The Role of Raf Kinase Inhibitor Protein in Cell Motility

Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development. When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division. RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target. Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B. In order to begin to study the function of RKIP in D. discoideum and its role in cell motility, I created a mutant cell line which lacks a functional RKIP-A gene. In this paper, we show that removal of RKIP-A does not affect vegetative motility, but impairs chemotaxis and development in the presence of drug. Interestingly, RKIP-A knockout mutants appear more resistant to drug effects on vegetative motility than wild-type cells. More research is needed to reconcile these seemingly contrasting results, and to better develop a model for RKIP-A’s role in cell motility.

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^

Identification and analysis of novel mitotic inhibitors of the Caenorhabditis elegans Aurora B kinase

Proper execution of mitosis requires the accurate segregation of replicated DNA into each daughter cell. The highly conserved mitotic kinase AIR-2/Aurora B is a dynamic protein that interacts with subsets of cofactors and substrates to coordinate chromosome segregation and cytokinesis in Caenorhabdiris elegans. To identify components of the AIR-2 regulatory pathway, a genome-wide RNAi-based screen for suppressors of air-2 temperature-sensitive mutant lethality was conducted. Here, I present evidence that two classes of suppressors identified in this screen are bona fide regulators of the AIR-2 kinase. The strongest suppressor cdc-48.3, encodes an Afg2/Spaf-related Cdc48-like AAA+ ATPase that regulates AIR-2 kinase activity and stability during C. elegans embryogenesis. Loss of CDC-48.3 suppresses the lethality of air-2 mutant embryos, marked by the restoration of the dynamic behavior of AIR-2 and rescue of chromosome segregation and cytokinesis defects. Loss of CDC-48.3 leads to mitotic delays and abnormal accumulation of AIR-2 during late telophase/mitotic exit. In addition, AIR-2 kinase activity is significantly upregulated from metaphase through mitotic exit in CDC-48.3 depleted embryos. Inhibition of the AIR-2 kinase is dependent on (1) a direct physical interaction between CDC-48.3 and AIR-2, and (2) CDC-48.3 ATPase activity. Importantly, the increase in AIR-2 kinase activity does not correlate with the stabilization of AIR-2 in late mitosis. Hence, CDC-48.3 is a bi-functional inhibitor of AIR-2 that is likely to act via distinct mechanisms. The second class of suppressors consists of psy-2/smk-1 and pph-4.1, which encode two components of the conserved PP4 phosphatase complex that is essential for spindle assembly, chromosome segregation, and overall mitotic progression. AIR-2 and its substrates are likely to be targets of this complex since mitotic AIR-2 kinase activity is significantly increased during mitosis when either PSY-2/SMK-1 or PPH-4.l is depleted. Altogether, this study demonstrates that during the C. elegans embryonic cell cycle, regulators including the CDC-48.3 ATPase and PP4 phosphatase complex interact with and control the kinase activity, targeting behavior and protein stability of the Aurora B kinase to ensure accurate and timely progression of mitosis. ^

ANALYSIS OF ESTROGEN-INDUCED ANEUPLOIDY AND CHROMOSOME DISPLACEMENT

Diethylstilbestrol (DES) is a known human carcinogen and teratogen whose mechanism of action remains undetermined. As essentially diploid Chinese hamster cell line (Don) was used to test diethylstilbestrol (DES), dienestrol, hexestrol and the naturally occurring estrogens, estradiol and estriol for their ability to cause metaphase arrest and to induce aneuploidy. These compounds arrest mitosis within a narrow range of high concentrations and induce aneuploidy in recovering cell populations. DES was the most effective arrestant on a comparative molar basis. Estradiol and estriol were less potent as arrestants but were effective inducers of aneuploidy. Aneuploidy was induced in a non-random manner. The smallest chromosomes were most frequently recorded in aneuploid cells. Using anti-tubulin antibody and indirect immunofluorescence, it was found that DES inhibits bi-polar spindle assembly and disrupts the cytoplasmic microtubule complex (CMTC). Estradiol arrests mitosis in a manner that allows spindle assembly. Estradiol has no apparent effect on the CMTC. The naturally occurring estrogens caused chromosome displacement during mitotic arrest. Electron microscopy confirmed that the displaced chromosomes appeared at the polar regions of arrested cells. The arresting effect of estradiol, and to some extent DES, was reduced by the addition of dibutyryl cyclic adenosine monophosphate (db-cAMP). Aneuploidy induction by DES and similar compounds may be related to their carcinogenic and/or teratogenic potential. ^

Characterization of a Novel Role for the Tousled- like Kinase in Kinetochore Assembly and Function in Caenorhabditis elegans

Chromosome segregation is a critical step during cell division to avoid aneuploidy and promote proper organismal development. Correct sister chromatid positioning and separation during mitosis helps to achieve faithful transmission of genetic material to daughter cells. This prevents improper chromosome partitioning that can potentially result in extrachromosomal fragments, increasing the tumorigenic potential of the cells. The kinetochore is a protenaicious structure responsible for the initiation and orchestration of chromosome movement during mitosis. This highly conserved structure among eukaryotes is required for chromosome attachment to the mitotic spindle and failure to assemble the kinetochore results in aberrant chromosome segregation. Thus elucidating the mechanism of kinetochore assembly is important to have a better understanding of the regulation that controls chromosome segregation. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as a mitotic kinase and depletion of TLK-1 results in embryonic lethality, characterized by nuclei displaying poor mitotic chromosome alignment, lagging chromosome, and chromosome bridges during anaphase. Additionally, previous studies from our group revealed that TLK-1 is phosphorylated independently by Aurora B at serine 634, and by CHK-1 at threonine T610. The research presented herein reveals that both phosphorylated forms of TLK-1 associate with the kinetochore during mitosis. Moreover, by systematic depletion of kinetochore proteins, I uncovered that pTLK-1 is bona fide kinetochore component that is located at the outer kinetochore layer, influencing the microtubule-binding interface. I also demonstrated that TLK-1 is necessary for the kinetochore localization of the microtubule interacting proteins CLS-2 and LIS-1 and I show that embryos depleted of TLK-1 presented an aberrant twisted kinetochore pattern. Furthermore, I established that the inner kinetochore protein KNL-2 is an in vitro substrate of TLK-1 indicating a possible role of TLK-1 in regulating centromeric assembly. Collectively, these results suggest a novel role for the Tousled-like kinase in regulation of kinetochore assembly and microtubule dynamics and demonstrate the necessity of TLK-1 for proper chromosome segregation in C. elegans.

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

Chemical composition of Fe-Mn manifestations from the Laptev Sea

In sediments of the Laptev Sea unknown earlier ferromanganese manifestations have been found. On the basis of structural-textural external signs they have been divided to five groups: 1) tube- and spindle-shaped pseudomorphs after and within invertebrates; 2) nuclear and non-nuclear nodules; 3) flagellum- and tube-like skeletons of polychaetes; 4) flat and flattened crustate nodules and crusts; 5) micronodules. All types of ferromanganese manifestations have been sorted in three main genetic series: eigenferrous formations of autochthonous (polychaetes, goethite micronodules) and allochthonous (nuclear nodules) nature; ferromanganese nodules formed under mild hydro-geodynamic conditions at the sediment-seawater geochemical barrier; and ferromanganese manifestations formed under conditions of the variable physico-chemical environment. Ferromanganese manifestations of allochthonous type have signs of littoral zones. They contain both ferrous and ferric iron and have low oxidation degree of manganese in comparison with the autochthonous type manifestations. Manganese minerals with moderate oxidation degree are represented by vernadite and buserite. Such features of iron and manganese indicate different conditions of their formation and occurrence. The main distinctive feature of ferromanganese mineralisation in the Laptev Sea is the redox barrier: the oxidized water layer enriched in oxygen and reduced sediments. This barrier provides favorable conditions for bacterial formation of ferromanganese ores. Understanding of the genesis of ferromanganese manifestations should be found in a study of organic matter reworking by bacteria.

Transductive neurofuzzy-based torque control of a milling process: results of a case study

This paper presents the design and implementation of an intelligent control system based on local neurofuzzy models of the milling process relayed through an Ehternet-based application. Its purpose is to control the spindle torque of a milling process by using an internal model control paradigm to modify the feed rate in real time. The stabilization of cutting cutting torque is especially necessary in milling processes such as high-spedd roughing of steel moulds and dies tha present minor geometric uncertainties. Thus, maintenance of the curring torque increaes the material removal rate and reduces the risk of damage due to excessive spindle vibration, a very sensitive and expensive component in all high-speed milling machines. Torque control is therefore an interesting challenge from an industrial point of view.

Caracterización de un robot para aplicaciones de mecanizado con requerimientos de tolerancias

El presente Trabajo fin Fin de Máster, versa sobre una caracterización preliminar del comportamiento de un robot de tipo industrial, configurado por 4 eslabones y 4 grados de libertad, y sometido a fuerzas de mecanizado en su extremo. El entorno de trabajo planteado es el de plantas de fabricación de piezas de aleaciones de aluminio para automoción. Este tipo de componentes parte de un primer proceso de fundición que saca la pieza en bruto. Para series medias y altas, en función de las propiedades mecánicas y plásticas requeridas y los costes de producción, la inyección a alta presión (HPDC) y la fundición a baja presión (LPC) son las dos tecnologías más usadas en esta primera fase. Para inyección a alta presión, las aleaciones de aluminio más empleadas son, en designación simbólica según norma EN 1706 (entre paréntesis su designación numérica); EN AC AlSi9Cu3(Fe) (EN AC 46000) , EN AC AlSi9Cu3(Fe)(Zn) (EN AC 46500), y EN AC AlSi12Cu1(Fe) (EN AC 47100). Para baja presión, EN AC AlSi7Mg0,3 (EN AC 42100). En los 3 primeros casos, los límites de Silicio permitidos pueden superan el 10%. En el cuarto caso, es inferior al 10% por lo que, a los efectos de ser sometidas a mecanizados, las piezas fabricadas en aleaciones con Si superior al 10%, se puede considerar que son equivalentes, diferenciándolas de la cuarta. Las tolerancias geométricas y dimensionales conseguibles directamente de fundición, recogidas en normas como ISO 8062 o DIN 1688-1, establecen límites para este proceso. Fuera de esos límites, las garantías en conseguir producciones con los objetivos de ppms aceptados en la actualidad por el mercado, obligan a ir a fases posteriores de mecanizado. Aquellas geometrías que, funcionalmente, necesitan disponer de unas tolerancias geométricas y/o dimensionales definidas acorde a ISO 1101, y no capaces por este proceso inicial de moldeado a presión, deben ser procesadas en una fase posterior en células de mecanizado. En este caso, las tolerancias alcanzables para procesos de arranque de viruta se recogen en normas como ISO 2768. Las células de mecanizado se componen, por lo general, de varios centros de control numérico interrelacionados y comunicados entre sí por robots que manipulan las piezas en proceso de uno a otro. Dichos robots, disponen en su extremo de una pinza utillada para poder coger y soltar las piezas en los útiles de mecanizado, las mesas de intercambio para cambiar la pieza de posición o en utillajes de equipos de medición y prueba, o en cintas de entrada o salida. La repetibilidad es alta, de centésimas incluso, definida según norma ISO 9283. El problema es que, estos rangos de repetibilidad sólo se garantizan si no se hacen esfuerzos o éstos son despreciables (caso de mover piezas). Aunque las inercias de mover piezas a altas velocidades hacen que la trayectoria intermedia tenga poca precisión, al inicio y al final (al coger y dejar pieza, p.e.) se hacen a velocidades relativamente bajas que hacen que el efecto de las fuerzas de inercia sean menores y que permiten garantizar la repetibilidad anteriormente indicada. No ocurre así si se quitara la garra y se intercambia con un cabezal motorizado con una herramienta como broca, mandrino, plato de cuchillas, fresas frontales o tangenciales… Las fuerzas ejercidas de mecanizado generarían unos pares en las uniones tan grandes y tan variables que el control del robot no sería capaz de responder (o no está preparado, en un principio) y generaría una desviación en la trayectoria, realizada a baja velocidad, que desencadenaría en un error de posición (ver norma ISO 5458) no asumible para la funcionalidad deseada. Se podría llegar al caso de que la tolerancia alcanzada por un pretendido proceso más exacto diera una dimensión peor que la que daría el proceso de fundición, en principio con mayor variabilidad dimensional en proceso (y por ende con mayor intervalo de tolerancia garantizable). De hecho, en los CNCs, la precisión es muy elevada, (pudiéndose despreciar en la mayoría de los casos) y no es la responsable de, por ejemplo la tolerancia de posición al taladrar un agujero. Factores como, temperatura de la sala y de la pieza, calidad constructiva de los utillajes y rigidez en el amarre, error en el giro de mesas y de colocación de pieza, si lleva agujeros previos o no, si la herramienta está bien equilibrada y el cono es el adecuado para el tipo de mecanizado… influyen más. Es interesante que, un elemento no específico tan común en una planta industrial, en el entorno anteriormente descrito, como es un robot, el cual no sería necesario añadir por disponer de él ya (y por lo tanto la inversión sería muy pequeña), puede mejorar la cadena de valor disminuyendo el costo de fabricación. Y si se pudiera conjugar que ese robot destinado a tareas de manipulación, en los muchos tiempos de espera que va a disfrutar mientras el CNC arranca viruta, pudiese coger un cabezal y apoyar ese mecanizado; sería doblemente interesante. Por lo tanto, se antoja sugestivo poder conocer su comportamiento e intentar explicar qué sería necesario para llevar esto a cabo, motivo de este trabajo. La arquitectura de robot seleccionada es de tipo SCARA. La búsqueda de un robot cómodo de modelar y de analizar cinemática y dinámicamente, sin limitaciones relevantes en la multifuncionalidad de trabajos solicitados, ha llevado a esta elección, frente a otras arquitecturas como por ejemplo los robots antropomórficos de 6 grados de libertad, muy populares a nivel industrial. Este robot dispone de 3 uniones, de las cuales 2 son de tipo par de revolución (1 grado de libertad cada una) y la tercera es de tipo corredera o par cilíndrico (2 grados de libertad). La primera unión, de tipo par de revolución, sirve para unir el suelo (considerado como eslabón número 1) con el eslabón número 2. La segunda unión, también de ese tipo, une el eslabón número 2 con el eslabón número 3. Estos 2 brazos, pueden describir un movimiento horizontal, en el plano X-Y. El tercer eslabón, está unido al eslabón número 4 por la unión de tipo corredera. El movimiento que puede describir es paralelo al eje Z. El robot es de 4 grados de libertad (4 motores). En relación a los posibles trabajos que puede realizar este tipo de robot, su versatilidad abarca tanto operaciones típicas de manipulación como operaciones de arranque de viruta. Uno de los mecanizados más usuales es el taladrado, por lo cual se elige éste para su modelización y análisis. Dentro del taladrado se elegirá para acotar las fuerzas, taladrado en macizo con broca de diámetro 9 mm. El robot se ha considerado por el momento que tenga comportamiento de sólido rígido, por ser el mayor efecto esperado el de los pares en las uniones. Para modelar el robot se utiliza el método de los sistemas multicuerpos. Dentro de este método existen diversos tipos de formulaciones (p.e. Denavit-Hartenberg). D-H genera una cantidad muy grande de ecuaciones e incógnitas. Esas incógnitas son de difícil comprensión y, para cada posición, hay que detenerse a pensar qué significado tienen. Se ha optado por la formulación de coordenadas naturales. Este sistema utiliza puntos y vectores unitarios para definir la posición de los distintos cuerpos, y permite compartir, cuando es posible y se quiere, para definir los pares cinemáticos y reducir al mismo tiempo el número de variables. Las incógnitas son intuitivas, las ecuaciones de restricción muy sencillas y se reduce considerablemente el número de ecuaciones e incógnitas. Sin embargo, las coordenadas naturales “puras” tienen 2 problemas. El primero, que 2 elementos con un ángulo de 0 o 180 grados, dan lugar a puntos singulares que pueden crear problemas en las ecuaciones de restricción y por lo tanto han de evitarse. El segundo, que tampoco inciden directamente sobre la definición o el origen de los movimientos. Por lo tanto, es muy conveniente complementar esta formulación con ángulos y distancias (coordenadas relativas). Esto da lugar a las coordenadas naturales mixtas, que es la formulación final elegida para este TFM. Las coordenadas naturales mixtas no tienen el problema de los puntos singulares. Y la ventaja más importante reside en su utilidad a la hora de aplicar fuerzas motrices, momentos o evaluar errores. Al incidir sobre la incógnita origen (ángulos o distancias) controla los motores de manera directa. El algoritmo, la simulación y la obtención de resultados se ha programado mediante Matlab. Para realizar el modelo en coordenadas naturales mixtas, es preciso modelar en 2 pasos el robot a estudio. El primer modelo se basa en coordenadas naturales. Para su validación, se plantea una trayectoria definida y se analiza cinemáticamente si el robot satisface el movimiento solicitado, manteniendo su integridad como sistema multicuerpo. Se cuantifican los puntos (en este caso inicial y final) que configuran el robot. Al tratarse de sólidos rígidos, cada eslabón queda definido por sus respectivos puntos inicial y final (que son los más interesantes para la cinemática y la dinámica) y por un vector unitario no colineal a esos 2 puntos. Los vectores unitarios se colocan en los lugares en los que se tenga un eje de rotación o cuando se desee obtener información de un ángulo. No son necesarios vectores unitarios para medir distancias. Tampoco tienen por qué coincidir los grados de libertad con el número de vectores unitarios. Las longitudes de cada eslabón quedan definidas como constantes geométricas. Se establecen las restricciones que definen la naturaleza del robot y las relaciones entre los diferentes elementos y su entorno. La trayectoria se genera por una nube de puntos continua, definidos en coordenadas independientes. Cada conjunto de coordenadas independientes define, en un instante concreto, una posición y postura de robot determinada. Para conocerla, es necesario saber qué coordenadas dependientes hay en ese instante, y se obtienen resolviendo por el método de Newton-Rhapson las ecuaciones de restricción en función de las coordenadas independientes. El motivo de hacerlo así es porque las coordenadas dependientes deben satisfacer las restricciones, cosa que no ocurre con las coordenadas independientes. Cuando la validez del modelo se ha probado (primera validación), se pasa al modelo 2. El modelo número 2, incorpora a las coordenadas naturales del modelo número 1, las coordenadas relativas en forma de ángulos en los pares de revolución (3 ángulos; ϕ1, ϕ 2 y ϕ3) y distancias en los pares prismáticos (1 distancia; s). Estas coordenadas relativas pasan a ser las nuevas coordenadas independientes (sustituyendo a las coordenadas independientes cartesianas del modelo primero, que eran coordenadas naturales). Es necesario revisar si el sistema de vectores unitarios del modelo 1 es suficiente o no. Para este caso concreto, se han necesitado añadir 1 vector unitario adicional con objeto de que los ángulos queden perfectamente determinados con las correspondientes ecuaciones de producto escalar y/o vectorial. Las restricciones habrán de ser incrementadas en, al menos, 4 ecuaciones; una por cada nueva incógnita. La validación del modelo número 2, tiene 2 fases. La primera, al igual que se hizo en el modelo número 1, a través del análisis cinemático del comportamiento con una trayectoria definida. Podrían obtenerse del modelo 2 en este análisis, velocidades y aceleraciones, pero no son necesarios. Tan sólo interesan los movimientos o desplazamientos finitos. Comprobada la coherencia de movimientos (segunda validación), se pasa a analizar cinemáticamente el comportamiento con trayectorias interpoladas. El análisis cinemático con trayectorias interpoladas, trabaja con un número mínimo de 3 puntos máster. En este caso se han elegido 3; punto inicial, punto intermedio y punto final. El número de interpolaciones con el que se actúa es de 50 interpolaciones en cada tramo (cada 2 puntos máster hay un tramo), resultando un total de 100 interpolaciones. El método de interpolación utilizado es el de splines cúbicas con condición de aceleración inicial y final constantes, que genera las coordenadas independientes de los puntos interpolados de cada tramo. Las coordenadas dependientes se obtienen resolviendo las ecuaciones de restricción no lineales con el método de Newton-Rhapson. El método de las splines cúbicas es muy continuo, por lo que si se desea modelar una trayectoria en el que haya al menos 2 movimientos claramente diferenciados, es preciso hacerlo en 2 tramos y unirlos posteriormente. Sería el caso en el que alguno de los motores se desee expresamente que esté parado durante el primer movimiento y otro distinto lo esté durante el segundo movimiento (y así sucesivamente). Obtenido el movimiento, se calculan, también mediante fórmulas de diferenciación numérica, las velocidades y aceleraciones independientes. El proceso es análogo al anteriormente explicado, recordando la condición impuesta de que la aceleración en el instante t= 0 y en instante t= final, se ha tomado como 0. Las velocidades y aceleraciones dependientes se calculan resolviendo las correspondientes derivadas de las ecuaciones de restricción. Se comprueba, de nuevo, en una tercera validación del modelo, la coherencia del movimiento interpolado. La dinámica inversa calcula, para un movimiento definido -conocidas la posición, velocidad y la aceleración en cada instante de tiempo-, y conocidas las fuerzas externas que actúan (por ejemplo el peso); qué fuerzas hay que aplicar en los motores (donde hay control) para que se obtenga el citado movimiento. En la dinámica inversa, cada instante del tiempo es independiente de los demás y tiene una posición, una velocidad y una aceleración y unas fuerzas conocidas. En este caso concreto, se desean aplicar, de momento, sólo las fuerzas debidas al peso, aunque se podrían haber incorporado fuerzas de otra naturaleza si se hubiese deseado. Las posiciones, velocidades y aceleraciones, proceden del cálculo cinemático. El efecto inercial de las fuerzas tenidas en cuenta (el peso) es calculado. Como resultado final del análisis dinámico inverso, se obtienen los pares que han de ejercer los cuatro motores para replicar el movimiento prescrito con las fuerzas que estaban actuando. La cuarta validación del modelo consiste en confirmar que el movimiento obtenido por aplicar los pares obtenidos en la dinámica inversa, coinciden con el obtenido en el análisis cinemático (movimiento teórico). Para ello, es necesario acudir a la dinámica directa. La dinámica directa se encarga de calcular el movimiento del robot, resultante de aplicar unos pares en motores y unas fuerzas en el robot. Por lo tanto, el movimiento real resultante, al no haber cambiado ninguna condición de las obtenidas en la dinámica inversa (pares de motor y fuerzas inerciales debidas al peso de los eslabones) ha de ser el mismo al movimiento teórico. Siendo así, se considera que el robot está listo para trabajar. Si se introduce una fuerza exterior de mecanizado no contemplada en la dinámica inversa y se asigna en los motores los mismos pares resultantes de la resolución del problema dinámico inverso, el movimiento real obtenido no es igual al movimiento teórico. El control de lazo cerrado se basa en ir comparando el movimiento real con el deseado e introducir las correcciones necesarias para minimizar o anular las diferencias. Se aplican ganancias en forma de correcciones en posición y/o velocidad para eliminar esas diferencias. Se evalúa el error de posición como la diferencia, en cada punto, entre el movimiento teórico deseado en el análisis cinemático y el movimiento real obtenido para cada fuerza de mecanizado y una ganancia concreta. Finalmente, se mapea el error de posición obtenido para cada fuerza de mecanizado y las diferentes ganancias previstas, graficando la mejor precisión que puede dar el robot para cada operación que se le requiere, y en qué condiciones. -------------- This Master´s Thesis deals with a preliminary characterization of the behaviour for an industrial robot, configured with 4 elements and 4 degrees of freedoms, and subjected to machining forces at its end. Proposed working conditions are those typical from manufacturing plants with aluminium alloys for automotive industry. This type of components comes from a first casting process that produces rough parts. For medium and high volumes, high pressure die casting (HPDC) and low pressure die casting (LPC) are the most used technologies in this first phase. For high pressure die casting processes, most used aluminium alloys are, in simbolic designation according EN 1706 standard (between brackets, its numerical designation); EN AC AlSi9Cu3(Fe) (EN AC 46000) , EN AC AlSi9Cu3(Fe)(Zn) (EN AC 46500), y EN AC AlSi12Cu1(Fe) (EN AC 47100). For low pressure, EN AC AlSi7Mg0,3 (EN AC 42100). For the 3 first alloys, Si allowed limits can exceed 10% content. Fourth alloy has admisible limits under 10% Si. That means, from the point of view of machining, that components made of alloys with Si content above 10% can be considered as equivalent, and the fourth one must be studied separately. Geometrical and dimensional tolerances directly achievables from casting, gathered in standards such as ISO 8062 or DIN 1688-1, establish a limit for this process. Out from those limits, guarantees to achieve batches with objetive ppms currently accepted by market, force to go to subsequent machining process. Those geometries that functionally require a geometrical and/or dimensional tolerance defined according ISO 1101, not capable with initial moulding process, must be obtained afterwards in a machining phase with machining cells. In this case, tolerances achievables with cutting processes are gathered in standards such as ISO 2768. In general terms, machining cells contain several CNCs that they are interrelated and connected by robots that handle parts in process among them. Those robots have at their end a gripper in order to take/remove parts in machining fixtures, in interchange tables to modify position of part, in measurement and control tooling devices, or in entrance/exit conveyors. Repeatibility for robot is tight, even few hundredths of mm, defined according ISO 9283. Problem is like this; those repeatibilty ranks are only guaranteed when there are no stresses or they are not significant (f.e. due to only movement of parts). Although inertias due to moving parts at a high speed make that intermediate paths have little accuracy, at the beginning and at the end of trajectories (f.e, when picking part or leaving it) movement is made with very slow speeds that make lower the effect of inertias forces and allow to achieve repeatibility before mentioned. It does not happens the same if gripper is removed and it is exchanged by an spindle with a machining tool such as a drilling tool, a pcd boring tool, a face or a tangential milling cutter… Forces due to machining would create such big and variable torques in joints that control from the robot would not be able to react (or it is not prepared in principle) and would produce a deviation in working trajectory, made at a low speed, that would trigger a position error (see ISO 5458 standard) not assumable for requested function. Then it could be possible that tolerance achieved by a more exact expected process would turn out into a worst dimension than the one that could be achieved with casting process, in principle with a larger dimensional variability in process (and hence with a larger tolerance range reachable). As a matter of fact, accuracy is very tight in CNC, (its influence can be ignored in most cases) and it is not the responsible of, for example position tolerance when drilling a hole. Factors as, room and part temperature, manufacturing quality of machining fixtures, stiffness at clamping system, rotating error in 4th axis and part positioning error, if there are previous holes, if machining tool is properly balanced, if shank is suitable for that machining type… have more influence. It is interesting to know that, a non specific element as common, at a manufacturing plant in the enviroment above described, as a robot (not needed to be added, therefore with an additional minimum investment), can improve value chain decreasing manufacturing costs. And when it would be possible to combine that the robot dedicated to handling works could support CNCs´ works in its many waiting time while CNCs cut, and could take an spindle and help to cut; it would be double interesting. So according to all this, it would be interesting to be able to know its behaviour and try to explain what would be necessary to make this possible, reason of this work. Selected robot architecture is SCARA type. The search for a robot easy to be modeled and kinematically and dinamically analyzed, without significant limits in the multifunctionality of requested operations, has lead to this choice. Due to that, other very popular architectures in the industry, f.e. 6 DOFs anthropomorphic robots, have been discarded. This robot has 3 joints, 2 of them are revolute joints (1 DOF each one) and the third one is a cylindrical joint (2 DOFs). The first joint, a revolute one, is used to join floor (body 1) with body 2. The second one, a revolute joint too, joins body 2 with body 3. These 2 bodies can move horizontally in X-Y plane. Body 3 is linked to body 4 with a cylindrical joint. Movement that can be made is paralell to Z axis. The robt has 4 degrees of freedom (4 motors). Regarding potential works that this type of robot can make, its versatility covers either typical handling operations or cutting operations. One of the most common machinings is to drill. That is the reason why it has been chosen for the model and analysis. Within drilling, in order to enclose spectrum force, a typical solid drilling with 9 mm diameter. The robot is considered, at the moment, to have a behaviour as rigid body, as biggest expected influence is the one due to torques at joints. In order to modelize robot, it is used multibodies system method. There are under this heading different sorts of formulations (f.e. Denavit-Hartenberg). D-H creates a great amount of equations and unknown quantities. Those unknown quatities are of a difficult understanding and, for each position, one must stop to think about which meaning they have. The choice made is therefore one of formulation in natural coordinates. This system uses points and unit vectors to define position of each different elements, and allow to share, when it is possible and wished, to define kinematic torques and reduce number of variables at the same time. Unknown quantities are intuitive, constrain equations are easy and number of equations and variables are strongly reduced. However, “pure” natural coordinates suffer 2 problems. The first one is that 2 elements with an angle of 0° or 180°, give rise to singular positions that can create problems in constrain equations and therefore they must be avoided. The second problem is that they do not work directly over the definition or the origin of movements. Given that, it is highly recommended to complement this formulation with angles and distances (relative coordinates). This leads to mixed natural coordinates, and they are the final formulation chosen for this MTh. Mixed natural coordinates have not the problem of singular positions. And the most important advantage lies in their usefulness when applying driving forces, torques or evaluating errors. As they influence directly over origin variable (angles or distances), they control motors directly. The algorithm, simulation and obtaining of results has been programmed with Matlab. To design the model in mixed natural coordinates, it is necessary to model the robot to be studied in 2 steps. The first model is based in natural coordinates. To validate it, it is raised a defined trajectory and it is kinematically analyzed if robot fulfils requested movement, keeping its integrity as multibody system. The points (in this case starting and ending points) that configure the robot are quantified. As the elements are considered as rigid bodies, each of them is defined by its respectively starting and ending point (those points are the most interesting ones from the point of view of kinematics and dynamics) and by a non-colinear unit vector to those points. Unit vectors are placed where there is a rotating axis or when it is needed information of an angle. Unit vectors are not needed to measure distances. Neither DOFs must coincide with the number of unit vectors. Lengths of each arm are defined as geometrical constants. The constrains that define the nature of the robot and relationships among different elements and its enviroment are set. Path is generated by a cloud of continuous points, defined in independent coordinates. Each group of independent coordinates define, in an specific instant, a defined position and posture for the robot. In order to know it, it is needed to know which dependent coordinates there are in that instant, and they are obtained solving the constraint equations with Newton-Rhapson method according to independent coordinates. The reason to make it like this is because dependent coordinates must meet constraints, and this is not the case with independent coordinates. When suitability of model is checked (first approval), it is given next step to model 2. Model 2 adds to natural coordinates from model 1, the relative coordinates in the shape of angles in revoluting torques (3 angles; ϕ1, ϕ 2 and ϕ3) and distances in prismatic torques (1 distance; s). These relative coordinates become the new independent coordinates (replacing to cartesian independent coordinates from model 1, that they were natural coordinates). It is needed to review if unit vector system from model 1 is enough or not . For this specific case, it was necessary to add 1 additional unit vector to define perfectly angles with their related equations of dot and/or cross product. Constrains must be increased in, at least, 4 equations; one per each new variable. The approval of model 2 has two phases. The first one, same as made with model 1, through kinematic analysis of behaviour with a defined path. During this analysis, it could be obtained from model 2, velocities and accelerations, but they are not needed. They are only interesting movements and finite displacements. Once that the consistence of movements has been checked (second approval), it comes when the behaviour with interpolated trajectories must be kinematically analyzed. Kinematic analysis with interpolated trajectories work with a minimum number of 3 master points. In this case, 3 points have been chosen; starting point, middle point and ending point. The number of interpolations has been of 50 ones in each strecht (each 2 master points there is an strecht), turning into a total of 100 interpolations. The interpolation method used is the cubic splines one with condition of constant acceleration both at the starting and at the ending point. This method creates the independent coordinates of interpolated points of each strecht. The dependent coordinates are achieved solving the non-linear constrain equations with Newton-Rhapson method. The method of cubic splines is very continuous, therefore when it is needed to design a trajectory in which there are at least 2 movements clearly differents, it is required to make it in 2 steps and join them later. That would be the case when any of the motors would keep stopped during the first movement, and another different motor would remain stopped during the second movement (and so on). Once that movement is obtained, they are calculated, also with numerical differenciation formulas, the independent velocities and accelerations. This process is analogous to the one before explained, reminding condition that acceleration when t=0 and t=end are 0. Dependent velocities and accelerations are calculated solving related derivatives of constrain equations. In a third approval of the model it is checked, again, consistence of interpolated movement. Inverse dynamics calculates, for a defined movement –knowing position, velocity and acceleration in each instant of time-, and knowing external forces that act (f.e. weights); which forces must be applied in motors (where there is control) in order to obtain requested movement. In inverse dynamics, each instant of time is independent of the others and it has a position, a velocity, an acceleration and known forces. In this specific case, it is intended to apply, at the moment, only forces due to the weight, though forces of another nature could have been added if it would have been preferred. The positions, velocities and accelerations, come from kinematic calculation. The inertial effect of forces taken into account (weight) is calculated. As final result of the inverse dynamic analysis, the are obtained torques that the 4 motors must apply to repeat requested movement with the forces that were acting. The fourth approval of the model consists on confirming that the achieved movement due to the use of the torques obtained in the inverse dynamics, are in accordance with movements from kinematic analysis (theoretical movement). For this, it is necessary to work with direct dynamics. Direct dynamic is in charge of calculating the movements of robot that results from applying torques at motors and forces at the robot. Therefore, the resultant real movement, as there was no change in any condition of the ones obtained at the inverse dynamics (motor torques and inertial forces due to weight of elements) must be the same than theoretical movement. When these results are achieved, it is considered that robot is ready to work. When a machining external force is introduced and it was not taken into account before during the inverse dynamics, and torques at motors considered are the ones of the inverse dynamics, the real movement obtained is not the same than the theoretical movement. Closed loop control is based on comparing real movement with expected movement and introducing required corrrections to minimize or cancel differences. They are applied gains in the way of corrections for position and/or tolerance to remove those differences. Position error is evaluated as the difference, in each point, between theoretical movemment (calculated in the kinematic analysis) and the real movement achieved for each machining force and for an specific gain. Finally, the position error obtained for each machining force and gains are mapped, giving a chart with the best accuracy that the robot can give for each operation that has been requested and which conditions must be provided.

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.

Continuous and lurching traveling pulses in neuronal networks with delay and spatially decaying connectivity

Propagation of discharges in cortical and thalamic systems, which is used as a probe for examining network circuitry, is studied by constructing a one-dimensional model of integrate-and-fire neurons that are coupled by excitatory synapses with delay. Each neuron fires only one spike. The velocity and stability of propagating continuous pulses are calculated analytically. Above a certain critical value of the constant delay, these pulses lose stability. Instead, lurching pulses propagate with discontinuous and periodic spatio-temporal characteristics. The parameter regime for which lurching occurs is strongly affected by the footprint (connectivity) shape; bistability may occur with a square footprint shape but not with an exponential footprint shape. For strong synaptic coupling, the velocity of both continuous and lurching pulses increases logarithmically with the synaptic coupling strength gsyn for an exponential footprint shape, and it is bounded for a step footprint shape. We conclude that the differences in velocity and shape between the front of thalamic spindle waves in vitro and cortical paroxysmal discharges stem from their different effective delay; in thalamic networks, large effective delay between inhibitory neurons arises from their effective interaction via the excitatory cells which display postinhibitory rebound.

Vascular endothelial growth factor C induces angiogenesis in vivo

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.