Unia Europejska wobec nowych tworów państwowych (casus Kosowa, Abchazji i Osetii południowej)

W przypadku niejednolitego stanowiska państw członkowskich Unii Europejskiej wobec Kosowa, osłabieniu ulega prestiż i siła oddziaływania Unii na arenie międzynarodowej. W odniesieniu do problemu Abchazji i Osetii Południowej natomiast, działania UE obrazują, że samo wyrażenie zainteresowania określonym problemem oraz udzielanie ‘apolitycznej’ pomocy finansowej, którym nie towarzyszą wyraźnie i precyzyjnie zdefiniowane cele polityczne, działalność instytucji wyposażonych w silny mandat oraz proces aktywnych negocjacji, nie przyczynia się do rozwiązania palącego problemu międzynarodowego, narażając na szwank pozycję międzynarodową Unii Europejskiej. Po raz kolejny państwa członkowskie pokazują, iż koncepcja Wspólnej Polityki Zagranicznej i Bezpieczeństwa, wspierana między innymi przez Europejską Politykę Sąsiedztwa, jest pozbawiona stabilnych podstaw.

Konwencja dotycząca statusu uchodźców a praktyka turecka. Problem przybyszów zza wschodniej granicy

Celem publikacji jest podjęcie próby przeanalizowania polityki, jaką Turcja prowadzi wobec obcokrajowców poszukujących schronienia na jej terytorium. O ważkości zagadnienia zadecydowała w ostatnich latach przede wszystkim tocząca się w Syrii wojna domowa, w wyniku której na terytorium Turcji znalazło się ponad 700 tysięcy Syryjczyków. Szczególne w tym kontekście kontrowersje budzi fakt stosowania przez Turcję podwójnych standardów w przedmiocie nadawania imigrantom konwencyjnego statusu uchodźcy. Państwo to, jako jedno z czterech na świecie, w momencie przystępowania do Konwencji dotyczącej statusu uchodźców i Protokołu nowojorskiego zastrzegło sobie prawo do stosowania w tej materii tzw. kryterium geograficznego. W efekcie, o ile status uchodźcy nadany być może osobom przybywającym zza zachodnich granic Turcji, o tyle uciekinierzy z państw takich, jak Syria, Iran, czy Irak z formalnego punktu widzenia są „poszukującymi schronienia” (tur. sığınmacı). To zaś oznacza brak ich konwencyjnej ochrony. Celem artykułu jest jednak nie tylko przeanalizowanie prawnego i rzeczywistego położenia, w jakim znajdują się ofiary syryjskiej wojny domowej, przybywające na terytorium Turcji, a także próba przewidzenia scenariusza rozwoju tejże sytuacji. Celem uczynienia analizy możliwie najbardziej rzetelną, odwołano się zarówno do anglo, jak i tureckojęzycznych materiałów źródłowych.

Tectonics Inversions & Emerald Deposits

In spite of the great amount of emerald deposits throughout the world, the priorities in quality and volume of extracted rough material are the sites of Colombia (Muzo and Chivor emerald belts). This sites are know even before the Spanish conquistadores. Emeralds were extracted from Somondoco mine (today Chivor) since 1537 and from Muzo in 1567. Contrariwise to the majority of the emerald deposits of the world, which are associated with granitic rocks, the Colombian emerald deposits are associated with hydrofracturing (the main factor controlling emerald mineralization) and hydrothermal fluids, rich in beryl, chrome and vanadium, induced by a tectonic inversion of the deep Mesozoic backarc basin, which is also responsible of the majority of the petroleum systems of the foredeep and foldbelt areas (maturation of the source-rocks andcreation of structural traps). The host rocks of the emeralds are carbonaceous calsiltites (calcareous schists) rich in organic matter of Lower Cretaceous age, which are cut by calcite veins, which, often, contain emeralds, particularly when they are folded. Indeed, since long time (Cheilletz, A. and Giulliani, G., 1996) suggested a two-stage model for the formation of the Colombian emeralds : (i) Stage I is characterized by décollement planes (early compressional tectonic regime) within the carbonaceous calsiltites, hydrothermal fluid infiltration and wall-rock metasomatic alteration ; (ii) Stage II (late tectonic regime) deforms the previous veins by thrust-related folds (development of stratiform and hydraulic breccia), which are synchronous of the emerald mineralization. The resulting tectonic structures are complex fold patterns characterized by propagation anticlines with emerald veins and emerald hydraulic breccia in the apexes, as in Quipama, Tendenquema and Chivor mines. Otherwise stated, since all emerald exploitations are, presently underground, exhaustive geological and particularly structural studies are required to reduce the probability of disappointments. The color of emeralds is from light green to thick green with obvious pleochroism. They appears with different colors when observed at different angles, especially with polarized light. The emeralds from Coscuez deposits have a homogeneous intensive color and bluish tone. At Muzo deposit, the emeralds have middle or dark green color with yellowish tone. At the Chivor deposits, the emeralds have less intensive green color with slight bluish tone. The typical inclusions are albite and pyrite, as well as long bubbles with three phase-inclusions according the zones of growth and along the crystal shapes.

Vivências amorosas em adolescentes

Dissertação apresentada à Universidade Fernando Pessoa como parte dos requisitos para a obtenção do grau de Mestre em Psicologia, ramo de Psicologia Clínica e da Saúde

The Whitby life of Gregory the Great: exegesis and hagiography

The eight-century Whitby Vita Gregorii is one of the earliest examples of Anglo-Saxon hagiography, and is the earliest surviving life of Gregory the Great (590-604). The work has proved itself an anomaly in subject matter, style and approach, not least because of the writer’s apparently arbitrary insertion of an account of the retrieval of the relics of the Anglo-Saxon King Edwin (d.633). There has, however, been relatively little research on the document to date, the most recent concentrating on elements in the Gregorian material in the work. The present thesis adapts a methodology which identifies patristic exegetical themes and techniques in the Vita. That is not only in material originating from the pen of Gregory himself, which is freely quoted and cited by the writer, but also in the narrative episodes concerning the Pope. It also identifies related exegetical themes underlying the narrative of the Anglo-Saxon material in the document, and this suggests that the work is of much greater coherence then has previously been thought. In the course of the thesis some of the Vita Gregorii’s major patristic themes are compared with Bede and other insular writers in the presentation of topics that have been of considerable interest to insular historians in recent years. That is themes including: the conversion and salvation of the English people; the ideal pastor; monastic influence on formation of Episcopal spiritual authority; relations between king and bishop. The thesis also includes a re-evaluation of the possible historical context and purpose of the work, and demonstrates the value of a proper understanding of the Vita’s spiritual nature in order to achieve this. Finally the research is supported by a new structural analysis of the entire Vita Gregorii as an artefact formed within literary traditions.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.

Surface physics at the nano-scale via scanning probe microscopy and molecular dynamics simulations

Probe-based scanning microscopes, such as the STM and the AFM, are used to obtain the topographical and electronic structure maps of material surfaces, and to modify their morphologies on nanoscopic scales. They have generated new areas of research in condensed matter physics and materials science. We will review some examples from the fields of experimental nano-mechanics, nano-electronics and nano-magnetism. These now form the basis of the emerging field of Nano-technology. A parallel development has been brought about in the field of Computational Nano-science, using quantum-mechanical techniques and computer-based numerical modelling, such as the Molecular Dynamics (MD) simulation method. We will report on the simulation of nucleation and growth of nano-phase films on supporting substrates. Furthermore, a theoretical modelling of the formation of STM images of metallic clusters on metallic substrates will also be discussed within the non-equilibrium Keldysh Green function method to study the effects of coherent tunnelling through different atomic orbitals in a tip-sample geometry.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Dissipative scheme to approach the boundary of two-qubit entangled mixed states

We discuss the generation of states close to the boundary family of maximally entangled mixed states as defined by the use of concurrence and linear entropy. The coupling of two qubits to a dissipation-affected bosonic mode is able to produce a bipartite state having, for all practical purposes, the entanglement and mixedness properties of one of such boundary states. We thoroughly study the effects that thermal and squeezed characters of the bosonic mode have in such a process and we discuss tolerance to qubit phase-damping mechanisms. The nondemanding nature of the scheme makes it realizable in a matter-light-based physical setup, which we address in some details.

Controllable Gaussian-Qubit Interface for Extremal Quantum State Engineering

We study state engineering through bilinear interactions between two remote qubits and two-mode Gaussian light fields. The attainable two-qubit states span the entire physically allowed region in the entanglement-versus-global-purity plane. Two-mode Gaussian states with maximal entanglement at fixed global and marginal entropies produce maximally entangled two-qubit states in the corresponding entropic diagram. We show that a small set of parameters characterizing extremally entangled two-mode Gaussian states is sufficient to control the engineering of extremally entangled two-qubit states, which can be realized in realistic matter-light scenarios.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

How the ship of fools was ship-wrecked - The Irish crisis and response

For a decade and half the Irish economy was the poster-boy of Europe. With substantial growth rates, an open economy, flexible labour markets and low levels of taxation, Ireland was seen as evidence of the success of neoliberal policies. Yet in the matter of a few short years Ireland has turned into a one of the peripheral black-holes (along with Greece and Portugal) that are threatening to bring down the whole Eurozone project. Given this context the paper will address two key questions. Firstly how did the much eulogised Celtic Tiger fall so far and so fast? And, secondly, what has been the government’s response to the fall and crash of the Irish economy? These two questions will be addressed through both a general historical analysis of the developments of Irish society up to the crash in 2008 and then the responses to it. Secondly by an analysis of two specific elements of that development; namely the much discussed low corporation tax rate and the failure of social housing to deliver decent affordable homes for those at the bottom of society. The third element is a review of the banking and sovereign debt crisis that led to the IMF/EU deal in November 2010 and a brief outlining of its implications for public finances, especially the question of default. The paper concludes by placing the Irish crisis in a global context.

HIF-2alpha Associated Familial Erythrocytosis Supports the PHD2-HIF2-alpha-VHL Axis as the Major Regulator of Erythropoietin Production

Transcriptionally erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF), which is composed of one alpha and one beta subunit that are constitutively expressed. The beta subunit is non-variable, but three different alpha subunits give rise to three isoforms of HIF. The alpha subunit is proteasomally regulated in the presence of oxygen by hydroxylation of the proline in the LXXLAP motif of the oxygen dependent degradation (ODD) domain of HIFalpha, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the alpha subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the alpha subunit to escape proteasomal regulation leads to elevated expression of HIF target genes.

Recently mutations in both VHL and PHD2 have been identified in a cohort of patients with erythrocytosis, but no mutations were found in the ODD domain of HIF1alpha. Instead, investigation of the homologous region in HIF-2alpha revealed four different mutations, Pro534Leu, Met535Val, Gly537Arg and Gly537Trp in seven individuals/families. Affected individuals presented at a young age with elevated serum Epo. Several individuals have a clinical history of thrombosis, but no evidence of a von Hippel Lindau-like syndrome.

To define how the four mutations relate to the erythrocytosis phenotype functional assays were performed in vitro. Binding of PHD2 to the four HIF-2alpha mutants was impaired to varying degrees, with both the Gly537 mutants showing the greatest reduction. The association of VHL with the hydroxylated Met535Val mutant peptide was similar to wild type HIF- 2alpha, but was decreased in the other three HIF-2alpha mutants. Expression of three HIF- 2alpha target genes, adrenomedullin, NDRG1 and VEGF, was significantly up-regulated in cells stably transfected with the mutants under normoxia compared to wild type HIF-2alpha. Mutations in the ODD domain of HIF-2alpha disrupt proteasomal regulation by reducing the association with PHD2 and hence hydroxylation. Furthermore the binding of VHL is also impaired, even when HIF-2alpha is hydroxylated. Examination of the three-dimensional structure of hydroxylated HIF-1alpha bound to VHL confirms that amino acids close to site of hydroxylation (Pro-531 in isoform 2) are important for this association. These observations, together with recent studies utilising murine models of erythrocytosis, support the PHD2-HIF-2alpha-VHL axis as the major regulator of erythropoietin.

Proton radiography as an electromagnetic field and density perturbation diagnostic (invited)

Laser driven proton beams have been used to diagnose transient fields and density perturbations in laser produced plasmas. Grid deflectometry techniques have been applied to proton radiography to obtain precise measurements of proton beam angles caused by electromagnetic fields in laser produced plasmas. Application of proton radiography to laser driven implosions has demonstrated that density conditions in compressed media can be diagnosed with million electron volt protons. This data has shown that proton radiography can provide unique insight into transient electromagnetic fields in super critical density plasmas and provide a density perturbation diagnostics in compressed matter.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.