A simulation study using the hybrid statistic and the meta-analysis t-test for data with matched and unmatched subjects in the interim analysis of clinical trials

An interim analysis is usually applied in later phase II or phase III trials to find convincing evidence of a significant treatment difference that may lead to trial termination at an earlier point than planned at the beginning. This can result in the saving of patient resources and shortening of drug development and approval time. In addition, ethics and economics are also the reasons to stop a trial earlier. In clinical trials of eyes, ears, knees, arms, kidneys, lungs, and other clustered treatments, data may include distribution-free random variables with matched and unmatched subjects in one study. It is important to properly include both subjects in the interim and the final analyses so that the maximum efficiency of statistical and clinical inferences can be obtained at different stages of the trials. So far, no publication has applied a statistical method for distribution-free data with matched and unmatched subjects in the interim analysis of clinical trials. In this simulation study, the hybrid statistic was used to estimate the empirical powers and the empirical type I errors among the simulated datasets with different sample sizes, different effect sizes, different correlation coefficients for matched pairs, and different data distributions, respectively, in the interim and final analysis with 4 different group sequential methods. Empirical powers and empirical type I errors were also compared to those estimated by using the meta-analysis t-test among the same simulated datasets. Results from this simulation study show that, compared to the meta-analysis t-test commonly used for data with normally distributed observations, the hybrid statistic has a greater power for data observed from normally, log-normally, and multinomially distributed random variables with matched and unmatched subjects and with outliers. Powers rose with the increase in sample size, effect size, and correlation coefficient for the matched pairs. In addition, lower type I errors were observed estimated by using the hybrid statistic, which indicates that this test is also conservative for data with outliers in the interim analysis of clinical trials.^

(Appendix) Analysis of benthic foraminifera from ODP Leg 181 and DSDP Leg 90

Canonical correspondence analysis indicates that the distribution of Neogene benthic foraminiferal faunas (>63 µm) in seven DSDP and ODP sites (500-4500 m water depth) east of New Zealand (38-51°S, 170°E-170°W) is most strongly influenced by depth (water mass stratification), and secondly by age (palaeoceanographic changes influencing faunal composition and biotic evolution). Stratigraphic faunal changes are interpretted in terms of the pulsed sequential development of southern, and later northern, polar glaciation and consequent cooling of bottom waters, increased vertical and lateral stratification of ocean water masses, and increased overall and seasonal surface water productivity. Oligocene initiation of the Antarctic Circumpolar Current and Deep Western Boundary Current (DWBC), flowing northwards past New Zealand, resulted in extensive hiatuses throughout the Southwest Pacific, some extending through into the Miocene. Planktic foraminiferal fragmentation index values indicate that carbonate dissolution was significant at abyssal depths throughout most of the Neogene, peaking at upper abyssal depths in the late Miocene (11-7 Ma), with the lysocline progressively deepened thereafter. Miocene abyssal faunas are dominated by Globocassidulina subglobosa and Oridorsalis umbonatus, with increasing Epistominella exigua after 16 Ma at upper abyssal depths. Peak abundances of Epistominella umbonifera indicate increased input of cold Southern Component Water to the DWBC at 7-6 Ma. Faunal association changes imply establishment of the modern Oxygen Minimum Zone (upper Circumpolar Deep Water) in the latest Miocene. Significant latitudinal differences between the benthic foraminiferal faunas at lower bathyal depths indicate the existence of an oceanic front along the Chatham Rise (location of present Subtropical Front), since the early late Miocene at least, with more pulsed productivity (higher E. exigua) along the south side. Modern Antarctic Intermediate Water faunal associations were established north of the Chatham Rise at 10-9 Ma, and south of it at 3-1.5 Ma. Middle-upper bathyal faunas on the Campbell Plateau are dominated by reticulate bolivinids during the early and middle Miocene, indicative of sustained productivity above relatively sluggish, suboxic bottom waters. Faunal changes and hiatuses indicate increased current vigour over the Campbell Plateau from the latest Miocene on. Surface water productivity (food supply) appears to have increased in three steps (at times of enhanced global cooling) marked by substantially increased relative abundance of: (1) Abditodentrix pseudothalmanni, Alabaminella weddellensis, Cassidulina norvangi (16-15 Ma, increased pulsed productivity); (2) Bulimina marginata f. aculeata, Nonionella auris, Trifarina angulosa, Uvigerina peregrina (3-1.5 Ma, increased overall productivity); and (3) Cassidulina carinata (1-0.5 Ma, increased overall productivity). Three intervals of deep-sea benthic foraminiferal taxonomic turnover are recognised (16-15, 11.5-10, 2-0.5 Ma) corresponding to intervals of enhanced global cooling and possible productivity changes. The late Pliocene-middle Pleistocene extinction, associated with increasing Northern Hemisphere glaciation, culminating in the middle Pleistocene climatic transition, was more significant in the study area than the earlier Neogene turnovers.

Contribution to the Analysis, Design and Assessment of Compact Antenna Test Ranges At Millimeter Wavelengths - Contribución al Análisis, Diseño y Caracterización de Sistemas de Medida de Antenas en Rango Compacto para Frecuencias Milimétricas

This Doctoral Thesis entitled Contribution to the analysis, design and assessment of compact antenna test ranges at millimeter wavelengths aims to deepen the knowledge of a particular antenna measurement system: the compact range, operating in the frequency bands of millimeter wavelengths. The thesis has been developed at Radiation Group (GR), an antenna laboratory which belongs to the Signals, Systems and Radiocommunications department (SSR), from Technical University of Madrid (UPM). The Radiation Group owns an extensive experience on antenna measurements, running at present four facilities which operate in different configurations: Gregorian compact antenna test range, spherical near field, planar near field and semianechoic arch system. The research work performed in line with this thesis contributes the knowledge of the first measurement configuration at higher frequencies, beyond the microwaves region where Radiation Group features customer-level performance. To reach this high level purpose, a set of scientific tasks were sequentially carried out. Those are succinctly described in the subsequent paragraphs. A first step dealed with the State of Art review. The study of scientific literature dealed with the analysis of measurement practices in compact antenna test ranges in addition with the particularities of millimeter wavelength technologies. Joint study of both fields of knowledge converged, when this measurement facilities are of interest, in a series of technological challenges which become serious bottlenecks at different stages: analysis, design and assessment. Thirdly after the overview study, focus was set on Electromagnetic analysis algorithms. These formulations allow to approach certain electromagnetic features of interest, such as field distribution phase or stray signal analysis of particular structures when they interact with electromagnetic waves sources. Properly operated, a CATR facility features electromagnetic waves collimation optics which are large, in terms of wavelengths. Accordingly, the electromagnetic analysis tasks introduce an extense number of mathematic unknowns which grow with frequency, following different polynomic order laws depending on the used algorithmia. In particular, the optics configuration which was of our interest consisted on the reflection type serrated edge collimator. The analysis of these devices requires a flexible handling of almost arbitrary scattering geometries, becoming this flexibility the nucleus of the algorithmia’s ability to perform the subsequent design tasks. This thesis’ contribution to this field of knowledge consisted on reaching a formulation which was powerful at the same time when dealing with various analysis geometries and computationally speaking. Two algorithmia were developed. While based on the same principle of hybridization, they reached different order Physics performance at the cost of the computational efficiency. Inter-comparison of their CATR design capabilities was performed, reaching both qualitative as well as quantitative conclusions on their scope. In third place, interest was shifted from analysis - design tasks towards range assessment. Millimetre wavelengths imply strict mechanical tolerances and fine setup adjustment. In addition, the large number of unknowns issue already faced in the analysis stage appears as well in the on chamber field probing stage. Natural decrease of dynamic range available by semiconductor millimeter waves sources requires in addition larger integration times at each probing point. These peculiarities increase exponentially the difficulty of performing assessment processes in CATR facilities beyond microwaves. The bottleneck becomes so tight that it compromises the range characterization beyond a certain limit frequency which typically lies on the lowest segment of millimeter wavelength frequencies. However the value of range assessment moves, on the contrary, towards the highest segment. This thesis contributes this technological scenario developing quiet zone probing techniques which achieves substantial data reduction ratii. Collaterally, it increases the robustness of the results to noise, which is a virtual rise of the setup’s available dynamic range. In fourth place, the environmental sensitivity of millimeter wavelengths issue was approached. It is well known the drifts of electromagnetic experiments due to the dependance of the re sults with respect to the surrounding environment. This feature relegates many industrial practices of microwave frequencies to the experimental stage, at millimeter wavelengths. In particular, evolution of the atmosphere within acceptable conditioning bounds redounds in drift phenomena which completely mask the experimental results. The contribution of this thesis on this aspect consists on modeling electrically the indoor atmosphere existing in a CATR, as a function of environmental variables which affect the range’s performance. A simple model was developed, being able to handle high level phenomena, such as feed - probe phase drift as a function of low level magnitudes easy to be sampled: relative humidity and temperature. With this model, environmental compensation can be performed and chamber conditioning is automatically extended towards higher frequencies. Therefore, the purpose of this thesis is to go further into the knowledge of millimetre wavelengths involving compact antenna test ranges. This knowledge is dosified through the sequential stages of a CATR conception, form early low level electromagnetic analysis towards the assessment of an operative facility, stages for each one of which nowadays bottleneck phenomena exist and seriously compromise the antenna measurement practices at millimeter wavelengths.

Analysis of concurrent constraint logic programs with a fixed scheduling rule

The analysis of concurrent constraint programs is a challenge due to the inherently concurrent behaviour of its computational model. However, most implementations of the concurrent paradigm can be viewed as a computation with a fixed scheduling rule which suspends some goals so that their execution is postponed until some condition awakens them. For a certain kind of properties, an analysis defined in these terms is correct. Furthermore, it is much more tractable, and in addition can make use of existing analysis technology for the underlying fixed computation rule. We show how this can be done when the starting point is a framework for the analysis of sequential programs. The resulting analysis, which incorporates suspensions, is adequate for concurrent models where concurrency is localized, e.g. the Andorra model. We refine the analysis for this particular case. Another model in which concurrency is preferably encapsulated, and thus suspensions are local to parts of the computation, is that of CIAO. Nonetheless, the analysis scheme can be generalized to models with global concurrency. We also sketch how this could be done, and we show how the resulting analysis framework could be used for analyzing typical properties, such as suspensión freeness.

Using quantitative descriptive analysis and temporal dominance of sensations analysis as complementary methods for profiling commercial blackcurrant squashes

Quantitative descriptive analysis (QDA) is used to describe the nature and the intensity of sensory properties from a single evaluation of a product, whereas temporal dominance of sensation (TDS) is primarily used to identify dominant sensory properties over time. Previous studies with TDS have focused on model systems, but this is the first study to use a sequential approach, i.e. QDA then TDS in measuring sensory properties of a commercial product category, using the same set of trained assessors (n = 11). The main objectives of this study were to: (1) investigate the benefits of using a sequential approach of QDA and TDS and (2) to explore the impact of the sample composition on taste and flavour perceptions in blackcurrant squashes. The present study has proposed an alternative way of determining the choice of attributes for TDS measurement based on data obtained from previous QDA studies, where available. Both methods indicated that the flavour profile was primarily influenced by the level of dilution and complexity of sample composition combined with blackcurrant juice content. In addition, artificial sweeteners were found to modify the quality of sweetness and could also contribute to bitter notes. Using QDA and TDS in tandem was shown to be more beneficial than each just on its own enabling a more complete sensory profile of the products.

Controlled time of arrival feasibility analysis

Previous research studies and operational trials have shown that using the airborne Required Time of Arrival (RTA) function, an aircraft can individually achieve an assigned time to a metering or merge point accurately. This study goes a step further and investigates the application of RTA to a real sequence of arriving aircraft into Melbourne Australia. Assuming that the actual arrival times were Controlled Time of Arrivals (CTAs) assigned to each aircraft, the study examines if the airborne RTA solution would work. Three scenarios were compared: a baseline scenario being the actual flown trajectories in a two hour time-span into Melbourne, a scenario in which the sequential landing slot times of the baseline scenario were assigned as CTAs and a third scenario in which the landing slots could be freely redistributed to the inbound traffic as CTAs. The research found that pressure on the terminal area would sometimes require aircraft to lose more time than possible through the RTA capability. Using linear holding as an additional measure to absorb extensive delays, up to 500NM (5%) of total track reduction and 1300kg (3%) of total fuel consumption could be saved in the scenario with landing slots freely distributed as CTAs, compared to the baseline scenario. Assigning CTAs in an arrival sequence requires the ground system to have an accurate trajectory predictor to propose additional delay measures (path stretching, linear holding) if necessary. Reducing the achievable time window of the aircraft to add control margin to the RTA function, had a negative impact and increased the amount of intervention other than speed control required to solve the sequence. It was concluded that the RTA capability is not a complete solution but merely a tool to assist in managing the increasing complexity of air traffic.

Analysis of the impact of globalization and economic growth on food security in developing countries

A pesar de los importantes avances en la reducción del hambre, la seguridad alimentaria continúa siendo un reto de dimensión internacional. La seguridad alimentaria es un concepto amplio y multidimensional, cuyo análisis abarca distintas escalas y horizontes temporales. Dada su complejidad, la identificación de las causas de la inseguridad alimentaria y la priorización de las medias para abordarlas, son dos cuestiones que suscitan un intenso debate en la actualidad. El objetivo de esta tesis es evaluar el impacto de la globalización y el crecimiento económico en la seguridad alimentaria en los países en desarrollo, desde una perspectiva macro y un horizonte temporal a largo plazo. La influencia de la globalización se aborda de una manera secuencial. En primer lugar, se analiza la relación entre la inversión público-privada en infraestructuras y las exportaciones agrarias. A continuación, se estudia el impacto de las exportaciones agrarias en los indicadores de seguridad alimentaria. El estudio del impacto del crecimiento económico aborda los cambios paralelos en la distribución de la renta, y cómo la inequidad influye en el comportamiento de la seguridad alimentaria nacional. Además, se analiza en qué medida el crecimiento económico contribuye a acelerar el proceso de mejora de la seguridad alimentaria. Con el fin de conseguir los objetivos mencionados, se llevan a cabo varios análisis econométricos basados en datos de panel, en el que se combinan datos de corte transversal de 52 países y datos temporales comprendidos en el periodo 1991-2012. Se analizan tanto variables en niveles como variables en tasas de cambio anual. Se aplican los modelos de estimación de efectos variables y efectos fijos, ambos en niveles y en primeras diferencias. La tesis incluye cuatro tipos de modelos econométricos, cada uno de ellos con sus correspondientes pruebas de robustez y especificaciones. Los resultados matizan la importancia de la globalización y el crecimiento económico como mecanismos de mejora de la seguridad alimentaria en los países en desarrollo. Se obtienen dos conclusiones relativas a la globalización. En primer lugar, los resultados sugieren que la promoción de las inversiones privadas en infraestructuras contribuye a aumentar las exportaciones agrarias. En segundo lugar, se observa que las exportaciones agrarias pueden tener un impacto negativo en los indicadores de seguridad alimentaria. La combinación de estas dos conclusiones sugiere que la apertura comercial y financiera no contribuye por sí misma a la mejora de la seguridad alimentaria en los países en desarrollo. La apertura internacional de los países en desarrollo ha de ir acompañada de políticas e inversiones que desarrollen sectores productivos de alto valor añadido, que fortalezcan la economía nacional y reduzcan su dependencia exterior. En relación al crecimiento económico, a pesar del incuestionable hecho de que el crecimiento económico es una condición necesaria para reducir los niveles de subnutrición, no es una condición suficiente. Se han identificado tres estrategias adicionales que han de acompañar al crecimiento económico con el fin de intensificar su impacto positivo sobre la subnutrición. Primero, es necesario que el crecimiento económico sea acompañado de una distribución más equitativa de los ingresos. Segundo, el crecimiento económico ha de reflejarse en un aumento de inversiones en salud, agua y saneamiento y educación. Se observa que, incluso en ausencia de crecimiento económico, mejoras en el acceso a agua potable contribuyen a reducir los niveles de población subnutrida. Tercero, el crecimiento económico sostenible en el largo plazo parece tener un mayor impacto positivo sobre la seguridad alimentaria que el crecimiento económico más volátil o inestable en el corto plazo. La estabilidad macroeconómica se identifica como una condición necesaria para alcanzar una mayor mejora en la seguridad alimentaria, incluso habiéndose mejorado la equidad en la distribución de los ingresos. Por último, la tesis encuentra que los países en desarrollo analizados han experimentado diferentes trayectorias no lineales en su proceso de mejora de sus niveles de subnutrición. Los resultados sugieren que un mayor nivel inicial de subnutrición y el crecimiento económico son responsables de una respuesta más rápida al reto de la mejora de la seguridad alimentaria. ABSTRACT Despite the significant reductions of hunger, food security still remains a global challenge. Food security is a wide concept that embraces multiple dimensions, and has spatial-temporal scales. Because of its complexity, the identification of the drivers underpinning food insecurity and the prioritization of measures to address them are a subject of intensive debate. This thesis attempts to assess the impact of globalization and economic growth on food security in developing countries with a macro level scale (country) and using a long-term approach. The influence of globalization is addressed in a sequential way. First, the impact of public-private investment in infrastructure on agricultural exports in developing countries is analyzed. Secondly, an assessment is conducted to determine the impact of agricultural exports on food security indicators. The impact of economic growth focuses on the parallel changes in income inequality and how the income distribution influences countries' food security performance. Furthermore, the thesis analyzes to what extent economic growth helps accelerating food security improvements. To address the above mentioned goals, various econometric models are formulated. Models use panel data procedures combining cross-sectional data of 52 countries and time series data from 1991 to 2012. Yearly data are expressed both in levels and in changes. The estimation models applied are random effects estimation and fixed effects estimations, both in levels and in first differences. The thesis includes four families of econometric models, each with its own set of robustness checks and specifications. The results qualify the relevance of globalization and economic growth as enabling mechanisms for improving food security in developing countries. Concerning globalization, two main conclusions can be drawn. First, results showed that enhancing foreign private investment in infrastructures contributes to increase agricultural exports. Second, agricultural exports appear to have a negative impact on national food security indicators. These two conclusions suggest that trade and financial openness per se do not contribute directly to improve food security in development countries. Both measures should be accompanied by investments and policies to support the development of national high value productive sectors, to strengthen the domestic economy and reduce its external dependency. Referring to economic growth, despite the unquestionable fact that income growth is a pre-requisite for reducing undernourishment, results suggest that it is a necessary but not a sufficient condition. Three additional strategies should accompany economic growth to intensifying its impact on food security. Firstly, it is necessary that income growth should be accompanied by a better distribution of income. Secondly, income growth needs to be followed by investments and policies in health, sanitation and education to improve food security. Even if economic growth falters, sustained improvements in the access to drinking water may still give rise to reductions in the percentage of undernourished people. And thirdly, long-term economic growth appears to have a greater impact on reducing hunger than growth regimes that combine periods of growth peaks followed by troughs. Macroeconomic stability is a necessary condition for accelerating food security. Finally, the thesis finds that the developing countries analyzed have experienced different non-linear paths toward improving food security. Results also show that a higher initial level of undernourishment and economic growth result in a faster response for improving food security.

Three-dimensional distribution of cortical synapses: a replicated point pattern-based analysis

The biggest problem when analyzing the brain is that its synaptic connections are extremely complex. Generally, the billions of neurons making up the brain exchange information through two types of highly specialized structures: chemical synapses (the vast majority) and so-called gap junctions (a substrate of one class of electrical synapse). Here we are interested in exploring the three-dimensional spatial distribution of chemical synapses in the cerebral cortex. Recent research has showed that the three-dimensional spatial distribution of synapses in layer III of the neocortex can be modeled by a random sequential adsorption (RSA) point process, i.e., synapses are distributed in space almost randomly, with the only constraint that they cannot overlap. In this study we hypothesize that RSA processes can also explain the distribution of synapses in all cortical layers. We also investigate whether there are differences in both the synaptic density and spatial distribution of synapses between layers. Using combined focused ion beam milling and scanning electron microscopy (FIB/SEM), we obtained three-dimensional samples from the six layers of the rat somatosensory cortex and identified and reconstructed the synaptic junctions. A total volume of tissue of approximately 4500μm3 and around 4000 synapses from three different animals were analyzed. Different samples, layers and/or animals were aggregated and compared using RSA replicated spatial point processes. The results showed no significant differences in the synaptic distribution across the different rats used in the study. We found that RSA processes described the spatial distribution of synapses in all samples of each layer. We also found that the synaptic distribution in layers II to VI conforms to a common underlying RSA process with different densities per layer. Interestingly, the results showed that synapses in layer I had a slightly different spatial distribution from the other layers.

Dynamic analysis of physiological properties of Torulaspora delbrueckii in wine fermentations and its incidence on wine quality

This work examines the physiology of a new commercial strain of Torulaspora delbrueckii in the production of red wine following different combined fermentation strategies. For a detailed comparison, several yeast metabolites and the strains implantation were measured over the entire fermentation period. In all fermentations in which T. delbrueckii was involved, the ethanol concentration was reduced; some malic acid was consumed; more pyruvic acid was released, and fewer amounts of higher alcohols were produced. The sensorial properties of final wines varied widely, emphasising the structure of wine in sequential fermentations with T. delbrueckii. These wines presented the maximum overall impression and were preferred by tasters. Semi-industrial assays were carried out confirming these differences at a higher scale. No important differences were observed in volatile aroma composition between fermentations. However, differences in mouthfeel properties were observed in semi-industrial fermentations, which were correlated with an increase in the mannoprotein content of red wines fermented sequentially with T. delbrueckii.

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.

TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin β6 subunit (β6−/−), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Mathematical analysis of activation thresholds in enzyme-catalyzed positive feedbacks: application to the feedbacks of blood coagulation.

A hierarchy of enzyme-catalyzed positive feedback loops is examined by mathematical and numerical analysis. Four systems are described, from the simplest, in which an enzyme catalyzes its own formation from an inactive precursor, to the most complex, in which two sequential feedback loops act in a cascade. In the latter we also examine the function of a long-range feedback, in which the final enzyme produced in the second loop activates the initial step in the first loop. When the enzymes generated are subject to inhibition or inactivation, all four systems exhibit threshold properties akin to excitable systems like neuron firing. For those that are amenable to mathematical analysis, expressions are derived that relate the excitation threshold to the kinetics of enzyme generation and inhibition and the initial conditions. For the most complex system, it was expedient to employ numerical simulation to demonstrate threshold behavior, and in this case long-range feedback was seen to have two distinct effects. At sufficiently high catalytic rates, this feedback is capable of exciting an otherwise subthreshold system. At lower catalytic rates, where the long-range feedback does not significantly affect the threshold, it nonetheless has a major effect in potentiating the response above the threshold. In particular, oscillatory behavior observed in simulations of sequential feedback loops is abolished when a long-range feedback is present.