linfáticos e a rede linfática pulmonar profunda. A modulação dos nanorradiolipossomas permite que eles atinjam órgãos ou tecidos específicos, conferindo-lhes importantes potencialidades no âmbito do diagnóstico e/ou da terapêutica.Rev Port Pneumol 2009; XV (2): 261-293 Abstract: Lung deep lymphatic drainage (LDLD) plays an important role in the removal of foreign materials from lungs being alveolar macrophages the first line of phagocytic defence with high affinity for pathogenic microorganisms. Bacillus subtilis is a well-known genome-decoded saprophyte of the human respiratory tract used in research and in the biotechnology industry.Lung deep lymphatic chains (LDLC) constitute one of the first sites of lung tumoursâ dissemination. In this work we intended to develop and validate a non-invasive method for assessing LDLC by nanoradioliposomes aerosolised modulated on the Bacillus subtilis spore wall. The final goal was to produce a nanoradioliposome formulation that can mimics the dynamics of preferential removal of spores by LDLD and present the ideal properties as a tracer for molecular imaging studies.Seven different liposomal formulations were tested, and the formulation-F demonstrated physicochemical and radiopharmaceutical properties that make it an ideal candidate as an in vivo probe for molecular imaging studies of the LDLC.Nanoradioliposomes of the formulation-F after labelling with 99mTc-HMPAO were administered as aerosols to 20 Sus scrofa. Hilar and interpulmonary communications were visualized in first 5 minutes post-inhalation, infradiaphragmatic chains between 10 and 20 minutes, the ganglia of the aortic chain at 20 minutes and those of the renal hilar region at 30 minutes.Conclusion: the proposed method enables visualization of deep lymphatic lung network and lymph nodes. Besides, this technique involving the modulation of nanoradioliposomes targeting specific organs or tissues may be an important tool for diagnostic or even for therapeutic purposes.Rev Port Pneumol 2009; XV (2): 261-293 Palavras-chave: Nanorradiolipossomas, modulação molecular, drenagem linfática pulmonar, imagem nuclear funcional, Key-words: Nanoradioliposomes, molecular modulation, lung lymphatic drainage, functional nuclear imaging

A autora discute as dificuldades do término das análises como equivalente a um estado de luto que tem características específicas, considerando, dentre outros conteúdos teóricos, o que diz Melanie Klein (1991): “o término de uma análise reativa no paciente situações mais arcaicas de separação e tem a natureza de uma experiência de desmame” (p. 65). Além disso, estuda também o luto pelo término no mundo dos analistas, considerando as análises didáticas e a especificidade dessa relação: os candidatos podem esperar manter relacionamento de colegas com seus analistas depois do término; mas os “pacientes comuns” típicos não podem contar com qualquer tipo de contato e, assim, é provável que sofram uma reação de luto muito mais intensa que os candidatos. Todas essas considerações contribuem para demonstrar que o luto é tema relevante, a ser considerado nos estudos psicanalíticos sobre o término e o pós-término das análises.

A evolução da infecção VIH é caracterizada por uma grande variabilidade individual. Na verdade, omo em outros processos da mesma natureza, depende largamente das complexas inter-relacções que num dado momento se estabelecem entre o hospedeiro e o agente agressor. Contudo, nesta infecção, essa correlação assume um papel determinante. Desde o início da pandemia que o pulmão se assumiu como alvo preferencial de complicações, quer de origem infecciosa quer de outras etiologias. A esta inevitabilidade biológica diríamos não serem de facto estranhas as características anatomo-funcionais do órgão, enquanto interface privilegiada entre o meio interno e o ambiente exterior, aliadas a particularidades de ordem imunológica que o tornam, sob muitos aspectos, um órgão único. Cedo se constatou que esta infecção se acompanhava de uma disfunção imunológica progressiva que culminava na completa exaustão deste sistema nas fases terminais da doença. Desde o reconhecimento da SIDA até à presente data foram sendo adquiridos enormes conhecimentos não só em relação ao vírus, como aos seus mecanismos patogénicos, no entanto subsistem ainda numerosas questões para as quais o estado da arte ainda não dispõe de respostas. Nessas incluíriamos os efeitos do VIH na dinâmica celular do pulmão. Vários estudos efectuados, nos quais tivemos oportunidade de participar, demonstraram a apresença de uma alveolite linfocitária durante a fase assintomática da infecção. Desde essa altura têm-se vindo a adquirir novos conhecimentos relativos aos mecanismos imunológicos e bioquímicos subjacentes à entrada do VIH nas células, às células-alvo, ao microambiente citocínico, assim como de outros mediadores celulares envolvidos. Neste contexto, a descoberta de que receptores específicos de quimiocinas actuavam como co-receptores para o VIH abriu definitivamente um novo capítulo na investigação dirigida aos mecanismos responsáveis pelo tropismo viral e infecção celular. Neste âmbito, vários autores têm salientado a importância, para além da molécula CD4, dos receptores quimiocínicos CCR5 e CXCR4 na ligação e, posteriormente, na entrada do vírus nas células, reconhecendo-se em relação ao primeiro uma importância fundamental na transmissão da infecção, enquanto que o CXCR4 parece ser utilizado por estirpes virais que emergem tardiamente no decurso da doença, quer isoladamente, quer em associação com o CCR5.The course of HIV infection is accompanied by a wide individual variability. The complex and large interplay between host and viral factors is crucial in the disease’s evolution. The lung has been recognised from the beginning of the disease as one of the main targets of infectious and non-infectious complications of AIDS. In this setting both anatomic and immunologic particularities of this organ play an important role. The hallmark of HIV is progressive immune dysfunction. Despite the intensive research into the pathogenesis, several questions remain to be answered on the dynamic effects of HIV on pulmonary cells. Previous studies in which we have participated showed the early presence of lymphocytic alveolitis from the asymptomatic phase of infection. Since then, many collected data has brought new insights into the immune and biochemical mechanisms involving HIV cell entry, as well as target cells, cytokines and other cellular mediators. In this context, the discovery that specific chemokine receptors could act as co-receptors for HIV, allowed a better understanding of the mechanisms underlying viral cellular entry and tropism. On this issue several authors have reported that in addition to the CD4 molecule, most strains of HIV use the chemokine receptor CCR5 for viral attachment and entry into the host cells. This receptor seems to be very important in disease transmission, whereas CXCR4 receptor tends to be used by the viral strains that emerge later in the disease in addition to or instead of the CCR5.

Resumo: A evolução da infecção VIH é caracterizada por uma grande variabilidade individual. Na verdade, como em outros processos da mesma natureza, depende largamente das complexas inter-relacções que num dado momento se esta-belecem entre o hospedeiro e o agente agressor. Contudo, nesta infecção, essa correlação assume um papel determinante. Desde o início da pandemia que o pulmão se assumiu como alvo preferencial de complicações, quer de origem infecciosa quer de outras etiologias. A esta inevitabilidade biológica diríamos não serem de facto estranhas as carac-terísticas anatomo-funcionais do órgão, enquanto interface privilegiada entre o meio interno e o ambiente exterior, aliadas a particularidades de ordem imunológica que o tor-nam, sob muitos aspectos, um órgão único. Cedo se constatou que esta infecção se acompanhava de uma disfunção imunológica progressiva que culminava na completa exaustão deste sistema nas fases terminais da doença. Desde o reconhecimento da SIDA até à presente data foram sendo adquiridos enormes conhecimentos não só em relação ao vírus, como aos seus mecanis mos patogénicos, no entanto subsistem ainda numerosas questões para as quais o estado da arte ainda não dispõe de respostas. Nessas incluíriamos os efeitos do VIH na dinâmica celular do pulmão. Vários estudos efectuados, nos quais tivemos oportunidade de participar, demonstraram a apresença de uma alveolite linfocitária durante a fase assintomática da infecção. Desde essa altura têm-se vindo a adquirir novos conhecimentos relativos aos mecanismos imunológicos e bioquímicos subja-centes à entrada do VIH nas células, à s células-alvo, ao microambiente citocínico, assim como de outros media-dores celulares envolvidos. Neste contexto, a descoberta de que receptores específicos de quimiocinas actuavam como co-receptores para o VIH abriu definitivamente um novo capítulo na investigação dirigida aos mecanismos responsáveis pelo tropismo viral e infecção celular. Neste âmbito, vários autores têm salientado a importância, para além da molécula CD4, dos receptores quimiocínicos CCR5 e CXCR4 na ligação e, posterior-mente, na entrada do vírus nas células, reconhecendo-se em relação ao primeiro uma importância fundamental na transmissão da infecção, enquanto que o CXCR4 parece ser utilizado por estirpes virais que emergem tardia-mente no decurso da doença, quer isoladamente, quer em associação com o CCR5. Objectivo: O nosso estudo dirigiu-se fundamentalmente à avaliação da dinâmica celular do pulmão profundo em doentes com SIDA, abarcando a determinação das cargas virais no líquido de lavagem broncoalveolar (LLBA), a celularidade envolvida nos mecanismos de defesa desse território e tropismo viral, através dos receptores quimiocínicos CCR5 e CXCR4. Material: Estudámos 14 doentes portadores de SIDA, com uma média de idades de 39±14,3 anos (9 homens e 5 mulheres) todos infectados pelo VIH1, heterossexuais, dos quais 6 eram fumadores e 8 não fumadores, e sem antecedentes de toxicodependência. Estes doentes foram referenciados pelo Deparamento de Doenças Infecciosas dos Hospitais da Universidade de Coimbra para a realiza-ção de broncofibroscopia e lavagem broncoalveolar (LBA), por suspeita clínica de infecção pulmonar oportunista. Posteriormente, este conjunto de doentes seria dividido e analisado em dois grupos: Grupo I, designado por diag-nóstico recente e Grupo II, englobando os de diagnóstico não recente. De facto, embora todos estes doentes fossem portadores de SIDA, a Amostra Clínica era constituída por indivíduos com infecções oportunistas de diagnóstico recente, nos quais ainda não tinha sido instituída terapêutica anti-retroviral, primeiro grupo, enquanto o segundo grupo incluía doentes com doença mais prolongada, com vários episódios infecciosos oportunistas e tratamento anti-retroviral, para além de outros esquemas terapêuticos. Métodos: Todos os doentes após, consentimento informado, foram submetidos a exame broncoendoscó-pico e LBA, após anestesia local da orofaringe e cordas vocais com lidocaína a 2%, sob monitorização cárdio-respiratória. O LBA foi efectuado com base nas alterações evidenciadas pela telerradiografia do tórax: lobo médio nas lesões difusas, ou outro brônquio segmentar correspondente a alterações radiológicas localizadas A determinação das cargas virais no líquido de lavagem broncoalveolar (LLBA), em 9 doentes, foi efectuada pela quantificação do ARN-VIH1, através de PCR-RT, nas primeiras 4horas após a colheita (sendo cada amostra centrifugada), procedendose posteriomente nas aliquo-tas do sobrenadante à congelação a menos 80 º C, até à extracção e amplificação dos ácidos nucleicos. Realizou-se igualmente, na totalidade da amostra, a determinação das viremias plasmáticas (14 doentes), a partir de amostras de sangue colhido em EDTA, com processa-mento ulterior (separação do plasma e congelamento a menos 80 ºC). A extração e amplificação dos ácidos nucleicos foi efectuada por um método automatizado Cobas Ampliprep/Cobas Amplicor HIV1 Monitor TM Test, version 1.5 Roche Diagnostic Systems. Este método permite a obtenção duma sequência de 155 nucleótidos situada numa região altamente conservada do gene gag. Os resultados foram observados numa escala numérica com uma variação dinâmica situada entre as 50 e 750 000 cópias de ARN VIH1/cm3, procedendo-se posterior-mente à sua conversão logarítmica. No LLBA (10 doentes) e no sangue (13 doentes) foi efectuado um estudo imunológico, dirigido à quantificação das populações e subpopulações linfocitárias: CD3, CD4, CD8, CD19, CD56 e CD56CD8, assim como dos receptores: CD3CCR5, CD4CCR5, CD8CCR5, CCR5Mø, CXCR4, CD3CXCR4, CXCR4CD14 e da molécula de co-esti-mulação CD28, CD3CD28, CD4CD28, CD8CD28 através de anticorpos monoclonais â CD8FITC, CD19FITC, CD3PE, CD56PE, CD4PECY5-Lymphogram Cytognos; CCR5PE, CXCRFITC-R & D Systems; CD8Cy5 e CD3Cy5 - DaKo, CD4PE, CD14PE, CD28FITC â Immunotech; CD4FITC-CLB, CD8Percp â Beckton Dickinson e CD3 APC â Beckton Dickinson, por cito-metria de fluxo (Facs Calibur-Beckton-Dickinson) a 3 ou 4 fluorescências â FL1-FITC, FL2-PE, FL3-PECY, FL4-APC. Na análise estatística dos resultados foram utilizados os testes t -Student e de correlação linear. Resultados: Salientamos os seguintes: presença do vírus no LLBA (2,95 log±3,08 log), embora em quantidades inferiores à s detectadas no sangue(5,89 log±5,90)e grande varibilidade das cargas virais, tanto no sangue como no LLBA, independente do período de tempo entre o diagnóstico e a realização do exame; as populações e subpopulações linfocitárias no sangue (Quadro V), em 13 doentes, mostraram uma queda acentuada, tanto dos linfócitos totais como das suas populações e subpopulações, que atingia maior expressão nas células T-CD4. Apenas em 2 casos (doentes n.os 12 e 13) esta subpopulação era superior a 250 células/mm3 e em 42,9% era inferior a 50 células/mm3. A população CD19 encontrava-se igualmente em níveis inferiores ao normal com uma distribuição individual se-melhante à da subpopulação CD4. Na maior parte dos casos, a diminuição dos CD8 acompanhava a redução dos CD4 e CD19(doentes n.os 7 e 8); na LLBA (Quadro VI), o estudo das populações e subpopulações linfocitárias (10 doentes) revelou uma distribuição percentual seme-lhante à que foi observada no sangue(Quadro VII) em relação à s células CD3, CD19, CD4 e CD8, embora no LLBA a percentagem de linfócitos-T fosse superior neste meio em relação ao sangue(94,5±5 /84,1±10,4), ao contrário do que se observou nos linfócitos-B (2,2±3 /10,4±9,6). Também no LLBA se verificou uma percentagem mais elevada de linfócitos-T CD8 do que no sangue (77,7±17,6 /67,6±4,2), o que não se observou em relação à s células T-CD4 (8,1±9,5 LLBA vs.10,4±9,6 no sangue); a actividade natural killer (NK) expressa pelas células T CD56 apresentava importantes variações individuais em ambos os meios, mas com valores mais elevados no sangue do que no LLBA (9,1±8/2,9±1,9); quanto à actividade citotóxica avaliada através das células CD56CD8, era similar no LLBA e no sangue (2,2±2/1,7±1,2), parecendo, contudo, que a distribuição individual era mais homogénea no primeiros destes meios (Quadros VI e VII); as céulas duplamente negativas (DN) apresentavam-se com valores ligeiramente mais elevados no LLBA (7,6±4,5 vs 5,6±± 5,3). Curiosamente, neste meio observa-se uma percentagem elevada das células menos diferenciadas (13±13,6) (Quadro VI). A análise dos receptores celulares CCR5 e CXCR4 mostrou, em termos globais e médios, diferentes comportamentos entre si e em relação aos meios biológicos (Quadros VI e VII). Assim, o CCR5 CD3 era mais elevado no sangue (10,9±13,2) do que no LLBA (8,4±± 3,5); no entanto, o CCR5 CD4 e a CCR5 CD8 en-contravam-se em maior percentagem no LLBA do que no sangue (2±2,3 e 4,9±3,7/0,9±0,7 e 4,1±4,0 respec-tivamente). No que diz respeito aos valores deste receptor na linha monocítica-macrofágica ele apresentava-se muito mais elevados no LLBA (77,8±41 no LLBA vs. 18,7±± 15 no sangue). Pelo contrário, o CXCR4 total era mais elevado no LLBA 31±19,9) do que no sangue (16,4±± 8,1); esta tendência manisfestava-se igualmente em relação aos linfócitos-T (26,6±19,8 vs. 10,7±7,6) e à linha monocítica-macrofágica, aliás, de uma forma exuberante (84,5±30,2 / 4,8±4,6). A actividade co-estimulatória CD28 mostrou-se superior no sangue (22,8±16,2)em comparação com o LLBA(15,9±10,1)para as células T totais, CD4 e CD8(22,5 16,7; 7,8±8,3; 13,3±8,3 / / 16,5±10,5; 2,9±2,8; 10,8±8,0, respectivamente). Conclusões: 1. A infecção VIH é reponsável por extensas e diversificadas alterações nos mecanismos de defesa do pulmão profundo. 2. A complexa interacçãoo entre o hospedeiro e o agente agressor, bem como a resposta imunológica, particularmente a natural killer e citotóxica, apoptose e doenças oportunistas, ou outras, podem dificultar a obtenção de amostras médicas homogéneas. Para além disso, considerações de ordem ética restringem, natu-ralmente, a abordagem destes doentes com finalidades exclusivamente científicas. 3. Os resultados obtidos apontam tendencialmente para uma resposta pulmonar compartimentada à agressão VIH, com base na dinâmica celular envolvida e no perfil dos receptores encontrado nos líquidos biológicos estudados. Neste contexto, esta compartimentação parece estar particularmente na dependência da actividade dos macrófagos alveolares que constituem, desde o início, o eixo fundamental destes processos, representando o últi-mo mecanismo celular de defesa deste território, quando os outros estão já profundamente alterados. 4. O estudo da dinâmica dos receptores quimiocínicos pode vir a ter importantes implicações terapêu-ticas. De facto, foi já demonstrado que o bloqueio do receptor CCR5 não foi acompanhado por um aumento da expressão do receptor CXCR4. Neste estudo tivemos oportunidade de verificar que o CXCR4 se manteve elevado nas células monocíticas-macrofágicas durante a doença, aumentando a sua expressão nos linfócitos T nos doentes do Grupo II, ao contrário do comportamento do CCR5 no LLBA, que dimi-nuía significativamente. No sangue, porém, a expressão do CCR5 aumentava. 5. A elevada co-existência de infecções oportunistas (71,4%) leva-nos a colocar a hipótese de que a modulação induzida pelos germes possa contribuir para a elevada expressão do CXCR4. 6. Finalmente, esta tão marcada variabilidade individual tem indiscutivelmente implicações clínicas. Em termos gerais, podemos dizer que, para uma mais correcta abordagem terapêutica, cada doente deve ser prévia e individualmente analisado neste contexto. Rev Port Pneumol 2007; XIII (2): 175-212 Abstract: The course of HIV infection is accompanied by a wide individual variability. The complex and large in-terplay between host and viral factors is crucial in the diseaseâs evolution. The lung has been recognised from the beginning of the disease as one of the main targets of infectious and non-infectious complications of AIDS. In this setting both anatomic and immunologic particularities of this organ play an important role. The hallmark of HIV is progressive immune dys-function. Despite the intensive research into the pathogenesis, several questions remain to be answered on the dynamic effects of HIV on pulmonary cells. Previous studies in which we have participated sho-wed the early presence of lymphocytic alveolitis from the asymptomatic phase of infection. Since then, many collected data has brought new insights into the immune and biochemical mechanisms involving HIV cell entry, as well as target cells, cytokines and other cellular mediators. In this context, the discovery that specific chemokine receptors could act as co-receptors for HIV, allowed a better understanding of the mechanisms underlying viral cellular entry and tropism. On this issue several authors have reported that in addition to the CD4 molecule, most strains of HIV use the chemokine receptor CCR5 for viral attachment and entry into the host cells. This receptor seems to be very important in disease transmission, whereas CXCR4 receptor tends to be used by the viral strains that emerge later in the disease in addition to or instead of the CCR5. Aims: To evaluate the pulmonary cellular dynamics in AIDS patients regarding the viral load in bronchoalveolar lavage fluid (LLBA), as well as cellularity and tropism through CCR5 and CXCR4 receptors. Material: 14 AIDS patients were enrolled in this study, with a mean age of 39±14.3 years (9 males and 5 females) all HIV1, heterosexuals, 6 smokers and 8 non-smokers, none of them drug addicts. These patients were referred to bronchoscopy with BAL, for clinical suspicion of opportunistic lung infections. These patients were later divided into two groups: Group I (recent diagnosis) and Group II (non-recent diagnosis). While all patients had AIDS, group I had also recent diagnosis of oportunistic infections and had not received yet anti-retroviral therapy whilst group II had a long-term disease evolution with several opportunistic episodes and anti-retroviral therapeutic. Methods: BAL was performed both in the middle bronchus in diffuse or in other segmentar bronchus, depending on radiographic abnormalities. Plasma viral load was performed through PCR-RT in blood samples with EDTA, centrifuged and frozen (-80º Cel-sius) in the first 4 hours after being collected. The viral load in BALf was quantified in 9 patients using the automatized Cobas Ampliprep/Cobas Amplicor HIV1 Monitor TM Test, version 1.5 Roche Diagnostic Systems. The results were expressed in a numeric scale, with a dynamic variation of 50-750.000 copies of RNA HIV1/cm3 and later converted into a logarithmic scale. In 10 patients an immunological study was carried out in BALf and blood to quantify the lymphocyte populations and subsets (CD3, CD4, CD8, CD19, CD56 and CD56CD8) as well as the receptors CD3CCR5, CD4CCR5, CD8CCR5, CCR5Mø, CXCR4, CD3CXCR4, CXCR4CD14 and co-stimulatory molecule CD28, CD3CD28, CD4CD28, CD8CD28 through monoclonal antibodies â CD8FITC, CD19FITC, CD3PE, CD56PE, CD4PECY5-Lymphogram Cytognos; CCR5PE, CXCRFITC-R & D Systems; CD8Cy5 and CD3Cy5-DaKo, CD4PE, CD14PE, CD28FITC-Immunotech; CD4FITC-CLB, CD8Percp- Beckton Dickinson and CD3 APC â Beckton Dickinson, by flow cytometry (Facs Calibur-Be-ckton-Dickinson) with 3 or 4 fluorescences â FL1-FITC, FL2-PE, FL3-PECY, FL4-APC. In the statistical analysis, we used the Student t-test, and linear correlation. Results: Presence of HIV1 in BALf (2.95 log±3.08 log), in small levels compared with plasma viral loads (5.89 log ±5.90 log) (Table IV). There was great variability of viral loads in BALf as there was in blood independent of the time elapsed between diagnosis and the exam. As for the lymphocytic populations and subsets in blood (Table V) determined in 13 patients, there was a significant fall of total lymphocytes as well as of their subsets, although more marked in CD4 cells; 42.9% had CD4 levels < 50 cels/mm3 and only 2 patients (nº 12, 13) had CD4> 250 cels/mm3. The CD19 was reduced with an individual distribution similar to the CD4 subset. In most cases, the fall of CD8 accompanied the decrease of CD4 and CD19 (patients-nº 7 and 8). The lymphocyte populations and subsets in BALf (10 patients) (Table VI) showed a percentual distribution similar to that observed in blood (Table VII) for CD3, CD19, CD4 and CD8 lymphocytes, although the percentage of T cells was higher than in blood (94.5±5 /84.1±10.4) as opposed to B cells (2.2±3 /10.4±9.6). In BALf CD8T cells were higher than in blood (77.7±17.6 /67.6±4.2), which was not observed for the CD4 lymphocytes (8.1±9.5 BALf vs.10.4±9.6 in blood). The natural killer activity expressed by CD56T cells had important indivi-dual variations in both biological fluids: higher levels in blood than in BALf (9.1±8 /2.9±1.9). The cytotoxic activity of CD56CD8 was similar in blood and in BALf (2.2±2 / 1.7±1.2) while the individual distribution seemed more homogeneous in BALf (Table VI) than in blood (Table VII). The double-negative (DN) cells had slightly higher values in BALf (7.6±4.5 vs 5.6±5.3). Curiously, in BALf we observed a higher percentage of less differenciated cells (13±13.6) (Table VI). The analysis of the receptors CCR5 and CXCR4 showed in general terms different behaviour concerning the two biological means (Tables VI and VII). Thus, the CCR5 CD3 was hi-gher in blood (10.9±13.2) than in BALf (8.4±± 3.5) while the CCR5 CD4 and CCR5 CD8 had an increased expression in BALf in relation to blood (2±2.3 and 4.9±3.7 / 0.9±0.7 and 4.1±4.0 respectively). Concerning the expression of this receptor on monocyte macrophage lineage a marked higher value was attained in BALft (77.8±± 41 in BALf vs. 18.7±15 in blood). On the contrary the total expression of CXCR4 was higher in BALf (31±19.9) than in blood (16.4±± 8.1). This tendency extended equally to the T lymphocytes (26.6±19.8 vs. 10.7±7.6) and also to the monocyte-macrophage lineage in an exuberant manner (84.5±30.2 / 4.8±4.6). The co-stimulatory activity of CD28 showed higher values in blood (22.8±16.2) than in BALf (15.9±± 10.1) for total T cells, CD4 and CD8 lymphocytes 22.5±16.7; 7.8±8.3; 13.3±8.3 / 16.5±± 10.5; 2.9±2.8; 10.8±8.0 respectively). Conclusions:1. HIV infection is responsible for important and extensive abnormalities in lung host defences.2. The complex interaction between host and aggressor as well as the immune response particularly represented by natural killer and cytotoxic activities, apoptosis, and opportunistic diseases or others, therapeutics and other factors may contibute to the difficulty in obtaining homogenous medical samples within research. There are also ethical issues that restrict a purely scientific approach to these patients.3. These results point to a pulmonary response to HIV in a compartmentalised fashion according to the dynamic cellular elements involved and receptors in which the latter had distinct profiles related to the biological fluids. In this context, the lung compartimental response is particularly dependent on alveolar macrophages activity which is from the beginning the cornerstone of this process and is the last cellular defense mechanism in this territory when all others are profoundly affected.4. The dynamics of chemokines receptors may be very important in therapeutic approach as the blockage of the CCR5 receptor does not seem to trigger an increased expression of CXCR4 strains. In fact, we found that CXCR4 remained high in monocyte-macrophage cells throughout infection and its expression was increased in T-lymphocytes in Group II patients as opposed to CCR5 behavior in BALf which significantly decreases. However, in blood, CCR5 expression increased, unlike CXCR4.5. Due to high co-existing opportunistic infections (71.4%) we cannot ignore the hypothesis that this increased expression of CXCR4 was a result of the modulation induced by opportunistic agents.6. Finally, this striking individual variability undoubtly has clinical implications. This makes a case-by-case management strategy the correct approach. Rev Port Pneumol 2007; XIII (2): 175-212 Palavras-chave: Lavagem broncoalveolar, SIDA, celularidade, receptores CCR5 e CXCR4, Key-words: Bronchoalveolar lavage, AIDS, cellularity, CCR5 and CXCR4 receptors

A point source of DDT pollution of industrial origin, discovered in 1996 in Lago Maggiore, the second largest (212 km2) and deepest (373 m) lake in Italy, created concern for wildlife and human health because contamination in some fish species exceeded the threshold for human consumption, even though the concentrations measured in the water were much lower than the legal requirements for drinking purposes. Some precautionary measures were undertaken to prevent DDT runoff from the industrial site to the lake but soil restoration had not yet been carried out. The first study to quantify land based sources of DDT homologue pollution was performed in 1998 by sampling all the main tributaries monthly in order to evaluate the annual load to the lake. From May 2001 to May 2002, the study was repeated in order to evaluate the degree of recovery. In this work we compared the results of that survey with those of a more recent campaign carried out from May 2001 to May 2002, concluding that land based sources are still relevant for pp’DDE, the most stable metabolite of pp’DDT, while they are negligible for the parent compound. Furthermore, the Toce River, receiving the soil runoff of the industrial area, was found to give the greatest contribution of DDT and DDE load to the lake in the past campaign, while its load drastically decreased in 2001-2002 in the case of pp’DDT, being comparable to that of Ticino River(the second biggest river of the Lago Maggiore basin, which rises in the St. Gotthard Massif of the Swiss Alpes). Air transport and cold condensation are very likely responsible for trapping DDT and DDE in high mountain glaciers, which represent a secondary pollution source.

Receptor tyrosine kinases (RTKs) play an important role in the control of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulated RTK signaling is critically involved in the development and progression of human cancer. Here, we present an assay for monitoring RTK activities in yeast, which provides an ideal heterologous cellular system to study these mammalian proteins in a null background environment. With our system, we have reconstituted aspects of the epidermal growth factor receptor (EGFR) signaling pathway as a model. Our approach is based on the Ras-recruitment system, in which membrane localization of a constitutively active human Ras achieved through protein-protein interactions can rescue growth of a temperature-sensitive yeast strain (cdc25-2). We show that co-expression of a dimerizing membrane-bound EGFR variant with specific adaptor proteins fused to the active Ras rescues growth of the cdc25-2 mutant yeast strain at the nonpermissive temperature. Using kinase-defective RTK mutants and selective EGFR kinase inhibitors, we demonstrate that growth rate of this yeast strain correlates with kinase activity of the EGFR derivatives. The RTK cellular assay presented here can be applied in high-throughput screens for selecting RTK-specific inhibitors that must be able to permeate the membrane and to function in an eukaryotic intrecellular environment.

Resumo: A sarcoidose é uma doença granulomatosa crónica de etiologia desconhecida. Atinge todos os órgãos e sistemas, particularmente o pulmão. O doseamento sérico da enzima de conversão da angiotensina (SACE) e da lisozima são exames complementares que contribuem para o seu diagnóstico e monitorização laboratorial. à desejável que outros marcadores possam optimizar a informação obtida com estes parâmetros. As granzimas A e B, produzidas por diversas células, poderão modular o turnover dos granulomas sarcoidóticos, tornando-se úteis como marcadores da doença.Objectivos: Dosear as granzimas A e B e avaliar o seu interesse como marcadores laboratoriais de sarcoidose. Paralelamente, dosear a SACE e a lisozima, marcadores reconhecidos da doença.Material e métodos: Indivíduos de ambos os sexos: Controlo normal (CN), n=30; controlo-doente (CD), n=21 (patologia pulmonar não granulomatosa); grupo-doente (D), n=11 (doentes com sarcoidose pulmonar).Recolheram-se amostras de sangue periférico para obter soro que se separou por tubos identificados e guardados a â30 ºC.Doseou-se a SACE por espectrofotometria e a lisozima por turbidimetria; as granzimas A e B por ELISA.Resultados: A actividade de SACE está significa-tivamente aumentada em D, comparativamente com CN e CD. A actividade da lisozima está significati-vamante aumentada nos grupos D e CD comparativamente com CN. A granzima B está significati-vamente diminuída nos grupos CD e D relativamente ao CN; a granzima A demonstrou diminuição significativa em D comparativamente com CN. Sugere-se que a diminuição das granzimas, na sarcoidose, poderá relacionar-se com resposta imunoin-flamatória local ineficaz relacionada com a formação do granuloma. Há necessidade de alargar o estudo também ao LLBA.Rev Port Pneumol 2005; XI (2): 111-133 Abstract: Sarcoidosis is a systemic disease of unknown aetiology, morphologically characterized by well-formed epithelioid granulomas, which show little or no cen-tral necrosis. These may be present in any organ or tissue. The lung is the most frequently and promi-nently involved target.The granuloma is often very sharply demarcated from the adjacent tissue and is surrounded by a mantle of lymphocytes, which mediate lysis of target cells by various mechanisms, including exocytosis of lytic proteins, perforins and granzymes.Sarcoidosis laboratorial diagnosis is usually made by SACE and Lisozyme dosages. The granzymes A and B could be two other markers of the disease, since the sarcoidosis granuloma is rich in cytotoxic and NK cells.An ELISA Kit was used to measure Granzyme A and B in serum of a normal control group (NC) (n=30), and in two groups with lung pathology: one without sarcoidosis, disease control (DC) (n=21) and other with sarcoidosis (S) (n=11).Our results showed that SACE activity is significantly augmented in S group comparing with NC and DC, respectively: 82,6±32,7/31,9±17,8 - p=0,00017 and 82,6±32,7/31,9±17,8 - p=0,00024. Lisozyme activity is significantly augmented in S and DC groups comparing with NC. Granzyme B showed a significant decrease in DC and S groups comparing with NC. Granzyme A showed a significant decrease between S/NC groups.Our results suggest that the decrease of Granzyme A and B in sarcoidotic patients could be related to an ineffective inflammatory local response related to the formation of sarcoidosis granulomas. More studies are needed, particularly in BAL.Rev Port Pneumol 2005; XI (2): 111-133 Palavras-chave: Sarcoidose, diagnóstico laboratorial, granzima A, granzima B, Key words: Sarcoidosis, laboratorial diagnosis, granzyme A, granzyme B

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.

Ground-based measurements of atmospheric parameters have been carried out for more than 20 years at the Network for the Detection of Atmospheric Composition Change (NDACC) station at Thule Air Base (76.5°N, 68.8°W), on the north-western coast of Greenland. Various instruments dedicated to the study of the lower and middle polar atmosphere are installed at Thule in the framework of a long standing collaboration among Danish, Italian, and US research institutes and universities. This effort aims at monitoring the composition, structure and dynamics of the polar stratosphere, and at studying the Arctic energy budget and the role played by different factors, such as aerosols, water vapour, and surface albedo. During the International Polar Year (IPY), in winter 2008-2009, an intensive measurement campaign was conducted at Thule within the framework of the IPY project “Ozone layer and UV radiation in a changing climate evaluated during IPY” (ORACLE-O3) which sought to improve our understanding of the complex mechanisms that lead to the Arctic stratospheric O3 depletion. The campaign involved a lidar system, measuring aerosol backscatter and depolarization ratios up to 35 km and atmospheric temperature profiles from 25 to 70 km altitude, a ground-based millimeter-wave spectrometer (GBMS) used to derive stratospheric mixing ratio profiles of different chemical species involved in the stratospheric ozone depletion cycle, and then ground-based radiometers and a Cimel sunphotometer to study the Arctic radiative budget at the surface. The observations show that the surface radiation budget is mainly regulated by the longwave component throughout most of the year. Clouds have a significant impact contributing to enhance the role of longwave radiation. Besides clouds, water vapour seasonal changes produce the largest modification in the shortwave component at the surface, followed by changes in surface albedo and in aerosol amounts. For what concerns the middle atmosphere, during the first part of winter 2008-2009 the cold polar vortex allowed for the formation of polar stratospheric clouds (PSCs) which were observed above Thule by means of the lidar. This period was also characterized by GBMS measurements of low values of O3 due to the catalytic reactions prompted by the PSCs. In mid-January, as the most intense Sudden Stratospheric Warming event ever observed in the Arctic occurred, GBMS and lidar measurements of O3, N2O, CO and temperature described its evolution as it propagated from the upper atmosphere to the lower stratosphere.

The RNA polymerase III-based two-hybrid system has been developed to detect interactions between proteins such as RNA polymerase II transcription factors and regulators that cannot be studied by the original RNA polymerase II two-hybrid system. This novel method appears to be most useful for a refined analysis of already known protein-protein interactions. However, the application of this system in library screenings has been impaired by the lack of a suitable assay for the selection of the activated pol III reporter gene in yeast. Here, we describe a novel selection assay for the pol III-based two-hybrid system that makes it readily usable for screening expression libraries to search for interacting partners. Our system utilizes a temperature-sensitive (ts) U6 snRNA, which is synthesized by RNA polymerase III from a mutated SNR6 gene in yeast. In this ts strain, interactions between hybrid proteins activate an artificial pol III reporter construct (UASG-SNR6), which controls expression of wild-type U6 snRNA. This wild-type U6 snRNA can suppress the ts phenotype and allow growth at the nonpermissive temperature of 37°C, thus providing a positive selection system for interacting proteins.

Since 1963 Lake Orta has been an emblematic case of industrial pollution by heavy metals and acidifying compounds (ammonium sulphate), to the extent that up to 1989 it was the largest acidified deep lake in the world. The low pH values of between 3.9 and 4.4 helped to keep high the levels of toxic compounds in solution, such as copper, aluminium, zinc and nickel. The liming performed in 1989-1990 brought the pH back to neutral values, determining the precipitation of the metals and the recovery of normal chemical conditions. The main results of researches conducted continuously on the lake water chemistry from 1988 to March 2000 are as follows. The whole water mass has been completely neutralised since the beginning of 1991; pH subsequently rose until in 1999-2000 it reached the values (6.7-6.9 units) of the years when the lake was in a natural condition. The alkaline reserve showed a continuous increase after the lake water was neutralised, until March 2000, when total alkalinity values levelled off at 0.19 meq l-1. The increase in pH has allowed a full recovery of nitrification processes; in fact, during the liming period the concentration of ammonium was drastically reduced, by over 80%; ammonium has been practically absent since the end of 1992, and it may be affirmed that the primary cause of the acidification of the lake has been completely removed. The nitrate content in the lake has almost halved compared with the mean concentrations measured before the liming; in March 2000 mean values of 2.0 mg N l-1 were measured, and it is likely that these values will fall further in the next few years, to below 1.5 mg N l-1. The concentrations of toxic metals have shown a progressive reduction, to the extent that in 1999 the content of copper and aluminium was close to zero in the whole water mass. The situation of Lake Orta has therefore improved enormously, and is now very similar to the original condition of the lake before it was polluted. However, this situation cannot be regarded as definitive, as a stable balance between the input and output of some important chemical species, such as bicarbonate and nitrogen compounds has not yet been achieved. Moreover, recent investigations have revealed that insufficiently treated industrial and urban waste is once more being dumped in the lake. In this situation there can be no doubts as to the necessity for continuing research on Lake Orta, not only to document the evolution of the chemical and biological characteristics of the lake, but also to verify the effectiveness of the treatment measures adopted and to determine the need for extending these measures to the whole watershed. In particular, the object of this research should be to quantify, control and reduce to an acceptable level the incoming loads of a) ammoniacal nitrogen, to eliminate the danger of the lake water becoming re-acidified; b) phosphorus, to prevent the onset of eutrophication processes; c) heavy metals, to minimise their toxic effects on the whole aquatic ecosystem.