Metabolism in cancer cells is adapted to meet the proliferative needs of these cells, with notable changes such as enhanced lactate secretion and glucose uptake rates. In this work, we use the Ensemble Modeling (EM) framework to gain insight and predict potential drug targets for tumor cells. A metabolic network consisting of 58 reactions is considered which accounts for glycolysis, the pentose phosphate pathway, lipid metabolism, amino acid metabolism, and includes allosteric regulation. Experimentally measured metabolite concentrations are used for developing the ensemble of models along with information on established drug targets. The resulting models predicted transaldolase (TALA) and succinate-CoA ligase (SUCOAS1m) to display a significant reduction in growth rate when repressed relative to currently known drug targets. Furthermore, the synergetic repression of transaldolase and glycine hydroxymethyltransferase (GHMT2r) showed a three fold decrease in growth rate compared to the repression of single enzyme targets.

Metabolism in cancer cells is adapted to meet the proliferative needs of these cells, with notable changes such as enhanced lactate secretion and glucose uptake rates. In this work, we use the Ensemble Modeling (EM) framework to gain insight and predict potential drug targets for tumor cells. A metabolic network consisting of 58 reactions is considered which accounts for glycolysis, the pentose phosphate pathway, lipid metabolism, amino acid metabolism, and includes allosteric regulation. Experimentally measured metabolite concentrations are used for developing the ensemble of models along with information on established drug targets. The resulting models predicted transaldolase (TALA) and succinate-CoA ligase (SUCOAS1m) to display a significant reduction in growth rate when repressed relative to currently known drug targets. Furthermore, the synergetic repression of transaldolase and glycine hydroxymethyltransferase (GHMT2r) showed a three fold decrease in growth rate compared to the repression of single enzyme targets.

Conductive anodic filament (CAF) is a failure mode in printed wiring boards (PWBs) which occurs under high humidity and high voltage gradient conditions. The filament, a copper salt, grows from anode to cathode along the epoxy-glass interface. Ready and Turbini (2000) identified this copper salt as the Cu2(OH)3Cl, atacamite compound. This work has investigated the influence of polyethylene glycol (PEG) and polyethylene propylene glycol (PEPG) fluxing agents on the chemical nature of CAF. For coupons processed with PEPG flux, with and without chloride, a copper-chloride containing compound was formed in the polymer matrix. This compound was characterized using x-ray photoelectron spectroscopy (XPS) as CuCl and an electrochemical mechanism for the formation of the chloride-containing CAF has been proposed. For PEG flux, with and without chloride, it has been shown that CAF only formed, but no copper containing compound formed in the matrix. It appears for PEG fluxed coupons, a PEG-Cu-Cl complex forms, binds the available Cu and acts as a barrier to the formation of CuCl in the polymer matrix. Meeker and Lu Valle (1995) have previously proposed that CAF failure is best represented by two competing reactions – the formation of a copper chloride corrosion compound (now identified as Cu2(OH)3Cl) and the formation of innocuous trapped chlorine compounds. Since no evidence of any trapped chloride compounds has been found, we propose that the formation of CAF is best represented by a single non-reversible reaction. For coupons processed with a high bromide-containing flux, bromide containing CAF was created and characterized using transmission electron microscopy (TEM) to be Cu2(OH)3Br. In addition, a copper-containing compound was formed in the polymer matrix and characterized using XPS as CuBr. An electrochemical mechanism for the formation of bromide-containing CAF has been proposed based on the XPS data. .

Conductive anodic filament (CAF) is a failure mode in printed wiring boards (PWBs) which occurs under high humidity and high voltage gradient conditions. The filament, a copper salt, grows from anode to cathode along the epoxy-glass interface. Ready and Turbini (2000) identified this copper salt as the Cu2(OH)3Cl, atacamite compound. This work has investigated the influence of polyethylene glycol (PEG) and polyethylene propylene glycol (PEPG) fluxing agents on the chemical nature of CAF. For coupons processed with PEPG flux, with and without chloride, a copper-chloride containing compound was formed in the polymer matrix. This compound was characterized using x-ray photoelectron spectroscopy (XPS) as CuCl and an electrochemical mechanism for the formation of the chloride-containing CAF has been proposed. For PEG flux, with and without chloride, it has been shown that CAF only formed, but no copper containing compound formed in the matrix. It appears for PEG fluxed coupons, a PEG-Cu-Cl complex forms, binds the available Cu and acts as a barrier to the formation of CuCl in the polymer matrix. Meeker and Lu Valle (1995) have previously proposed that CAF failure is best represented by two competing reactions – the formation of a copper chloride corrosion compound (now identified as Cu2(OH)3Cl) and the formation of innocuous trapped chlorine compounds. Since no evidence of any trapped chloride compounds has been found, we propose that the formation of CAF is best represented by a single non-reversible reaction. For coupons processed with a high bromide-containing flux, bromide containing CAF was created and characterized using transmission electron microscopy (TEM) to be Cu2(OH)3Br. In addition, a copper-containing compound was formed in the polymer matrix and characterized using XPS as CuBr. An electrochemical mechanism for the formation of bromide-containing CAF has been proposed based on the XPS data. .

La tesi té com a objectiu l'obtenció d'una sèrie d'equips automatitzats per a l'anàlisi de paràmetres d'interès mediambiental. Les bases de partida, fonamentades en els coneixements del GSB (UAB), són les tècniques de flux continu (FIA), com manera d'una senzilla automatització, i l'acoblament d'un tipus de sensor com són els àmpliament emprats ESI. L'objectiu final implica l'aplicació final dels equips obtinguts dintre d'un programa de monitorització de la qualitat de l'aigua de riu. L'empresa Adasa Sistemas és la que durà a terme l'aplicació. Per això, els equips desenvolupats han de complir uns requisits de caràcter tècnic i funcional: robustesa, règim autònom d'una setmana, precisió del 10%, exactitud del 20%, mínim manteniment, autocalibració, ...Visant la primera fita, i en base a estudis anteriors al GSB, l'equip d'amoni (AQUAMONIA®) incorpora un procés de difusió gasosa que permet que l'ESI d'amoni assoleixi un elevat grau de selectivitat i sensibilitat. Una vegada desenvolupat l'equip i realitzada la seva validació en camp (rius Ebre i Llobregat) s'ha instal·lat en diverses estacions del programa SAICA, i posteriorment s'incoporà al programa XACQA. Per desenvolupar l'analitzador de nitrat, i a fi d'assolir la segona fita marcada, l'estudi s'inicia amb el disseny i l'optimització del sensor potenciomètric (ESI). S'avança amb la seva integració en un sistema de flux. Degut a les característiques de la matriu d'aplicació, l'estudi d'interferències adquireix una especial rellevància. Els resultats d'aquests ens porten a concloure la necessitat d'incorporar en sèrie un sensor de clorur. L'ESI de clorur ens permetrà la senzilla compensació de la mesura interferida del nitrat. La correcció és matemàtica seguint l'equació de Nikolskii-Einseman. L'equip AQUANITRA® determina de forma simultània nitrat i clorur. Superada la seva validació en camp s'integra en les xarxes SAICA i XACQA.En el disseny del tercer analitzador, s'assoleix la construcció d'un primer prototipus que ha estat validat en condicions de laboratori amb mostres reals. L'equip integra l'anterior determinació dels ions amoni, nitrat-clorur, i la determinació de fosfat mitjançant un ESI. Però, presenta un elevat límit de detecció respecte aquesta última espècie, per tant, en un futur es planteja la incorporació d'un sistema de preconcentració.Dintre de la mateixa filosofia però donant un pas endavant cap a la miniaturització, s'estudia l'escalat (reducció) dels diferents equips. Això s'assoleix amb els microsistemes. Les condicions hidrodinàmiques de funcionament no mostren cap influencia de l'escalat a aquest nivell. Les millores respecte a la integració del sistema desbombollador i l'elèctrode de terra són evidents, però resta per superar la integració dels diferents sensors. / The objective of this thesis is the development of various automatic analysers for the determination of relevant environmental parameters. The scientific bases, coming from GSB's (UAB) knowledge, are the continuous flow techniques (FIA) as an easy automation procedure, and potentiometric sensor (ISE) as detection system. Adasa Sistemas, S.A enterprise, will apply in real world all the analysers developed at the final step to the monitoring of river water quality. So, the technical and operative requirements for this application are: robustness, unattended operation, 10% of accuracy, 20% of precision, minimum maintenance, autocalibration, .Some preliminary studies carried out in our laboratories allows the fast design and construction of ammonium analyser (AQUAMONIA®) with an ammonium electrode (flow-through configuration) and gaseous diffusion cell to attain a great sensibility and selectivity. After its validation in field conditions (Ebro and Llobregat Rivers) it was installed inside automatic alert stations belonging SAICA (national) and XACQA (regional) monitoring networks.At the first stage of nitrate analyser development, AQUANITRA®, we design and optimise the potentiometric sensor (ISE). Due to the high content of chloride ion in river water it has an important interference in nitrate determination. So it is necessary to incorporate a second serial ISE (chloride electrode) to determine its concentration and correct mathematically nitrate concentration by means of Nikolskii-Einseman's equation: the analyser carry out the simultaneous determination of nitrate and chloride ions. It was also installed in SAICA and XACQA networks.The third automatic analyser integrates previously described systems for the monitoring of ammonium and nitrate-chloride ions, plus the potentiometric determination of phosphate. And it has been validated in laboratory conditions with real samples. Owing to the high detection limit of phosphate electrode for a real application will be necessary a preliminary preconcentration step.An other objective is the miniaturisation of all analyser developed, mainly the third one. The first studies show no influence on the hidrodynamic conditions of the mircroflow system designed. A debubbling system and the grounding electrode have been successfully and easily implemented, but it must be greatly improved the sensor's integration.

Introducció: El carcinoma de pulmó és un dels problemes de salut prioritari de la societat actual, amb una major presència en homes entre els 55 i 65 anys. És una neoplàsia molt agressiva i malgrat els esforços de crear noves teràpies de QT amb diferents combinacions de fàrmacs, o RT i QT conjuntament, s'han millorat poc les taxes de supervivència d'aquesta malaltia. Sabem, per altre banda, que el cicle cel·lular amb les seves diferents fases són objecte d'una regulació bioquímica molt complexa. En aquesta tesi ens vam centrar en l'estudi d'unes quantes proteïnes implicades en el cicle (PCNA, Ki-67, p53, ciclines D1 i E) i veure quin paper tenien. Objectius de la tesi: 1) Determinar mitjançant tècniques de citometria de flux (CMF) l'índex de DNA, la fase S i el percentatge de cèl·lules PCNA positives i també el grau d'heterogeneïtat per a cadascuna en el carcinoma de pulmó de cèl·lula no petita (CPNCP), tant en els tumors primaris com en les seves metàstasis ganglionars. 2) Investigar si existeix un patró o patrons d'índexs de DNA i/o de fase S i/o de percentatge de PCNA relacionables amb el fenomen metastàtic. 3) Determinar l'expressió de l'antígen de proliferació cel·lular PCNA a les diferents fases del cicle cel·lular i avaluar si hi ha alguna relació estadística amb altres paràmetres estudiats. 4) Quantificar per mitja de tècniques d'immunohistoquímica el grau d'expressió de marcadors de proliferació cel·lular (PCNA, Ki-67), els reguladors negatius (p53) i les ciclines (D i E). 5) Avaluar les possibles relacions entre les variables següents: clíniques, anatomopatològiques, variables de CMF i variables immunohistoquímiques. 6) Analitzar estadísticament les variables que puguin ser valorades com a factor pronòstic independent. Material i mètodes: Es varen seleccionar 99 mostres de CPCNP entre 1993-1997, 89 homes i 10 dones. Es van recollir 4 tipus de dades diferents i per separat, per garantir l'objectivitat en l'obtenció de les variables. dades clíniques, anatomopatològiques, immunohistoquímiques i de citometria de flux. Les dades clíniques es van recollir per calcular la supervivència lliure i la global de la malaltia. Les dades anatomopatològiques, immunohistoquímiques i de CMF s'obtingueren de les anàlisis de les recessions quirúrgiques remeses al laboratori durant la intervenció dels malalts. Cada mostra fou dividida en dues parts: una per a CMF per l'estudi del cicle cel·lular i expressió de l'antígen PCNA a les diferents fases del cicle i l'altre per a l'estudi histològic rutinari i per l'anàlisi amb tècniques d'immunohistoquímica. Resultats: Un 70% dels adenocarcinomes es van diagnosticar a l'estadi IIIA. Ki-67, PCNA i ciclina E estaven relacionades amb el tipus histològic de carcinoma indiferenciat de cèl·lula gran (CICG). La ciclina E també estava relacionada amb el CE. La ciclina D1 amb el grau de diferenciació. PCNA i ciclina E també estaven relacionades significativament amb Ki-67. A l'anàlisi univariable en relació amb la supervivència global de la malaltia, l'estadi, CV de l'íDNA i de la fase S assoleixen la significació estadística, com a variables relacionades amb l'evolució de la malaltia. Finalment a l'anàlisi multivariable per la supervivència global de la malaltia, l'estadi i el CV de la fase S assoleixen un valor pronòstic independent. Conclusions: Hi ha una relació directa entre l'estadi i la supervivència global dels malalts. Les variables determinades per immunohistoquímica no tenen implicacions pronostiques. Hi ha una relació entre el CICG i l'expressió augmentada de marcadors de proliferació. La sobreexpressió de ciclina D1 es relaciona directament amb els CPNCP pobrament diferenciats. La fase S es relaciona amb l'expressió augmentada del PCNA per CMF. L'heterogeneïtat en l'expressió del PCNA per CMF es relaciona amb el grau d'heterogeneïtat de l'íDNA i fase S. / Introduction: Lung carcinoma is one of the priority health problems in our society. It shows mainly in men from 55 to 65 years. Is a very aggressive neoplasm and in spite of the efforts done in chemotherapy or combined with radiotherapy, the survival rate has not improved much. On the other hand, we know that the cellular cycle with its different phases is object of a very complex biochemical regulation. In this thesis we pay attention to the study of some cell cycle proteins (PCNA, KI-67, P53, cyclin D1 and E) and to try to see their functions. Objects of thesis: 1) To determine by flow cytometry (FCM) DNA index, S phase, percentage of positive PCNA cells and the heterogeneity degree of every one in non-small-cell-lung carcinomas (NSCLC), in primary tumors and ganglionary metastases. 2) To research the existence of a model or models of DNA index and/or S phase and/or a percentage of PCNA related with metastatic phenomenon. 3) To determine the proliferating cell nuclear antigen (PCNA) expression during the cell cycle and to evaluate if there are any other statistic relation with other study parameters. 4) To calculate by immunohistochemistry the degree of expression of cellular proliferating markers (PCNA, Ki-67), negative regulators (p53) and cyclins (D and E). 5) To assess the potential relations between the following values: clinics, anatomopathologics, FCM and immunohistochemistry. 6) To analyze statistically the values that might be important as an independent prognostic factor. Material and methods: We colleted 99 samples of NSCLC from 1993 to 1997, 89 men and 10 women. We collected four different types of data, in order to guarantee the objectivity when we obtain the values: clinical data, anatomopathologic, immunohistochemistry and FCM data. The clinical data was obtained to calculate the free survival and global survival of disease. The anatomopathologic, immunohistochemistry and FCM data was obtained by the analysis of surgically resected lung carcinomas that were sent to laboratory. Every sample was divided in two parts: one to study cell cycle and PCNA expression in different phases of cycle by FCM and the other to study the sample by histological study and immunohistochemical techniques. Results: 70% of adenocarcinomas were diagnosed as stage IIIA. Ki-67, PCNA and cyclin E were related with large cell carcinoma histological type, cyclin E was related too with squamous cell carcinoma type. Cyclin D1 was related with the degree of cell differentiation. PCNA and cyclin E were significantly correlated with Ki-67. A univariate analysis of global survival showed that study, variation coeficient of DNA index and variation coeficient of S phase were the most significant prognostic factors as variables related with disease evolution. Finally in a multivariate analysis to global survival disease, study and variability of S phase showed an independent prognostic value. Conclusions: There is a direct relationship between study and global survival of patients. The variables detected immunohistochemically do not have prognostic factors. There is a relationship between large cell carcinoma and high value of proliferative markers. Cyclin D1 over-expression is directly related with poorly differentiated NSCLC. S phase is related with a high PCNA expression by flow cytometry. Heterogeneity in PCNA expression by flow cytometry is related with heterogeneity degree of DNA index and S phase.

La leucemia mieloide aguda (LMA) es una proliferación neoplásica de células inmaduras de estirpe mieloide que se produce por una alteración en la regulación del crecimiento y la diferenciación de las células hematopoyéticas. Durante las décadas de los 80 y 90 se produjo un avance muy importante en el diagnóstico de la leucemia aguda que ha permitido un mayor refinamiento diagnóstico y por tanto un tratamiento más individualizado de los enfermos. Aproximadamente el 60-80% de los adultos con LMA consiguen remisión completa, pero sólo el 30-40% de estos pacientes está vivo y libre de enfermedad a los 5 años. Estos datos indican que a pesar del avance conseguido en los últimos años es necesario continuar estudiando esta enfermedad tan heterogénea. Los principales factores pronósticos en la LMA pueden dividirse entre los relacionados con características del paciente y los relacionados con características de la leucemia. En la actualidad la citogenética en el momento del diagnóstico constituye el factor pronóstico más importante para predecir la evolución clínica de los pacientes. La identificación de diferentes alteraciones moleculares está permitiendo refinar el pronóstico de determinados grupos de LMA, como el de citogenética normal. Los datos inmunofenotípicos obtenidos por citometría de flujo también son de utilidad pronóstica, especialmente durante el seguimiento de los pacientes mediante la detección de la enfermedad residual mínima (ERM). En los trabajos incluidos en esta tesis se analiza el significado pronóstico de determinados aspectos inmunofenotípicos y moleculares. En el primer trabajo se analiza la inestabilidad de microsatélites (MSI) en pacientes jóvenes (<60 años) con LMA de novo. La MSI es un fenómeno que provoca inestabilidad genética y que juega un papel muy importante en la patogénesis de las neoplasias sólidas. Su implicación en la leucemogénesis es controvertida. En nuestro estudio se observa que la MSI no es infrecuente en pacientes jóvenes con LMA de novo. No se ha observado una asociación entre la presencia de la MSI y el pronóstico de los pacientes. La MSI no se asocia de forma significativa con ninguna lesión molecular concreta lo que sugiere que esta alteración no es dependiente de ninguna vía molecular específica. En el segundo trabajo se analiza el seguimiento de la ERM mediante citometría de flujo y PCR cuantitativa en pacientes con LMA y citogenética de buen pronóstico [t(8;21)/inv(16)/t(16;16)]. Hemos observado que ambos métodos son útiles y eficaces para el seguimiento de la ERM en este grupo de pacientes. Se han podido definir unos valores críticos de ERM, tanto por citometría de flujo como por PCR cuantitativa, que permiten identificar pacientes con mayor riesgo de recaída al final del tratamiento quimioterápico y que serían, por tanto, candidatos a recibir un tratamiento más intensivo. Por último, se han analizado las características inmunofenotípicas al diagnóstico de pacientes jóvenes con LMA de novo y se ha estudiado la correlación de la expresión de determinados antígenos con las características clínicas, biológicas y con el pronóstico. CD2 es un marcador linfoide que se expresa con mayor frecuencia en LMA asociadas a la inv(16)/t(16;16), tal y como está descrito en la literatura. La expresión de CD2 no modifica el buen pronóstico de este tipo de leucemias, pero por el contrario, se asocia a una mayor tasa de refractariedad al tratamiento de inducción y por tanto, a una menor supervivencia cuando se expresa en otros tipos de LMA. CD36 se expresa en LMA con participación monocítica y, en nuestro estudio, se asocia con una menor supervivencia y un mayor riesgo de recaída. Este peor pronóstico es independiente de otros factores como la citogenética o la presencia de la FLT3-ITD. Por tanto, el perfil inmunofenotípico al diagnóstico puede aportar información pronóstica complementaria. / Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by a neoplastic proliferation of immature myeloid lineage cells produced by an alteration in the regulation of growth and differentiation of hematopoietic cells. This alteration will cause the accumulation of immature myeloid precursors capable of replication, but has lost its ability to differentiate into mature hematopoietic cells. During the decades of 80 and 90 there was a major advance in the diagnosis of AML that has allowed a more refined diagnosis and therefore a more individualized treatment of patients. About 60-80% of adults with AML achieve complete remission, but only 30-40% of these patients are alive and free of disease at 5 years. These data indicate that despite the progress achieved in recent years in diagnosis and treatment of AML, it is necessary to continue studying this disease. Prognostic factors in AML can be divided into those related to patient characteristics and those related to leukemia features. Today cytogenetics at the time of diagnosis is the most important prognostic factor for predicting clinical outcome of patients. In recent years the identification of different molecular alterations are allowing to refine the prognosis of certain groups of AML, like normal cytogenetics. Immunophenotypic data obtained by flow cytometry are also of prognostic value, especially for the study of minimal residual disease (MRD) in patients in complete remission. The papers included in this thesis examine the prognostic significance of certain immunophenotypic and molecular aspects. In the first paper we analyzed the microsatellite instability (MSI) in young patients (<60 years) with de novo AML. The MSI is a phenomenon that causes genetic instability and plays an important role in the pathogenesis of solid tumors. Their involvement in leukemogenesis is controversial. Our study shows that MSI is not an uncommon finding in young patients with de novo AML. It was observed that the presence of the MSI does not affect the prognosis of patients. The MSI was not significantly associated with any particular molecular lesion suggesting that this alteration is not dependent on any specific molecular pathway. The second paper discusses the monitoring of MRD by flow cytometry and quantitative PCR in AML patients with good prognosis cytogenetic [t(8;21) and inv(16)]. We observed that both methods are useful and effective for monitoring MRD in these patients. It was possible to define a threshold of ERM, both by flow cytometry and by quantitative PCR, which can identify patients at increased risk of relapse at the end of chemotherapy. These patients would candidates for more intensive treatment. Finally, we have analyzed the immunophenotypic characteristics at diagnosis of young patients (<60 years) with de novo AML. We studied the correlation of the expression of specific antigens with the clinical and biological characteristics and prognosis. CD2 is a lymphoid marker that is expressed more frequently in AML associated with inv(16), as described in the literature. The expression of CD2 does not alter the good prognosis of this type of leukemia, but instead is associated with decreased survival when expressed in other types of AML due to a higher rate of refractory to induction therapy. CD36 is frequently expressed in AML with monocytic involvement and, in our study is associated with decreased survival and increased risk of relapse. A multivariate study shows that the prognostic value of CD36 is independent of other factors such as cytogenetics and FLT3-ITD. Therefore, the immunophenotypic profile of diagnosis may provide additional prognostic information in AML.

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