Seawater carbonate chemistry and biological processes during experiments with the spider crab Hyas araneus in Kongsfjorden, Svalbard, 2009

With global climate change, ocean warming and acidification occur concomitantly. In this study, we tested the hypothesis that increasing CO2 levels affect the acid-base balance and reduce the activity capacity of the Arctic spider crab Hyas araneus, especially at the limits of thermal tolerance. Crabs were acclimated to projected oceanic CO2 levels for 12 days (today: 380, towards the year 2100: 750 and 1,120 and beyond: 3,000 ?atm) and at two temperatures (1 and 4 °C). Effects of these treatments on the righting response (RR) were determined (1) at acclimation temperatures followed by (2) righting when exposed to an additional acute (15 min) heat stress at 12 °C. Prior to (resting) and after the consecutive stresses of combined righting activity and heat exposure, acid-base status and lactate contents were measured in the haemolymph. Under resting conditions, CO2 caused a decrease in haemolymph pH and an increase in oxygen partial pressure. Despite some buffering via an accumulation of bicarbonate, the extracellular acidosis remained uncompensated at 1 °C, a trend exacerbated when animals were acclimated to 4 °C. The additional combined exposure to activity and heat had only a slight effect on blood gas and acid-base status. Righting activity in all crabs incubated at 1 and 4 °C was unaffected by elevated CO2 levels or acute heat stress but was significantly reduced when both stressors acted synergistically. This impact was much stronger in the group acclimated at 1 °C where some individuals acclimated to high CO2 levels stopped responding. Lactate only accumulated in the haemolymph after combined righting and heat stress. In the group acclimated to 1 °C, lactate content was highest under normocapnia and lowest at the highest CO2 level in line with the finding that RR was largely reduced. In crabs acclimated to 4 °C, the RR was less affected by CO2 such that activity caused lactate to increase with rising CO2 levels. In line with the concept of oxygen and capacity limited thermal tolerance, all animals exposed to temperature extremes displayed a reduction in scope for performance, a trend exacerbated by increasing CO2 levels. Additionally, the differences seen between cold- and warm-acclimated H. araneus after heat stress indicate that a small shift to higher acclimation temperatures also alleviates the response to temperature extremes, indicating a shift in the thermal tolerance window which reduces susceptibility to additional CO2 exposure.

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.

Damage reduction of the laser drilling process on back contact solar cells by chemical treatment

Production of back contact solar cells requires holes generations on the wafers to keep both positive and negative contacts on the back side of the cell. This drilling process weakens the wafer mechanically due to the presence of the holes and the damage introduced during the process as microcracks. In this study, several chemical processes have been applied to drilled wafers in order to eliminate or reduce the damage generated during this fabrication step. The treatments analyzed are the followings: alkaline etching during 1, 3 and 5 minutes, acid etching for 2 and 4 minutes and texturisation. To determine mechanical strength of the samples a common mechanical study has been carried out testing the samples by the Ring on Ring bending test and obtaining the stress state in the moment of failure by FE simulation. Finally the results obtained for each treatment were fitted to a three parameter Weibull distribution

Robustness and Recoverability in Transport Logistics

Abstract Transport is the foundation of any economy: it boosts economic growth, creates wealth, enhances trade, geographical accessibility and the mobility of people. Transport is also a key ingredient for a high quality of life, making places accessible and bringing people together. The future prosperity of our world will depend on the ability of all of its regions to remain fully and competitively integrated in the world economy. Efficient transport is vital in making this happen. Operations research can help in efficiently planning the design and operating transport systems. Planning and operational processes are fields that are rich in combinatorial optimization problems. These problems can be analyzed and solved through the application of mathematical models and optimization techniques, which may lead to an improvement in the performance of the transport system, as well as to a reduction in the time required for solving these problems. The latter aspect is important, because it increases the flexibility of the system: the system can adapt in a faster way to changes in the environment (i.e.: weather conditions, crew illness, failures, etc.). These disturbing changes (called disruptions) often enforce the schedule to be adapted. The direct consequences are delays and cancellations, implying many schedule adjustments and huge costs. Consequently, robust schedules and recovery plans must be developed in order to fight against disruptions. This dissertation makes contributions to two different fields: rail and air applications. Robust planning and recovery methods are presented. In the field of railway transport we develop several mathematical models which answer to RENFE’s (the major railway operator in Spain) needs: 1. We study the rolling stock assignment problem: here, we introduce some robust aspects in order to ameliorate some operations which are likely to fail. Once the rolling stock assignment is known, we propose a robust routing model which aims at identifying the train units’ sequences while minimizing the expected delays and human resources needed to perform the sequences. 2. It is widely accepted that the sequential solving approach produces solutions that are not global optima. Therefore, we develop an integrated and robust model to determine the train schedule and rolling stock assignment. We also propose an integrated model to study the rolling stock circulations. Circulations are determined by the rolling stock assignment and routing of the train units. 3. Although our aim is to develop robust plans, disruptions will be likely to occur and recovery methods will be needed. Therefore, we propose a recovery method which aims to recover the train schedule and rolling stock assignment in an integrated fashion all while considering the passenger demand. In the field of air transport we develop several mathematical models which answer to IBERIA’s (the major airline in Spain) needs: 1. We look at the airline-scheduling problem and develop an integrated approach that optimizes schedule design, fleet assignment and passenger use so as to reduce costs and create fewer incompatibilities between decisions. Robust itineraries are created to ameliorate misconnected passengers. 2. Air transport operators are continuously facing competition from other air operators and different modes of transport (e.g., High Speed Rail). Consequently, airline profitability is critically influenced by the airline’s ability to estimate passenger demands and construct profitable flight schedules. We consider multi-modal competition including airline and rail, and develop a new approach that estimates the demand associated with a given schedule; and generates airline schedules and fleet assignments using an integrated schedule design and fleet assignment optimization model that captures the impacts of schedule decisions on passenger demand.

Water scarcity and climate change impact and vulnerability in irrigated agriculture in Mediterranean river basins

Actualmente, la escasez de agua constituye un importante problema en muchos lugares del mundo. El crecimiento de la población, la creciente necesidad de alimentos, el desarrollo socio-económico y el cambio climático ejercen una importante y cada vez mayor presión sobre los recursos hídricos, a la que muchos países van a tener que enfrentarse en los próximos anos. La región Mediterránea es una de las regiones del mundo de mayor escasez de recursos hídricos, y es además una de las zonas más vulnerables al cambio climático. La mayoría de estudios sobre cambio climático prevén mayores temperaturas y una disminución de las precipitaciones, y una creciente escasez de agua debida a la disminución de recursos disponibles y al aumento de las demandas de riego. En el contexto actual de desarrollo de políticas se demanda cada vez más una mayor consideración del cambio climático en el marco de las políticas sectoriales. Sin embargo, los estudios enfocados a un solo sector no reflejan las múltiples dimensiones del los efectos del cambio climático. Numerosos estudios científicos han demostrado que el cambio climático es un fenómeno de naturaleza multi-dimensional y cuyos efectos se transmiten a múltiples escalas. Por tanto, es necesaria la producción de estudios y herramientas de análisis capaces de reflejar todas estas dimensiones y que contribuyan a la elaboración de políticas robustas en un contexto de cambio climático. Esta investigación pretende aportar una visión global de la problemática de la escasez de agua y los impactos, la vulnerabilidad y la adaptación al cambio climático en el contexto de la región mediterránea. La investigación presenta un marco integrado de modelización que se va ampliando progresivamente en un proceso secuencial y multi-escalar en el que en cada etapa se incorpora una nueva dimensión. La investigación consta de cuatro etapas que se abordan a lo largo de cuatro capítulos. En primer lugar, se estudia la vulnerabilidad económica de las explotaciones de regadío del Medio Guadiana, en España. Para ello, se utiliza un modelo de programación matemática en combinación con un modelo econométrico. A continuación, en la segunda etapa, se utiliza un modelo hidro-económico que incluye un modelo de cultivo para analizar los procesos que tienen lugar a escala de cultivo, explotación y cuenca teniendo en cuenta distintas escalas geográficas y de toma de decisiones. Esta herramienta permite el análisis de escenarios de cambio climático y la evaluación de posibles medidas de adaptación. La tercera fase consiste en el análisis de las barreras que dificultan la aplicación de procesos de adaptación para lo cual se analizan las redes socio-institucionales en la cuenca. Finalmente, la cuarta etapa aporta una visión sobre la escasez de agua y el cambio climático a escala nacional y regional mediante el estudio de distintos escenarios de futuro plausibles y los posibles efectos de las políticas en la escasez de agua. Para este análisis se utiliza un modelo econométrico de datos de panel para la región mediterránea y un modelo hidro-económico que se aplica a los casos de estudio de España y Jordania. Los resultados del estudio ponen de relieve la importancia de considerar múltiples escalas y múltiples dimensiones en el estudio de la gestión de los recursos hídricos y la adaptación al cambio climático en los contextos mediterráneos de escasez de agua estudiados. Los resultados muestran que los impactos del cambio climático en la cuenca del Guadiana y en el conjunto de España pueden comprometer la sostenibilidad del regadío y de los ecosistemas. El análisis a escala de cuenca hidrográfica resalta la importancia de las interacciones entre los distintos usuarios del agua y en concreto entre distintas comunidades de regantes, así como la necesidad de fortalecer el papel de las instituciones y de fomentar la creación de una visión común en la cuenca para facilitar la aplicación de los procesos de adaptación. Asimismo, los resultados de este trabajo evidencian también la capacidad y el papel fundamental de las políticas para lograr un desarrollo sostenible y la adaptación al cambio climático es regiones de escasez de agua tales como la región mediterránea. Especialmente, este trabajo pone de manifiesto el potencial de la Directiva Marco del Agua de la Unión Europea para lograr una efectiva adaptación al cambio climático. Sin embargo, en Jordania, además de la adaptación al cambio climático, es preciso diseñar estrategias de desarrollo sostenible más ambiciosas que contribuyan a reducir el riesgo futuro de escasez de agua. ABSTRACT Water scarcity is becoming a major concern in many parts of the world. Population growth, increasing needs for food production, socio-economic development and climate change represent pressures on water resources that many countries around the world will have to deal in the coming years. The Mediterranean region is one of the most water scarce regions of the world and is considered a climate change hotspot. Most projections of climate change envisage an increase in temperatures and a decrease in precipitation and a resulting reduction in water resources availability as a consequence of both reduced water availability and increased irrigation demands. Current policy development processes require the integration of climate change concerns into sectoral policies. However, sector-oriented studies often fail to address all the dimensions of climate change implications. Climate change research in the last years has evidenced the need for more integrated studies and methodologies that are capable of addressing the multi-scale and multi-dimensional nature of climate change. This research attempts to provide a comprehensive view of water scarcity and climate change impacts, vulnerability and adaptation in Mediterranean contexts. It presents an integrated modelling framework that is progressively enlarged in a sequential multi-scale process in which a new dimension of climate change and water resources is addressed at every stage. It is comprised of four stages, each one explained in a different chapter. The first stage explores farm-level economic vulnerability in the Spanish Guadiana basin using a mathematical programming model in combination with an econometric model. Then, in a second stage, the use of a hydro-economic modelling framework that includes a crop growth model allows for the analysis of crop, farm and basin level processes taking into account different geographical and decision-making scales. This integrated tool is used for the analysis of climate change scenarios and for the assessment of potential adaptation options. The third stage includes the analysis of barriers to the effective implementation of adaptation processes based on socioinstitutional network analysis. Finally, a regional and country level perspective of water scarcity and climate change is provided focusing on different possible socio-economic development pathways and the effect of policies on future water scarcity. For this analysis, a panel-data econometric model and a hydro-economic model are applied for the analysis of the Mediterranean region and country level case studies in Spain and Jordan. The overall results of the study demonstrate the value of considering multiple scales and multiple dimensions in water management and climate change adaptation in the Mediterranean water scarce contexts analysed. Results show that climate change impacts in the Guadiana basin and in Spain may compromise the sustainability of irrigation systems and ecosystems. The analysis at the basin level highlights the prominent role of interactions between different water users and irrigation districts and the need to strengthen institutional capacity and common understanding in the basin to enhance the implementation of adaptation processes. The results of this research also illustrate the relevance of water policies in achieving sustainable development and climate change adaptation in water scarce areas such as the Mediterranean region. Specifically, the EU Water Framework Directive emerges as a powerful trigger for climate change adaptation. However, in Jordan, outreaching sustainable development strategies are required in addition to climate change adaptation to reduce future risk of water scarcity.

Analysing the complexity of the model-based decision making processes within the industrial management context

PURPOSE The decision-making process plays a key role in organizations. Every decision-making process produces a final choice that may or may not prompt action. Recurrently, decision makers find themselves in the dichotomous question of following a traditional sequence decision-making process where the output of a decision is used as the input of the next stage of the decision, or following a joint decision-making approach where several decisions are taken simultaneously. The implication of the decision-making process will impact different players of the organization. The choice of the decision- making approach becomes difficult to find, even with the current literature and practitioners’ knowledge. The pursuit of better ways for making decisions has been a common goal for academics and practitioners. Management scientists use different techniques and approaches to improve different types of decisions. The purpose of this decision is to use the available resources as well as possible (data and techniques) to achieve the objectives of the organization. The developing and applying of models and concepts may be helpful to solve managerial problems faced every day in different companies. As a result of this research different decision models are presented to contribute to the body of knowledge of management science. The first models are focused on the manufacturing industry and the second part of the models on the health care industry. Despite these models being case specific, they serve the purpose of exemplifying that different approaches to the problems and could provide interesting results. Unfortunately, there is no universal recipe that could be applied to all the problems. Furthermore, the same model could deliver good results with certain data and bad results for other data. A framework to analyse the data before selecting the model to be used is presented and tested in the models developed to exemplify the ideas. METHODOLOGY As the first step of the research a systematic literature review on the joint decision is presented, as are the different opinions and suggestions of different scholars. For the next stage of the thesis, the decision-making process of more than 50 companies was analysed in companies from different sectors in the production planning area at the Job Shop level. The data was obtained using surveys and face-to-face interviews. The following part of the research into the decision-making process was held in two application fields that are highly relevant for our society; manufacturing and health care. The first step was to study the interactions and develop a mathematical model for the replenishment of the car assembly where the problem of “Vehicle routing problem and Inventory” were combined. The next step was to add the scheduling or car production (car sequencing) decision and use some metaheuristics such as ant colony and genetic algorithms to measure if the behaviour is kept up with different case size problems. A similar approach is presented in a production of semiconductors and aviation parts, where a hoist has to change from one station to another to deal with the work, and a jobs schedule has to be done. However, for this problem simulation was used for experimentation. In parallel, the scheduling of operating rooms was studied. Surgeries were allocated to surgeons and the scheduling of operating rooms was analysed. The first part of the research was done in a Teaching hospital, and for the second part the interaction of uncertainty was added. Once the previous problem had been analysed a general framework to characterize the instance was built. In the final chapter a general conclusion is presented. FINDINGS AND PRACTICAL IMPLICATIONS The first part of the contributions is an update of the decision-making literature review. Also an analysis of the possible savings resulting from a change in the decision process is made. Then, the results of the survey, which present a lack of consistency between what the managers believe and the reality of the integration of their decisions. In the next stage of the thesis, a contribution to the body of knowledge of the operation research, with the joint solution of the replenishment, sequencing and inventory problem in the assembly line is made, together with a parallel work with the operating rooms scheduling where different solutions approaches are presented. In addition to the contribution of the solving methods, with the use of different techniques, the main contribution is the framework that is proposed to pre-evaluate the problem before thinking of the techniques to solve it. However, there is no straightforward answer as to whether it is better to have joint or sequential solutions. Following the proposed framework with the evaluation of factors such as the flexibility of the answer, the number of actors, and the tightness of the data, give us important hints as to the most suitable direction to take to tackle the problem. RESEARCH LIMITATIONS AND AVENUES FOR FUTURE RESEARCH In the first part of the work it was really complicated to calculate the possible savings of different projects, since in many papers these quantities are not reported or the impact is based on non-quantifiable benefits. The other issue is the confidentiality of many projects where the data cannot be presented. For the car assembly line problem more computational power would allow us to solve bigger instances. For the operation research problem there was a lack of historical data to perform a parallel analysis in the teaching hospital. In order to keep testing the decision framework it is necessary to keep applying more case studies in order to generalize the results and make them more evident and less ambiguous. The health care field offers great opportunities since despite the recent awareness of the need to improve the decision-making process there are many opportunities to improve. Another big difference with the automotive industry is that the last improvements are not spread among all the actors. Therefore, in the future this research will focus more on the collaboration between academia and the health care sector.

Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25–3 hr) and followed sequentially by c-jun (0.5–3 hr), JunB (0.5–6 hr), NRF-1 (1–12 hr), Cyt c (12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, β-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Glial reduction in the subgenual prefrontal cortex in mood disorders

Mood disorders are among the most common neuropsychiatric illnesses, yet little is known about their neurobiology. Recent neuroimaging studies have found that the volume of the subgenual part of Brodmann’s area 24 (sg24) is reduced in familial forms of major depressive disorder (MDD) and bipolar disorder (BD). In this histological study, we used unbiased stereological techniques to examine the cellular composition of area sg24 in two different sets of brains. There was no change in the number or size of neurons in area sg24 in mood disorders. In contrast, the numbers of glia were reduced markedly in both MDD and BD. The reduction in glial number was most prominent in subgroups of subjects with familial MDD (24%, P = 0.01) or BD (41%, P = 0.01). The glial reduction in subjects without a clear family history was lower in magnitude and not statistically significant. Consistent with neuroimaging findings, cortical volume was reduced in area sg24 in subjects with familial mood disorders. Schizophrenic brains studied as psychiatric controls had normal neuronal and glial numbers and cortical volume. Glial and neuronal numbers also were counted in area 3b of the somatosensory cortex in the same group of brains and were normal in all psychiatric groups. Glia affect several processes, including regulation of extracellular potassium, glucose storage and metabolism, and glutamate uptake, all of which are crucial for normal neuronal activity. We thus have identified a biological marker associated with familial mood disorders that may provide important clues regarding the pathogenesis of these common psychiatric conditions.

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription–PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500–15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.