Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

Pseudomonas syringae pv. syringae strain B728a (P.s.s. B728a) is an economically significant plant pathogen that is capable of successful epiphytic colonization of leaf surfaces. Although the virulence factors associated with this pathogen’s ability to cause disease have been well studied, the transition from epiphyte to pathogen is not well understood. The research described in this dissertation utilizes high throughput sequencing transcriptome analyses to define an iron regulatory network that is predicted to be utilized during the epiphytic portion of the P.s.s. B728a lifecycle. This dissertation also describes a collaborative microarray analysis that analyzes the P.s.s. B728a transcriptome at a global level. An iron associated sigma factor, AcsS, encoded within a peptide synthesis rich region of the P.s.s. B728a genome is shown to regulate the citrate siderophore achromobactin. RNA-seq transcriptome analysis reveals that this sigma factor regulates expression of genes predicted to be involved in functions that are important during the epiphytic stage of P.s.s. B728a, including genes involved in iron response, secretion, extracellular polysaccharide production, and cell motility. As part of a collaboration, the transcriptomes of the P.s.s. B728a genome and nine deletion mutants in regulatory genes were analyzed by microarray analayses using seven treatment conditions, including epiphytic and in planta conditions. As part of these microarray analyses, results are described for the global regulator, GacS, and a downstream transcription factor, SalA. This study confirms the role of GacS and SalA in the regulation of major virulence components of P.s.s. B728a such as phytotoxin production and Type III secretion. This study also elucidates a role for GacS and SalA regulation of genes important for epiphytic survival and function, including the Type VI secretion system, iron acquisition, and EPS production.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

La neumonía nosocomial es la complicación infecciosa más frecuente en los pacientes que ingresan en las Unidades de Cuidados Intensivos (UCI). Sin embargo, esta entidad no se presenta con igual frecuencia en todos los grupos de pacientes, existiendo mayor riesgo en algunas poblaciones. Sin embargo, la calificación de nosocomial ha excluido aquellos pacientes con neumonía dentro de las primeras 48 a 72 horas. La hipótesis de este trabajo ha sido que los factores de riesgo para el desarrollo de neumonía en el período inmediatamente posterior a la intubación son diferentes al resto de períodos que el paciente permanece en ventilación mecánica. El objetivo principal del estudio fue identificar los factores de riesgo para el desarrollo de neumonía en pacientes intubados desde la instauración de la ventilación mecánica hasta las primeras 48 horas y comparar si estos factores de riesgo que pueden influir en otros períodos. Los objetivos secundarios fueron investigar si la etiología es diferente entre los períodos, investigar si el uso de un sistema de aspiración continua de secreciones subglóticas (ACSS) y el uso de antibioterapia o la combinación de ambas influye en la aparición de neumonía en las primeras 48 horas.Para responder a esta hipótesis se realizó un estudio prospectivo y observacional durante 24 meses en una UCI polivalente médico-quirúrgica. Se incluyeron todos los pacientes intubados y con más de 24 horas de ventilación mecánica y se excluyeron aquellos pacientes intubados durante menos de 24 horas y aquellos de los que no se pudo completar datos. Se evaluaron diversos grupos de variables: demográficas, comorbilidades, tratamiento usado y presencia o no de infección previa. Las variables continuas se compararon mediante el test de t y análisis de la varianza o el test U de Mann-Whitney para variables no paramétricas. Para comparar las diferencias entre grupos con variables discretas se utilizó el test exacto de Fisher de dos colas.La variable dependiente fue la presencia o no de neumonía las primeras 48 horas de ventilación mecánica. Las variables identificadas en el análisis univariante con p< 0,1 y presente en más del 10% de los pacientes fueron elegidas para el análisis de regresión logística multiple. La etiología de la neumonía en el período de las 48 horas iniciales estuvo formada mayoritariamente por Staphylococcus aureus sensible a oxacilina, Streptococcus pneumoniae y Haemophilus influenzae, mientras que en el resto de períodos Pseudomonas aeruginosa fue el agente etiológico más frecuente. Las variables asociadas al desarrollo de neumonía en el análisis univariante fueron la presencia de reanimación cardiopulmonar, aspiración, coma, intubación en situación de emergencia, sedación continua, intubación por personal no experimentado, infección previa, administración previa de antibiótico, edad inferior a 55 años. La ACSS no influyó en la incidencia de neumonía en este período. El análisis multivariado seleccionó como factores de riesgo independientes para el desarrollo de neumonía en las primeras 48 horas de ventilación mecánica, la presencia de RCP (ods ratio 4,4; IC 95%: 2,2;9), aspiración (OR: 3,3; IC 95%: 1,5;7,5) y sedación (OR:2,9; IC 95%: 1,5; 5,9). La administración previa de antibiótico, en cambio, se presentó como factor protector para la presencia de neumonía en los primeros dos días de ventilación mecánica (OR: 0,2; IC 95%: 0,09; 0,4). Ninguna de las variables analizadas se encontró relacionada con la presencia de neumonía en los períodos posteriores.En conclusión, los pacientes en los cuales se ha realizado reanimación cardiopulmonar, han presentado broncoaspiración o han estado bajo tratamiento con sedación continua presentan un mayor riesgo de presentar neumonía en el período inmediatamente posterior a la instauración de la ventilación mecánica. Al contrario, la administración de antibioterapia aparece como un factor protector. / In Intensive Care Unit patients nosocomial pneumonia is the most frequent infectious complication. In addition, nosocomial pneumonia presents very differents rates on incidence between patients subgroups. Several risk factors for pneumonia have been investigated for two decades. However, classical nosocomial pneumonia definition have excluded the first 48-72 hours of mechanical ventilation.Study hypothese was that risk factors for pneumonia in the period following intubation are differents from the other periods under mechanical ventilation. Main study objectives were to identify risk factors for penumonia in intubated patients from starting mechanical ventilation and to compare the influence of these risk factors on other periods. Secondary objectives were to investigate the etiology between differents periods, to investigate if continuous aspiration of subglottic secretions system and antibiotic use or both have effect on the occurrence of pneumonia in the first 48 hours.We performed a prospective and observational study for 24 months in medical-surgical ICU. Inclussion criteria were all patients intubated for more than 24 hours. Exclussion criteria were mechanical ventilation time less than 24 hours and absence of important data. Several variables were analized including demographical, comorbidities, treatment and previous infection.Continuous variables were compared with t test and analysis of variance or U Mann-Whitney test for nonparametric variables. Differences between groups for discretes variables were analized with Fisher exact test. Dependent variable was the presence of pneumonia within the first 48 hours of mechanical ventilation. Significant variables at the univariate analysis with p<0,1 and present in more than 10% of patients were elegible for multivariate analysis. Odds ratio and confidence intervals were calculated with standandardized methods.Etiologies within the first 48 hours were mostly due to oxacillin-sensitive Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae, whereas in later periods the microorganism most frequently isolated was Pseudomonas aeruginosa. Univariate analysis showed as associated variables with pneumonia the presence of cardiopulmonary resuscitation, aspiration, coma, emergence intubation, continuous sedation, intubation performed by a physician with low training, age lower than 55 years, whereas prior infection, and prior antibiotic exposure acted as protective. Continuous aspiration of subglottic secretions did not have impact in the incidence of pneumonia in this period. Multivariate analysis selected as independent risk factors for pneumonia within the first 48 hours of mechanical ventilation the presence of cardiopulmonary resuscitation (ods ratio 4,4; CI 95%: 2,2;9), aspiration (OR: 3,3; IC 95%: 1,5;7,5) and continuous sedation (OR:2,9; IC 95%: 1,5; 5,9). Prior antibiotic exposure were protective in this period (OR: 0,2; IC 95%: 0,09; 0,4). None of these variables were associated with pneumonia in later periods.In summary, patients requiring cardiopulmonary resuscitation, evidence of aspiration of gastric contents into airway or treated with continuous sedation are at higher risk for pneumonia within the first 48 hours of mechanical ventilation. In contrast, prior antibiotic exposure decrease the risk of pneumonia within the first 48 hours.

El cáncer de mama es una enfermedad heterogénea y solo el 20-30% de las pacientes con cáncer de mama ganglios negativos desarrollarán una recurrencia de su enfermedad en los 10 años posteriores al tratamiento local inicial. La información sobre la tasa de recurrencia a lo largo del seguimiento, y el efecto de los factores pronósticos sobre el riesgo de recurrencia, pueden tener implicaciones importantes en el tipo y la duración del tratamiento adecuado tras el tratamiento local. METODOS: Se analizan las variaciones en la tasa anual de recurrencia en 271 pacientes consecutivas con cáncer de mama ganglios negativos, tratadas solo con tratamiento local y seguidas por una mediana de 12.2 años. La distribución del riesgo se estudió en referencia a la edad, el tamaño tumoral, el grado tumoral, los receptores hormonales, la citometría de flujo y la expresión de p53 . RESULTADOS: Para todo el grupo hay un pico de recurrencia en el periodo de 2 a 3 años tras la cirugía; la tasa decrece notablemente en el intervalo 3 a 6 años, y entonces disminuye lentamente hasta el año 15, con un pequeño segundo pico aproximadamente en el décimo año del seguimiento. La tasa media de recurrencia es del 5,6% por año en los primeros 5 años postcirugía, y del 1,9% por año entre los años 5 y 15. El estudio de los factores pronósticos demuestra que su efecto cambia a lo largo del tiempo. La edad al diagnóstico, el grado tumoral, los receptores de progesterona, la ploidía y la fase S son factores pronósticos importantes en los primeros 5 años de seguimiento, pero se encuentra una importante pérdida de influencia en el periodo que va de los 5 a 15 años, sin hallar diferencias entre los subgrupos de riesgo en las recurrencias que aparecen en este periodo. La expresión de p53 es un factor pronóstico muy importante en los primeros 5 años (p=0.0008), disminuyendo su significación (p=0.07) tras cinco años de seguimiento. El tamaño tumoral es el factor que mejor mantiene su poder pronóstico durante el periodo de observación; la tasa de recurrencia para los tumores con un diámetro igual o menor a 2 cm. es mucho más baja que los tumores de mayor tamaño en todos los periodos de seguimiento; el tamaño tumoral es el mejor factor pronóstico (p=0.015) para las recaídas aparecidas tras los primeros 5 años postcirugía. CONCLUSIONES: En esta serie, la persistencia del poder pronóstico a lo largo del tiempo de los diferentes factores pronósticos es marcadamente diferente. De hecho, el poder predictivo del tamaño tumoral y la expresión de p53 se mantiene en todo el periodo de seguimiento, mientras que el de la edad, grado, receptores hormonales y ploidía se pierde tras los primeros años. / Breast carcinoma is a hetereogeneus disease and only 20% to 30% of Node¬negative breast cancer will develop recurrent disease and risk death within 10 years after initial local therapy.Information regarding recurrence rates in late follow-up, and the effect of prognostic factors on the hazard of recurrence, may have important inplications concerning the type and duration of therapy appropriate after primary local therapy. METHODS: 271 consecutive NNBC patients, treated with local therapy alone and followed for a median of 12.2 years, were analyzed in terms of annual hazard of recurrence of breast cancer. The risk distribution was assessed relative to age, tumor size, tumor grade, hormonal receptors, DNA flow cytometry and p53 expression. RESULTS: For the entire group, the peak hazard of recurrence ocurred in the interval of 2 to 3 years; the hazard decreased consistently in the interval 3 to 6 years, and then decreased slowly through year 15, with a second peak at about year 10. The average hazard of recurrence in the first 5 years postsurgery is 5.6% per year, and between years 5 and 15 the average hazard of recurrence was 1.9% per year. The study of the prognostic factors demonstrates that their prognostic effect changed over time. Age at diagnostic, tumor grade, progesterone receptors, DNA ploidy and S-phase were important prognostic factors in the first 5 years of follow-up, but a significant loss of strength was found for the period 5 to 15 years, with no difference between the risk subsets in the recurrences ocurred in this period. p53 expression was a very important prognostic factor in the first 5 years (p=0.0008), decreasing its significance (p=0.07) after 5 years of follow-up. Tumor size was the factor best maintaining prognostic power during the observation period; the hazad of recurrence for tumors less than 2 cm in diameter was much lower in all time periods than for larger tumors; tumor size was the best prognostic predictor (p=0.015) for recurrences after the first 5 years postsurgery. CONCLUSIONS: In this serie, the persistence over time of the prognostic power of the differents prognostic factors were markedly different. In fact, predictivity of tumor size and p53 expression was maintained for the entire observation time, whereas that of age, tumor grade, hormone receptors and DNA cytometry were lost within the first years.

La Diabetis Mellitus tipus 1 i tipus 2 es caracteritzen pel desenvolupament d'hiperglucèmia, la qual apareix com a conseqüència d'una disminució de la massa de cèl.lula ß i/o d'una pèrdua de funció dels illots. Així doncs, és necessari conèixer els mecanismes implicats en el creixement i la funció del pàncrees endocrí pel seu tractament.Es va demostrar que l'expressió del factor de creixement IGF-I a les cèl·lules ß de ratolins transgènics (RIP-I/IGF-I) era capaç de contrarrestar la hiperglucèmia diabètica i recuperar la massa de cèl.lula ß, després de la inducció de diabetis experimental (George, 2002). En la part I d'aquest treball, vam estudiar els mecanismes mitjançant els quals l'IGF-I era capaç d'induir aquesta recuperació. Els ratolins transgènics IGF-I tractats amb estreptozotocina (STZ) presentaven un increment en la taxa de replicació de les cèl.lules ß. Aquest augment no es donava en els animals IGF-I sense tractar. Això indicava que la regeneració es donava per una proliferació de les cèl.lules ß i, que s'activava principalment després de la inducció d'un dany. En els illots d'animals transgènics IGF-I, es va observar un increment en Cdk-4 (que activa el cicle cel.lular) i una disminució de l'inhibidor p27, suggerint una predisposició a replicar. No obstant, també es va observar un increment de l'expressió de p21, i uns nivells de ciclina D1 (reguladora de Cdk-4) similars als dels illots controls. D'aquesta manera, les cèl.lules ß dels ratolins transgènics IGF-I evitaven una sobreproliferació. Per contra, després del tractament amb STZ, els nivells de p21 disminuïen i els de ciclina D1 augmentaven, induïnt una major taxa de proliferació de les cèl.lules ß. Així doncs, això suggereix que IGF-I podria utilitzar-se com una teràpia per la regeneració del pàncrees endocrí. Per a la reversió de la Diabetis, a més de la regeneració de la massa de cèl.lula ß, és necessari una correcta funció. Recentment, s'ha descrit que la matriu extracel.lular de l'endoteli vascular dels illots promou l'expressió d'insulina i la proliferació de les cèl.lules ß (Nikolova, 2006) i que la microcirculació dels illots controla la secreció d'insulina (Ballian, 2007). Així doncs, en la part II es va estudiar el paper del factor pro-angiogènic VEGF i de l'endoteli en la funcionalitat del pàncrees endocrí i en la regulació de la massa de cèl.lula ß. Amb aquest objectiu, es van generar ratolins transgènics que sobreexpressaven VEGF a les cèl.lules ß (RIP-I/VEGF). Es van obtenir dues línies amb diferent nivell d'expressió del transgén: Tg VEGF 1, amb una elevada expressió i, Tg VEGF 2, amb una expressió més moderada. Els animals transgènics d'ambdues línies presentaven illots desorganitzats amb un increment en la vascularització i un engruixement de la membrana basal, però la massa de cèl.lula ß, i l'homeòstasi de la glucosa no es modificaven. No obstant, amb l'edat, els animals Tg VEGF 1, esdevenien intolerants a la glucosa i, posteriorment, diabètics oberts. El pàncrees endocrí d'aquests ratolins Tg VEGF 1 mostrava un increment en l'expressió de diverses citoquines i una infiltració de macròfags, que portava a la destrucció dels illots pancreàtics. Per la seva banda, els ratolins Tg VEGF 2 desenvolupaven, amb l'edat, una disminució en la secreció d'insulina, sense arribar a desenvolupar diabetis oberta. Els illots d'aquests animals mostraven una lleu infiltració de macròfags i un increment en la producció de citoquines pro-inflamatòries. Per tant, la sobreproducció de VEGF i l'increment en la vascularització dels illots no milloraven ni la viabilitat ni la funcionalitat dels illots. Contràriament, portaven, a llarg termini, a una inflamació crònica del pàncrees endocrí i a alteracions en la cèl.lula ß, induint intolerància a la glucosa i, fins i tot, diabetis oberta. / Both type 1 and type 2 diabetes are considered the most common metabolic disease, which is characterized by the development of hyperglycemia. Higher blood glucose levels are due to a decreased function and/or beta cell mass. That's why restoring a beta cell mass which colud be able to mantain normal blood glucose levels is one of the most important challanges in Regenerative Medicine for Diabetes treatment. Thus, it is necessary to further know mechanisms involved in endocrine pancreas growth and function.Transgenic mice that overexpress IGF-I in beta cells (RIP-I/IGF-I) showed that this growth factor is able to normalize blood glucose levels and restore beta cell mass after induction of diabetes (using STZ treatment) (George, 2002 and, Casellas, 2006). These results suggest that IGF-I could be a key gene for diabetes treatment. Thus, in part I of this work, we studied the putative mechanisms by which IGF-I can regenerate beta cell mass in these mice. STZ-treated transgenic mice presented an increased beta-cell replication comparing to STZ-treated wild-type and to healthy wild-type and transgenic mice. This fact suggested that regeneration of endocrine pancreas could be mainly due to beta-cell proliferation.Islets from transgenic mice showed increased levels of Cdk4 protein (a cell cyle activator) and decreased expression of p27 (a cell cycle inhibitor). However, in parallel, these islets showed increased expresion of p21 (an inhibitor) and normal levels of cyclin D1 (Cdk4 activator). Beta cells from IGF-I-transgenic animals had an equilibrium and did not show an enhanced replication rate. Nevertheless, after STZ treatment, p21 mRNA levels decreased and cyclin D1 expression increased. Consequently, cell cycle machinery could be activated. This would be consistent with increased beta cell proliferation rate in transgenic animals after STZ treatment. Thus, our study suggested that IGF-I could restore beta cell mass by inducing beta cell proliferation. To reverse Diabetes it is necessary not only to restore beta cell mass, but beta cell function as well. Extracellular matrix (ECM) and islet endothelium promote insulin gene expression and beta cell proliferation (Nikolova, 2006). Moreover, islet vascularization controls insulin secretion (Ballian, 2007). Thus, in the part II of this work, we studied the role of the angiogenic factor VEGF in the function of endocrine pancreas and in the control of beta cell mass. To this end, we generated transgenic mice that overexpressed VEGF in beta cells (RIP-I/VEGF). Two different lines were obtained: Tg VEGF 1 (with high levels of VEGF expression) and Tg VEGF 2 (with lower expression levels). Both lines showed disorganized islets with increased vascularization. No differences in glucose homeostasis and in beta cell mass were found. However, Tg VEGF 1 mice firstly developed glucose intolerance and finally became overt diabetic with age. Endocrine pancreas of these mice showed increased expression of proimflamatory cytoquines and macrophage infiltration and higly reduced beta cell mass. On the other hand, Tg VEGF 2 mice showed decreased insulina secretion with age, but never developed overt diabetes. Islets from Tg VEGF 2 mice showed cytoquine production and macrophage infiltration, but they were lower than in Tg VEGF 1 mice.Thus, VEGF overexpression and increased vascularization in islets did not improved beta cell survival and function. But, long-term overexpression of VEGF induced an inflamatory process that decreased insulin secretion and even could induced beta cell loss and overt diabetes.

La inestabilidad genómica presente en el tumor determina su evolución. Esta evolución puede ocurrir por diferentes vías de progresión tumoral que comportan unas características moleculares, cromosómicas y clínico-patológicas concretas. El estudio del daño genómico, consecuencia de la inestabilidad genómica, puede ayudar a caracterizar las vías de progresión tumoral y puede permitir la identificación de los grupos de tumores con peor pronóstico.Con el objetivo de caracterizar las distintas formas de daño genómico presentes en el cáncer colorectal y de determinar su relación con el comportamiento biológico del tumor se procedió a analizar el daño genómico de 131 tumores colorectales esporádicos mediante dos técnicas distintas: la citometría de flujo para medir aneuploidía, y la AP-PCR para cuantificar ganancias y pérdidas alélicas. A continuación se realizó la comparación de los dos tipos de daño genómico entre ellos y con las variables clínico-patológicas y moleculares de los tumores y se determinó el valor pronóstico de las medidas de daño genómico. También se analizó el papel de la aneuploidía en la diseminación metastásica.Con la intención de mejorar la cuantificación de la aneuploidía de los tumores decidimos crear un nuevo índice (Aneuploidy Index, AI) que tuviera en cuenta el grado y la extensión de la aneuploidía en el tumor. El AI tiene valor pronóstico independiente del estadío de Dukes y permite identificar un subgrupo de pacientes con tumores en estadíos tempranos, pero con alto riesgo de muerte. Por otra parte, el daño genómico medido por AP-PCR (GDF) cuantifica desequilibrios alélicos y también presenta valor pronóstico independiente. El alto GDF se asocia a mutaciones en p53, lo que indica que la inactivación de este gen podría ser una de las causas de producción de desequilibrios alélicos. Además, el GDF y el AI son independientes y por este motivo la combinación de las dos variables es el mejor predictor de supervivencia en los pacientes con resección quirúrgica radical. En cuanto al análisis de la ploidía en las metástasis, hemos observado que la mayoría presenta una población de células tumorales diploides, lo que indicaría que la diseminación ha sido llevada a cabo por este tipo de células. Además las metástasis reproducen el patrón de ploidía existente en el tumor primario.En base a los distintos tipos de daño genómico observado proponemos que éstos son la manifestación de 4 vías de progresión tumoral con factores pronósticos diferentes: vía de la inestabilidad de microsatélites, vía diploide sin inestabilidad de microsatélites (factor pronóstico: estadío de Dukes), vía aneuploide 'numérica' (factor pronóstico: AI) y vía aneuploide 'numérico-estructural' (factor pronóstico: GDF). / Genomic instability determines tumor evolution. This evolution takes place following different pathways of tumor progression that associate with specific molecular, chromosomic and clinicopathologic characteristics. The study of genomic damage, consequence of genomic instability, could help to characterize the pathways of tumor progression and to identify the subgroups of patients with worse prognosis.To characterize the different forms of genomic damage present in colorectal cancer and to determine their relationship with the biological behaviour of the tumor, we analized the genomic damage of 131 sporadic colorectal tumors using two different techniques: flow cytometry to mesure aneuploidy and AP-PCR to mesure allelic gains and losses. We made the comparison of the two types of genomic damage between them and with tumor molecular and clinicopathologic variables and we determined the prognostic value of genomic damage assessment. Furthermore we analyzed the role of aneuploidy in metastasic dissemination.To quantify tumor aneuploidy in a comprehensive way, we created a new index (Aneuploidy Index, AI) that considers both, the degree and the extension of aneuploidy in the tumor. AI showed prognostic value independent of Dukes stage and identified a subset of patients with early stage tumors but with high risk of death. On the other hand, genomic damage mesured by AP-PCR (GDF) quantified allelic imbalances and also showed independent prognostic value. High GDF associated with p53 mutations, indicating that the inactivation of this gene could be a possible cause of production of allelic imbalances. Furthermore, GDF and AI were independent and, therefore, the combination of both variables was the best predictor of survival in patients with absence of remnant disease. Ploidy analysis in metastasis revealed that most of them show a population of diploid tumoral cells, suggesting that dissemination is accomplished by diploid cells. Furthermore, most metastasis reproduced the ploidy pattern of the primary tumor.In base of the distinct types of genomic damage observed we propose that they are the manifestation of 4 pathways of tumor progression with different prognostic factors: microsatellite instability pathway, diploid without microsatellite instability pathway (prognostic factor: Dukes stage), 'numerical' aneuploid pathway (prognostic factor: AI) and 'numerical-structural' aneuploid pathway (prognostic factor: GDF).