Analysis of space-time environmental data in complex regions

This thesis develops a comprehensive and a flexible statistical framework for the analysis and detection of space, time and space-time clusters of environmental point data. The developed clustering methods were applied in both simulated datasets and real-world environmental phenomena; however, only the cases of forest fires in Canton of Ticino (Switzerland) and in Portugal are expounded in this document. Normally, environmental phenomena can be modelled as stochastic point processes where each event, e.g. the forest fire ignition point, is characterised by its spatial location and occurrence in time. Additionally, information such as burned area, ignition causes, landuse, topographic, climatic and meteorological features, etc., can also be used to characterise the studied phenomenon. Thereby, the space-time pattern characterisa- tion represents a powerful tool to understand the distribution and behaviour of the events and their correlation with underlying processes, for instance, socio-economic, environmental and meteorological factors. Consequently, we propose a methodology based on the adaptation and application of statistical and fractal point process measures for both global (e.g. the Morisita Index, the Box-counting fractal method, the multifractal formalism and the Ripley's K-function) and local (e.g. Scan Statistics) analysis. Many measures describing the space-time distribution of environmental phenomena have been proposed in a wide variety of disciplines; nevertheless, most of these measures are of global character and do not consider complex spatial constraints, high variability and multivariate nature of the events. Therefore, we proposed an statistical framework that takes into account the complexities of the geographical space, where phenomena take place, by introducing the Validity Domain concept and carrying out clustering analyses in data with different constrained geographical spaces, hence, assessing the relative degree of clustering of the real distribution. Moreover, exclusively to the forest fire case, this research proposes two new methodologies to defining and mapping both the Wildland-Urban Interface (WUI) described as the interaction zone between burnable vegetation and anthropogenic infrastructures, and the prediction of fire ignition susceptibility. In this regard, the main objective of this Thesis was to carry out a basic statistical/- geospatial research with a strong application part to analyse and to describe complex phenomena as well as to overcome unsolved methodological problems in the characterisation of space-time patterns, in particular, the forest fire occurrences. Thus, this Thesis provides a response to the increasing demand for both environmental monitoring and management tools for the assessment of natural and anthropogenic hazards and risks, sustainable development, retrospective success analysis, etc. The major contributions of this work were presented at national and international conferences and published in 5 scientific journals. National and international collaborations were also established and successfully accomplished. -- Cette thèse développe une méthodologie statistique complète et flexible pour l'analyse et la détection des structures spatiales, temporelles et spatio-temporelles de données environnementales représentées comme de semis de points. Les méthodes ici développées ont été appliquées aux jeux de données simulées autant qu'A des phénomènes environnementaux réels; nonobstant, seulement le cas des feux forestiers dans le Canton du Tessin (la Suisse) et celui de Portugal sont expliqués dans ce document. Normalement, les phénomènes environnementaux peuvent être modélisés comme des processus ponctuels stochastiques ou chaque événement, par ex. les point d'ignition des feux forestiers, est déterminé par son emplacement spatial et son occurrence dans le temps. De plus, des informations tels que la surface bru^lée, les causes d'ignition, l'utilisation du sol, les caractéristiques topographiques, climatiques et météorologiques, etc., peuvent aussi être utilisées pour caractériser le phénomène étudié. Par conséquent, la définition de la structure spatio-temporelle représente un outil puissant pour compren- dre la distribution du phénomène et sa corrélation avec des processus sous-jacents tels que les facteurs socio-économiques, environnementaux et météorologiques. De ce fait, nous proposons une méthodologie basée sur l'adaptation et l'application de mesures statistiques et fractales des processus ponctuels d'analyse global (par ex. l'indice de Morisita, la dimension fractale par comptage de boîtes, le formalisme multifractal et la fonction K de Ripley) et local (par ex. la statistique de scan). Des nombreuses mesures décrivant les structures spatio-temporelles de phénomènes environnementaux peuvent être trouvées dans la littérature. Néanmoins, la plupart de ces mesures sont de caractère global et ne considèrent pas de contraintes spatiales com- plexes, ainsi que la haute variabilité et la nature multivariée des événements. A cet effet, la méthodologie ici proposée prend en compte les complexités de l'espace géographique ou le phénomène a lieu, à travers de l'introduction du concept de Domaine de Validité et l'application des mesures d'analyse spatiale dans des données en présentant différentes contraintes géographiques. Cela permet l'évaluation du degré relatif d'agrégation spatiale/temporelle des structures du phénomène observé. En plus, exclusif au cas de feux forestiers, cette recherche propose aussi deux nouvelles méthodologies pour la définition et la cartographie des zones périurbaines, décrites comme des espaces anthropogéniques à proximité de la végétation sauvage ou de la forêt, et de la prédiction de la susceptibilité à l'ignition de feu. A cet égard, l'objectif principal de cette Thèse a été d'effectuer une recherche statistique/géospatiale avec une forte application dans des cas réels, pour analyser et décrire des phénomènes environnementaux complexes aussi bien que surmonter des problèmes méthodologiques non résolus relatifs à la caractérisation des structures spatio-temporelles, particulièrement, celles des occurrences de feux forestières. Ainsi, cette Thèse fournit une réponse à la demande croissante de la gestion et du monitoring environnemental pour le déploiement d'outils d'évaluation des risques et des dangers naturels et anthro- pogéniques. Les majeures contributions de ce travail ont été présentées aux conférences nationales et internationales, et ont été aussi publiées dans 5 revues internationales avec comité de lecture. Des collaborations nationales et internationales ont été aussi établies et accomplies avec succès.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium.

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 micromol/L), as well as by glutamate (100 micromol/L) and the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (10 micromol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10(-6) mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type-specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

The multipoint Morisita index for the analysis of spatial patterns

In many fields, the spatial clustering of sampled data points has many consequences. Therefore, several indices have been proposed to assess the level of clustering affecting datasets (e.g. the Morisita index, Ripley's Kfunction and Rényi's generalized entropy). The classical Morisita index measures how many times it is more likely to select two measurement points from the same quadrats (the data set is covered by a regular grid of changing size) than it would be in the case of a random distribution generated from a Poisson process. The multipoint version (k-Morisita) takes into account k points with k >= 2. The present research deals with a new development of the k-Morisita index for (1) monitoring network characterization and for (2) detection of patterns in monitored phenomena. From a theoretical perspective, a connection between the k-Morisita index and multifractality has also been found and highlighted on a mathematical multifractal set.

Functional effects of Na+,K+-ATPase gene mutations linked to familial hemiplegic migraine.

Familial hemiplegic migraine type 2, an autosomal dominant form of migraine with aura, has been associated with four distinct mutations in the alpha2-subunit of the Na+,K+-ATPase. We have introduced these mutations in the alpha2-subunit of the human Na+,K+-ATPase and the corresponding mutations in the Bufo marinus alpha1-subunit and studied these mutants by expression in Xenopus oocyte. Metabolic labeling studies showed that the mutants were synthesized and associated with the beta-subunit, except for the alpha2HW887R mutant, which was poorly synthesized, and the alpha1BW890R, which was partially retained in the endoplasmic reticulum. [3H]ouabain binding showed the presence of the alpha2HR689Q and alpha2HM731T at the membrane, whereas the alpha2HL764P and alpha2HW887R could not be detected. Functional studies with the mutants of the B. marinus Na+,K+-ATPase showed a reduced or abolished electrogenic activity and a low K+ affinity for the alpha1BW890R mutant. Through different mechanisms, all these mutations result in a strong decrease of the functional expression of the Na+,K+-pump. The decreased activity in alpha2 isoform of the Na+,K+-pump expressed in astrocytes seems an essential component of hemiplegic migraine pathogenesis and may be responsible for the cortical spreading depression, which is one of the first events in migraine attacks.

Nonvitamin-k-antagonist oral anticoagulants in patients with atrial fibrillation and previous stroke or transient ischemic attack: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND AND PURPOSE: To assess whether the combined analysis of all phase III trials of nonvitamin-K-antagonist (non-VKA) oral anticoagulants in patients with atrial fibrillation and previous stroke or transient ischemic attack shows a significant difference in efficacy or safety compared with warfarin. METHODS: We searched PubMed until May 31, 2012, for randomized clinical trials using the following search items: atrial fibrillation, anticoagulation, warfarin, and previous stroke or transient ischemic attack. Studies had to be phase III trials in atrial fibrillation patients comparing warfarin with a non-VKA currently on the market or with the intention to be brought to the market in North America or Europe. Analysis was performed on intention-to-treat basis. A fixed-effects model was used as more appropriate than a random-effects model when combining a small number of studies. RESULTS: Among 47 potentially eligible articles, 3 were included in the meta-analysis. In 14 527 patients, non-VKAs were associated with a significant reduction of stroke/systemic embolism (odds ratios, 0.85 [95% CI, 074-0.99]; relative risk reduction, 14%; absolute risk reduction, 0.7%; number needed to treat, 134 over 1.8-2.0 years) compared with warfarin. Non-VKAs were also associated with a significant reduction of major bleeding compared with warfarin (odds ratios, 0.86 [95% CI, 075-0.99]; relative risk reduction, 13%; absolute risk reduction, 0.8%; number needed to treat, 125), mainly driven by the significant reduction of hemorrhagic stroke (odds ratios, 0.44 [95% CI, 032-0.62]; relative risk reduction, 57.9%; absolute risk reduction, 0.7%; number needed to treat, 139). CONCLUSIONS: In the context of the significant limitations of combining the results of disparate trials of different agents, non-VKAs seem to be associated with a significant reduction in rates of stroke or systemic embolism, hemorrhagic stroke, and major bleeding when compared with warfarin in patients with previous stroke or transient ischemic attack.

The type of K-ras mutation determines prognosis in colorectal cancer.

BACKGROUND: Mutations involving the oncogene K-ras in colorectal cancer may be related to tumor aggressiveness. However, the value of K-ras gene determination as a prognostic marker has not been clearly established. PATIENTS AND METHODS: The results from 98 patients recruited in a prospective study analyzing the effect of a K-ras mutation as a prognostic factor in colorectal cancer are reported. RESULTS: Disease-free (P = 0.02) and overall survival (P = 0.03) were significantly reduced for patients harboring a K-ras mutation. Two specific mutations demonstrated a significantly increased risk of disease recurrence, namely, 12-TGT (P = 0.04) and 13-GAC substitutions (P = 0.002). Patients with either of these substitutions had a 2-year disease-free survival rate of 37% compared with that of 67% for the group of patients harboring any other mutation type or a wild-type status (P = 0.01). CONCLUSIONS: The results herein presented suggest that K-ras acts as a prognostic factor in colorectal cancer and that this effect is probably related to a limited number of defined mutations.

Structural and functional interaction sites between Na,K-ATPase and FXYD proteins.

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K(+) and Na(+) affinity. Little is known about the interaction sites between the Na,K-ATPase alpha subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase alpha subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe(956), Glu(960), Leu(964), and Phe(967) in TM9 of the Na,K-ATPase alpha subunit represent one face interacting with the three FXYD proteins. Leu(964) and Phe(967) contribute to the efficient association of FXYD proteins with the Na,K-ATPase alpha subunit, whereas Phe(956) and Glu(960) are essential for the transmission of the functional effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. The relative contribution of Phe(956) and Glu(960) to the K(+) effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. In contrast to the effect on the apparent K(+) affinity, Phe(956) and Glu(960) are not involved in the effect of FXYD2 and FXYD4 on the apparent Na(+) affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe(956), Glu(960), Leu(964), and Phe(967) in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase alpha subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Early vitamin K deficiency bleeding after maternal phenobarbital intake: management of massive intracranial haemorrhage by minimal surgical intervention.

Vitamin K deficiency bleeding within the first 24 h of life is caused in most cases by maternal drug intake (e.g. coumarins, anticonvulsants, tuberculostatics) during pregnancy. Haemorrhage is often life-threatening and usually not prevented by vitamin K prophylaxis at birth. We report a case of severe intracranial bleeding at birth secondary to phenobarbital-induced vitamin K deficiency and traumatic delivery. Burr hole trepanations of the skull were performed and the subdural haematoma was evacuated. Despite the severe prognosis, the infant showed an unexpected good recovery. At the age of 3 years, neurological examinations were normal as was the EEG at the age of 9 months. CT showed close to normal intracranial structures. CONCLUSION: This case report stresses the importance of antenatal vitamin K prophylaxis and the consideration of a primary Caesarean section in maternal vitamin K deficiency states and demonstrates the successful management of massive subdural haemorrhage by a limited surgical approach.

Spin-labeling coronary MR angiography with steady-state free precession and radial k-space sampling: initial results in healthy volunteers.

The purpose of this study was to prospectively compare free-breathing navigator-gated cardiac-triggered three-dimensional steady-state free precession (SSFP) spin-labeling coronary magnetic resonance (MR) angiography performed by using Cartesian k-space sampling with that performed by using radial k-space sampling. A new dedicated placement of the two-dimensional selective labeling pulse and an individually adjusted labeling delay time approved by the institutional review board were used. In 14 volunteers (eight men, six women; mean age, 28.8 years) who gave informed consent, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), vessel sharpness, vessel length, and subjective image quality were investigated. Differences between groups were analyzed with nonparametric tests (Wilcoxon, Pearson chi2). Radial imaging, as compared with Cartesian imaging, resulted in a significant reduction in the severity of motion artifacts, as well as an increase in SNR (26.9 vs 12.0, P < .05) in the coronary arteries and CNR (23.1 vs 8.8, P < .05) between the coronary arteries and the myocardium. A tendency toward improved vessel sharpness and vessel length was also found with radial imaging. Radial SSFP imaging is a promising technique for spin-labeling coronary MR angiography.

Purification and characterization of (Na+ + K+)-ATPase from toad kidney

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.