Using Electronic Documents for Knowledge Acquisition and Model Maintenance

Knowledge acquisition and model maintenance are key problems in knowledge engineering to improve the productivity in the development of intelligent systems. Although historically a number of technical solutions have been proposed in this area, the recent experience shows that there is still an important gap between the way end-users describe their expertise and the way intelligent systems represent knowledge. In this paper we propose an original way to cope with this problem based on electronic documents. We propose the concept of intelligent document processor as a tool that allows the end-user to read/write a document explaining how an intelligent system operates in such a way that, if the user changes the content of the document, the intelligent system will react to these changes. The paper presents the structure of such a document based on knowledge categories derived from the modern knowledge modeling methodologies together with a number of requirements to be understandable by end-users and problem solvers.

Task-based telemanipulation for maintenance in large scientific facilities

This thesis presents a task-oriented approach to telemanipulation for maintenance in large scientific facilities, with specific focus on the particle accelerator facilities at European Organization for Nuclear Research (CERN) in Geneva, Switzerland and GSI Helmholtz Centre for Heavy Ion Research (GSI) in Darmstadt, Germany. It examines how telemanipulation can be used in these facilities and reviews how this differs from the representation of telemanipulation tasks within the literature. It provides methods to assess and compare telemanipulation procedures as well a test suite to compare telemanipulators themselves from a dexterity perspective. It presents a formalisation of telemanipulation procedures into a hierarchical model which can be then used as a basis to aid maintenance engineers in assessing tasks for telemanipulation, and as the basis for future research. The model introduces a new concept of Elemental Actions as the building block of telemanipulation movements and incorporates the dependent factors for procedures at a higher level of abstraction. In order to gain insight into realistic tasks performed by telemanipulation systems within both industrial and research environments a survey of teleoperation experts is presented. Analysis of the responses is performed from which it is concluded that there is a need within the robotics community for physical benchmarking tests which are geared towards evaluating the dexterity of telemanipulators for comparison of their dexterous abilities. A three stage test suite is presented which is designed to allow maintenance engineers to assess different telemanipulators for their dexterity. This incorporates general characteristics of the system, a method to compare kinematic reachability of multiple telemanipulators and physical test setups to assess dexterity from a both a qualitative perspective and measurably by using performance metrics. Finally, experimental results are provided for the application of the proposed test suite onto two telemanipulation systems, one from a research setting and the other within CERN. It describes the procedure performed and discusses comparisons between the two systems, as well as providing input from the expert operator of the CERN system.

On the harmonic analysis of cup anemometer rotation speed: A principle to monitor performance and maintenance status of rotating meteorological sensors

The calibration results of one anemometer equipped with several rotors, varying their size, were analyzed. In each case, the 30-pulses pert turn output signal of the anemometer was studied using Fourier series decomposition and correlated with the anemometer factor (i.e., the anemometer transfer function). Also, a 3-cup analytical model was correlated to the data resulting from the wind tunnel measurements. Results indicate good correlation between the post-processed output signal and the working condition of the cup anemometer. This correlation was also reflected in the results from the proposed analytical model. With the present work the possibility of remotely checking cup anemometer status, indicating the presence of anomalies and, therefore, a decrease on the wind sensor reliability is revealed.

Tag detection for preventing unauthorized face image processing

A new technology is being proposed as a solution to the problem of unintentional facial detection and recognition in pictures in which the individuals appearing want to express their privacy preferences, through the use of different tags. The existing methods for face de-identification were mostly ad hoc solutions that only provided an absolute binary solution in a privacy context such as pixelation, or a bar mask. As the number and users of social networks are increasing, our preferences regarding our privacy may become more complex, leaving these absolute binary solutions as something obsolete. The proposed technology overcomes this problem by embedding information in a tag which will be placed close to the face without being disruptive. Through a decoding method the tag will provide the preferences that will be applied to the images in further stages.

Characterisation of the operation & maintenance phase in PV rural electrification programmes

Con 1.300 millones de personas en el mundo sin acceso a la electricidad (la mayoría en entornos rurales de países empobrecidos), la energía solar fotovoltaica constituye una solución viable técnica y económicamente para electrificar las zonas más remotas del planeta donde las redes eléctricas convencionales no llegan. Casi todos los países en el mundo han desarrollado algún tipo de programa de electrificación fotovoltaica rural durante los últimos 40 años, principalmente los países más pobres, donde a través de diferentes modelos de financiación, se han instalado millones de sistemas solares domiciliarios (pequeños sistemas fotovoltaicos para uso doméstico). Durante este largo período, se han ido superando muchas barreras, como la mejora de la calidad de los sistemas fotovoltaicos, la reducción de costes, la optimización del diseño y del dimensionado de los sistemas, la disponibilidad financiera para implantar programas de electrificación rural, etc. Gracias a esto, la electrificación rural descentralizada ha experimentado recientemente un salto de escala caracterizada por la implantación de grandes programas con miles de sistemas solares domiciliarios e integrando largos períodos de mantenimiento. Muchos de estos grandes programas se están llevando a cabo con limitado éxito, ya que generalmente parten de supuestos e hipótesis poco contrastadas con la realidad, comprometiendo así un retorno económico que permita el desarrollo de esta actividad a largo plazo. En este escenario surge un nuevo reto: el de cómo garantizar la sostenibilidad de los grandes programas de electrificación rural fotovoltaica. Se argumenta que la principal causa de esta falta de rentabilidad es el imprevisto alto coste de la fase de operación y mantenimiento. Cuestiones clave tales como la estructura de costes de operación y mantenimiento o la fiabilidad de los componentes del sistema fotovoltaico no están bien caracterizados hoy en día. Esta situación limita la capacidad de diseñar estructuras de mantenimiento capaces de asegurar la sostenibilidad y la rentabilidad del servicio de operación y mantenimiento en estos programas. Esta tesis doctoral tiene como objetivo responder a estas cuestiones. Se ha realizado varios estudios sobre la base de un gran programa de electrificación rural fotovoltaica real llevado a cabo en Marruecos con más de 13.000 sistemas solares domiciliarios instalados. Sobre la base de este programa se ha hecho una evaluación en profundidad de la fiabilidad de los sistemas solares a partir de los datos de mantenimiento recogidos durante 5 años con más de 80.000 inputs. Los resultados han permitido establecer las funciones de fiabilidad de los equipos tal y como se comportan en condiciones reales de operación, las tasas de fallos y los tiempos medios hasta el fallo para los principales componentes del sistema, siendo este el primer caso de divulgación de resultados de este tipo en el campo de la electrificación rural fotovoltaica. Los dos principales componentes del sistema solar domiciliario, la batería y el módulo fotovoltaico, han sido analizados en campo a través de una muestra de 41 sistemas trabajando en condiciones reales pertenecientes al programa solar marroquí. Por un lado se ha estudiado la degradación de la capacidad de las baterías y por otro la degradación de potencia de los módulos fotovoltaicos. En el caso de las baterías, los resultados nos han permitido caracterizar la curva de degradación en capacidad llegando a obtener una propuesta de nueva definición del umbral de vida útil de las baterías en electrificación rural. También sobre la base del programa solar de Marruecos se ha llevado a cabo un estudio de caracterización de los costes reales de operación y mantenimiento a partir de la base de datos de contabilidad del programa registrados durante 5 años. Los resultados del estudio han permitido definir cuáles son costes que más incidencia tienen en el coste global. Se han obtenido los costes unitarios por sistema instalado y se han calculado los montantes de las cuotas de mantenimiento de los usuarios para garantizar la rentabilidad de la operación y mantenimiento. Finalmente, se propone un modelo de optimización matemática para diseñar estructuras de mantenimiento basado en los resultados de los estudios anteriores. La herramienta, elaborada mediante programación lineal entera mixta, se ha aplicado al programa marroquí con el fin de validar el modelo propuesto. ABSTRACT With 1,300 million people worldwide deprived of access to electricity (mostly in rural environments), photovoltaic solar energy has proven to be a cost‐effective solution and the only hope for electrifying the most remote inhabitants of the planet, where conventional electric grids do not reach because they are unaffordable. Almost all countries in the world have had some kind of rural photovoltaic electrification programme during the past 40 years, mainly the poorer countries, where through different organizational models, millions of solar home systems (small photovoltaic systems for domestic use) have been installed. During this long period, many barriers have been overcome, such as quality enhancement, cost reduction, the optimization of designing and sizing, financial availability, etc. Thanks to this, decentralized rural electrification has recently experienced a change of scale characterized by new programmes with thousands of solar home systems and long maintenance periods. Many of these large programmes are being developed with limited success, as they have generally been based on assumptions that do not correspond to reality, compromising the economic return that allows long term activity. In this scenario a new challenge emerges, which approaches the sustainability of large programmes. It is argued that the main cause of unprofitability is the unexpected high cost of the operation and maintenance of the solar systems. In fact, the lack of a paradigm in decentralized rural services has led to many private companies to carry out decentralized electrification programmes blindly. Issues such as the operation and maintenance cost structure or the reliability of the solar home system components have still not been characterized. This situation does not allow optimized maintenance structure to be designed to assure the sustainability and profitability of the operation and maintenance service. This PhD thesis aims to respond to these needs. Several studies have been carried out based on a real and large photovoltaic rural electrification programme carried out in Morocco with more than 13,000 solar home systems. An in‐depth reliability assessment has been made from a 5‐year maintenance database with more than 80,000 maintenance inputs. The results have allowed us to establish the real reliability functions, the failure rate and the main time to failure of the main components of the system, reporting these findings for the first time in the field of rural electrification. Both in‐field experiments on the capacity degradation of batteries and power degradation of photovoltaic modules have been carried out. During the experiments both samples of batteries and modules were operating under real conditions integrated into the solar home systems of the Moroccan programme. In the case of the batteries, the results have enabled us to obtain a proposal of definition of death of batteries in rural electrification. A cost assessment of the Moroccan experience based on a 5‐year accounting database has been carried out to characterize the cost structure of the programme. The results have allowed the major costs of the photovoltaic electrification to be defined. The overall cost ratio per installed system has been calculated together with the necessary fees that users would have to pay to make the operation and maintenance affordable. Finally, a mathematical optimization model has been proposed to design maintenance structures based on the previous study results. The tool has been applied to the Moroccan programme with the aim of validating the model.

Las Redes de Petri Interpretadas en el Diagnóstico de Fallos de Sistemas Híbridos. Aplicación a un Helicóptero no Tripulado

La seguridad y fiabilidad de los procesos industriales son la principal preocupación de los ingenieros encargados de las plantas industriales. Por lo tanto, desde un punto de vista económico, el objetivo principal es reducir el costo del mantenimiento, el tiempo de inactividad y las pérdidas causadas por los fallos. Por otra parte, la seguridad de los operadores, que afecta a los aspectos sociales y económicos, es el factor más relevante a considerar en cualquier sistema Debido a esto, el diagnóstico de fallos se ha convertido en un foco importante de interés para los investigadores de todo el mundo e ingenieros en la industria. Los principales trabajos enfocados en detección de fallos se basan en modelos de los procesos. Existen diferentes técnicas para el modelado de procesos industriales tales como máquinas de estado, árboles de decisión y Redes de Petri (RdP). Por lo tanto, esta tesis se centra en el modelado de procesos utilizando redes de petri interpretadas. Redes de Petri es una herramienta usada en el modelado gráfico y matemático con la habilidad para describir información de los sistemas de una manera concurrente, paralela, asincrona, distribuida y no determinística o estocástica. RdP son también una herramienta de comunicación visual gráfica útil como lo son las cartas de flujo o diagramas de bloques. Adicionalmente, las marcas de las RdP simulan la dinámica y concurrencia de los sistemas. Finalmente, ellas tienen la capacidad de definir ecuaciones de estado específicas, ecuaciones algebraicas y otros modelos que representan el comportamiento común de los sistemas. Entre los diferentes tipos de redes de petri (Interpretadas, Coloreadas, etc.), este trabajo de investigación trata con redes de petri interpretadas principalmente debido a características tales como sincronización, lugares temporizados, aparte de su capacidad para procesamiento de datos. Esta investigación comienza con el proceso para diseñar y construir el modelo y diagnosticador para detectar fallos definitivos, posteriormente, la dinámica temporal fue adicionada para detectar fallos intermitentes. Dos procesos industriales, concretamente un HVAC (Calefacción, Ventilación y Aire Acondicionado) y un Proceso de Envasado de Líquidos fueron usados como banco de pruebas para implementar la herramienta de diagnóstico de fallos (FD) creada. Finalmente, su capacidad de diagnóstico fue ampliada en orden a detectar fallos en sistemas híbridos. Finalmente, un pequeño helicóptero no tripulado fue elegido como ejemplo de sistema donde la seguridad es un desafío, y las técnicas de detección de fallos desarrolladas en esta tesis llevan a ser una herramienta valorada, desde que los accidentes de las aeronaves no tripuladas (UAVs) envuelven un alto costo económico y son la principal razón para introducir restricciones de volar sobre áreas pobladas. Así, este trabajo introduce un proceso sistemático para construir un Diagnosticador de Fallos del sistema mencionado basado en RdR Esta novedosa herramienta es capaz de detectar fallos definitivos e intermitentes. El trabajo realizado es discutido desde un punto de vista teórico y práctico. El procedimiento comienza con la división del sistema en subsistemas para seguido integrar en una RdP diagnosticadora global que es capaz de monitorear el sistema completo y mostrar las variables críticas al operador en orden a determinar la salud del UAV, para de esta manera prevenir accidentes. Un Sistema de Adquisición de Datos (DAQ) ha sido también diseñado para recoger datos durante los vuelos y alimentar la RdP diagnosticadora. Vuelos reales realizados bajo condiciones normales y de fallo han sido requeridos para llevar a cabo la configuración del diagnosticador y verificar su comportamiento. Vale la pena señalar que un alto riesgo fue asumido en la generación de fallos durante los vuelos, a pesar de eso esto permitió recoger datos básicos para desarrollar el diagnóstico de fallos, técnicas de aislamiento, protocolos de mantenimiento, modelos de comportamiento, etc. Finalmente, un resumen de la validación de resultados obtenidos durante las pruebas de vuelo es también incluido. Un extensivo uso de esta herramienta mejorará los protocolos de mantenimiento para UAVs (especialmente helicópteros) y permite establecer recomendaciones en regulaciones. El uso del diagnosticador usando redes de petri es considerado un novedoso enfoque. ABSTRACT Safety and reliability of industrial processes are the main concern of the engineers in charge of industrial plants. Thus, from an economic point of view, the main goal is to reduce the maintenance downtime cost and the losses caused by failures. Moreover, the safety of the operators, which affects to social and economic aspects, is the most relevant factor to consider in any system. Due to this, fault diagnosis has become a relevant focus of interest for worldwide researchers and engineers in the industry. The main works focused on failure detection are based on models of the processes. There are different techniques for modelling industrial processes such as state machines, decision trees and Petri Nets (PN). Thus, this Thesis is focused on modelling processes by using Interpreted Petri Nets. Petri Nets is a tool used in the graphic and mathematical modelling with ability to describe information of the systems in a concurrent, parallel, asynchronous, distributed and not deterministic or stochastic manner. PNs are also useful graphical visual communication tools as flow chart or block diagram. Additionally, the marks of the PN simulate the dynamics and concurrence of the systems. Finally, they are able to define specific state equations, algebraic equations and other models that represent the common behaviour of systems. Among the different types of PN (Interpreted, Coloured, etc.), this research work deals with the interpreted Petri Nets mainly due to features such as synchronization capabilities, timed places, apart from their capability for processing data. This Research begins with the process for designing and building the model and diagnoser to detect permanent faults, subsequently, the temporal dynamic was added for detecting intermittent faults. Two industrial processes, namely HVAC (Heating, Ventilation and Air Condition) and Liquids Packaging Process were used as testbed for implementing the Fault Diagnosis (FD) tool created. Finally, its diagnostic capability was enhanced in order to detect faults in hybrid systems. Finally, a small unmanned helicopter was chosen as example of system where safety is a challenge and fault detection techniques developed in this Thesis turn out to be a valuable tool since UAVs accidents involve high economic cost and are the main reason for setting restrictions to fly over populated areas. Thus, this work introduces a systematic process for building a Fault Diagnoser of the mentioned system based on Petri Nets. This novel tool is able to detect both intermittent and permanent faults. The work carried out is discussed from theoretical and practical point of view. The procedure begins with a division of the system into subsystems for further integration into a global PN diagnoser that is able to monitor the whole system and show critical variables to the operator in order to determine the UAV health, preventing accidents in this manner. A Data Acquisition System (DAQ) has been also designed for collecting data during the flights and feed PN Diagnoser. Real flights carried out under nominal and failure conditions have been required to perform the diagnoser setup and verify its performance. It is worth noting that a high risk was assumed in the generation of faults during the flights, nevertheless this allowed collecting basic data so as to develop fault diagnosis, isolations techniques, maintenance protocols, behaviour models, etc. Finally, a summary of the validation results obtained during real flight tests is also included. An extensive use of this tool will improve preventive maintenance protocols for UAVs (especially helicopters) and allow establishing recommendations in regulations. The use of the diagnoser by using Petri Nets is considered as novel approach.

Systematic Process for Building a Fault Diagnoser Based on Petri Nets Applied to a Helicopter

This work presents a systematic process for building a Fault Diagnoser (FD), based on Petri Nets (PNs) which has been applied to a small helicopter. This novel tool is able to detect both intermittent and permanent faults. The work carried out is discussed from theoretical and practical point of view. The procedure begins with a division of the whole system into subsystems, which are the devices that have to be modeled by using PN, considering both the normal and fault operations. Subsequently, the models are integrated into a global Petri Net diagnoser (PND) that is able to monitor a whole helicopter and show critical variables to the operator in order to determine the UAV health, preventing accidents in this manner. A Data Acquisition System (DAQ) has been designed for collecting data during the flights and feeding PN diagnoser with them. Several real flights (nominal or under failure) have been carried out to perform the diagnoser setup and verify its performance. A summary of the validation results obtained during real flight tests is also included. An extensive use of this tool will improve preventive maintenance protocols for UAVs (especially helicopters) and allow establishing recommendations in regulations

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (Malus pumila)- and hawthorn (Crataegus spp.)-infesting races of Rhagoletis pomonella (Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect’s life cycle with the phenology of its host plant.

Maintenance of a somatotopic cortical map in the face of diminishing thalamocortical inputs

This study addresses the extent of divergence in the ascending somatosensory pathways of primates. Divergence of inputs from a particular body part at each successive synaptic step in these pathways results in a potential magnification of the representation of that body part in the somatosensory cortex, so that the representation can be expanded when peripheral input from other parts is lost, as in nerve lesions or amputations. Lesions of increasing size were placed in the representation of a finger in the ventral posterior thalamic nucleus (VPL) of macaque monkeys. After a survival period of 1–5 weeks, area 3b of the somatosensory cortex ipsilateral to the lesion was mapped physiologically, and the extent of the representation of the affected and adjacent fingers was determined. Lesions affecting less than 30% of the thalamic VPL nucleus were without effect upon the cortical representation of the finger whose thalamic representation was at the center of the lesion. Lesions affecting about 35% of the VPL nucleus resulted in a shrinkage of the cortical representation of the finger whose thalamic representation was lesioned, with concomitant expansion of the representations of adjacent fingers. Beyond 35–40%, the whole cortical representation of the hand became silent. These results suggest that divergence of brainstem and thalamocortical projections, although normally not expressed, are sufficiently great to maintain a representation after a major loss of inputs from the periphery. This is likely to be one mechanism of representational plasticity in the cerebral cortex.

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (≈103 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.