A systematic review of personalized therapy in patients with advanced cancer

Background: An increased understanding of the pathogenesis of cancer at the molecular level has led to the development of personalized cancer therapy based on the mutation status of the tumor. Tailoring treatments to genetic signatures has improved treatment outcomes in patients with advanced cancer. We conducted a meta-analysis to provide a quantitative summary of the response to treatment on a phase I clinical trial matched to molecular aberration in patients with advanced solid tumors. ^ Methods: Original studies that reported the results of phase I clinical trials in patients with advanced cancer treated with matched anti-cancer therapies between January 2006 and November 2011 were identified through an extensive search of Medline, Embase, Web of Science and Cochrane Library databases. Odds Ratio (OR) with 95% confidence interval (CI) was estimated for each study to assess the strength of an association between objective response rate (ORR) and mutation status. Random effects model was used to estimate the pooled OR and their 95% CI was derived. Funnel plot was used to assess publication bias. ^ Results: Thirteen studies published between January 2006 and November 2011that reported on responses to matched phase I clinical trials in patients with advanced cancer were included in the meta-analysis. Nine studies reported on the responses seen in 538 of the 835 patients with driver mutations responsive to therapy and seven studies on the responses observed in 234 of the 306 patients with mutation predictive for negative response. Random effects model was used to estimate pooled OR, which was 7.767(95% CI = 4.199 − 14.366; p-value=0.000) in patients with activating mutations that were responsive to therapy and 0.287 (95% CI = 0.119 − 0.694; p-value=0.009) in patients with mutation predictive of negative response. ^ Conclusion: It is evident from the meta-analysis that somatic mutations present in tumor tissue of patients are predictive of responses to therapy in patients with advanced cancer in phase I setting. Plethora of research and growing evidence base indicate that selection of patients based on mutation analysis of the tumor and personalizing therapy is a step forward in the war against cancer.^

Fasting blood glucose and 8-year mortality in the hypertension detection follow-up program population

This study analyzed the relationship between fasting blood glucose (FBG) and 8-year mortality in the Hypertension Detection Follow-up Program (HDFP) population. Fasting blood glucose (FBG) was examined both as a continuous variable and by specified FBG strata: Normal (FBG 60–100 mg/dL), Impaired (FBG ≥100 and ≤125 mg/dL), and Diabetic (FBG>125 mg/dL or pre-existing diabetes) subgroups. The relationship between type 2 diabetes was examined with all-cause mortality. This thesis described and compared the characteristics of fasting blood glucose strata by recognized glucose cut-points; described the mortality rates in the various fasting blood glucose strata using Kaplan-Meier mortality curves, and compared the mortality risk of various strata using Cox Regression analysis. Overall, mortality was significantly greater among Referred Care (RC) participants compared to Stepped Care (SC) {HR = 1.17; 95% CI (1.052,1.309); p-value = 0.004}, as reported by the HDFP investigators in 1979. Compared with SC participants, the RC mortality rate was significantly higher for the Normal FBG group {HR = 1.18; 95% CI (1.029,1.363); p-value = 0.019} and the Impaired FBG group, {HR = 1.34; 95% CI (1.036,1.734); p-value = 0.026,}. However, for the diabetic group, 8-year mortality did not differ significantly between the RC and SC groups after adjusting for race, gender, age, smoking status among Diabetic individuals {HR = 1.03; 95% CI (0.816,1.303); p-value = 0.798}. This latter finding is possibly due to a lack of a treatment difference of hypertension among Diabetic participants in both RC and SC groups. The largest difference in mortality between RC and SC was in the Impaired subgroup, suggesting that hypertensive patients with FBG between 100 and 125 mg/dL would benefit from aggressive antihypertensive therapy.^

Improving Cervical Cancer Nodal Boost Radiation Therapy by Quantifying Uncertainties and Exploring Advanced Radiation Therapy Modalities

Radiation therapy for patients with intact cervical cancer is frequently delivered using primary external beam radiation therapy (EBRT) followed by two fractions of intracavitary brachytherapy (ICBT). Although the tumor is the primary radiation target, controlling microscopic disease in the lymph nodes is just as critical to patient treatment outcome. In patients where gross lymphadenopathy is discovered, an extra EBRT boost course is delivered between the two ICBT fractions. Since the nodal boost is an addendum to primary EBRT and ICBT, the prescription and delivery must be performed considering previously delivered dose. This project aims to address the major issues of this complex process for the purpose of improving treatment accuracy while increasing dose sparing to the surrounding normal tissues. Because external beam boosts to involved lymph nodes are given prior to the completion of ICBT, assumptions must be made about dose to positive lymph nodes from future implants. The first aim of this project was to quantify differences in nodal dose contribution between independent ICBT fractions. We retrospectively evaluated differences in the ICBT dose contribution to positive pelvic nodes for ten patients who had previously received external beam nodal boost. Our results indicate that the mean dose to the pelvic nodes differed by up to 1.9 Gy between independent ICBT fractions. The second aim is to develop and validate a volumetric method for summing dose of the normal tissues during prescription of nodal boost. The traditional method of dose summation uses the maximum point dose from each modality, which often only represents the worst case scenario. However, the worst case is often an exaggeration when highly conformal therapy methods such as intensity modulated radiation therapy (IMRT) are used. We used deformable image registration algorithms to volumetrically sum dose for the bladder and rectum and created a voxel-by-voxel validation method. The mean error in deformable image registration results of all voxels within the bladder and rectum were 5 and 6 mm, respectively. Finally, the third aim explored the potential use of proton therapy to reduce normal tissue dose. A major physical advantage of protons over photons is that protons stop after delivering dose in the tumor. Although theoretically superior to photons, proton beams are more sensitive to uncertainties caused by interfractional anatomical variations, and must be accounted for during treatment planning to ensure complete target coverage. We have demonstrated a systematic approach to determine population-based anatomical margin requirements for proton therapy. The observed optimal treatment angles for common iliac nodes were 90° (left lateral) and 180° (posterior-anterior [PA]) with additional 0.8 cm and 0.9 cm margins, respectively. For external iliac nodes, lateral and PA beams required additional 0.4 cm and 0.9 cm margins, respectively. Through this project, we have provided radiation oncologists with additional information about potential differences in nodal dose between independent ICBT insertions and volumetric total dose distribution in the bladder and rectum. We have also determined the margins needed for safe delivery of proton therapy when delivering nodal boosts to patients with cervical cancer.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Adaptive clinical trial design for targeted therapy development

The development of targeted therapy involve many challenges. Our study will address some of the key issues involved in biomarker identification and clinical trial design. In our study, we propose two biomarker selection methods, and then apply them in two different clinical trial designs for targeted therapy development. In particular, we propose a Bayesian two-step lasso procedure for biomarker selection in the proportional hazards model in Chapter 2. In the first step of this strategy, we use the Bayesian group lasso to identify the important marker groups, wherein each group contains the main effect of a single marker and its interactions with treatments. In the second step, we zoom in to select each individual marker and the interactions between markers and treatments in order to identify prognostic or predictive markers using the Bayesian adaptive lasso. In Chapter 3, we propose a Bayesian two-stage adaptive design for targeted therapy development while implementing the variable selection method given in Chapter 2. In Chapter 4, we proposed an alternate frequentist adaptive randomization strategy for situations where a large number of biomarkers need to be incorporated in the study design. We also propose a new adaptive randomization rule, which takes into account the variations associated with the point estimates of survival times. In all of our designs, we seek to identify the key markers that are either prognostic or predictive with respect to treatment. We are going to use extensive simulation to evaluate the operating characteristics of our methods.^

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

Study on the effect of metformin in patients of metastatic renal cell cancer with diabetes receiving tyrosine kinase inhibitor therapy

Objective: The primary objective of our study was to study the effect of metformin in patients of metastatic renal cell cancer (mRCC) and diabetes who are on treatment with frontline therapy of tyrosine kinase inhibitors. The effect of therapy was described in terms of overall survival and progression free survival. Comparisons were made between group of patients receiving metformin versus group of patients receiving insulin in diabetic patients of metastatic renal cancer on frontline therapy. Exploratory analyses were also done comparing non-diabetic patients of metastatic renal cell cancer receiving frontline therapy compared to diabetic patients of metastatic renal cell cancer receiving metformin therapy. ^ Methods: The study design is a retrospective case series to elaborate the response rate of frontline therapy in combination with metformin for mRCC patients with type 2 diabetes mellitus. The cohort was selected from a database, which was generated for assessing the effect of tyrosine kinase inhibitor therapy associated hypertension in metastatic renal cell cancer at MD Anderson Cancer Center. Patients who had been started on frontline therapy for metastatic renal cell carcinoma from all ethnic and racial backgrounds were selected for the study. The exclusion criteria would be of patients who took frontline therapy for less than 3 months or were lost to follow-up. Our exposure variable was treatment with metformin, which comprised of patients who took metformin for the treatment of type 2 diabetes at any time of diagnosis of metastatic renal cell carcinoma. The outcomes assessed were last available follow-up or date of death for the overall survival and date of progression of disease from their radiological reports for time to progression. The response rates were compared by covariates that are known to be strongly associated with renal cell cancer. ^ Results: For our primary analyses between the insulin and metformin group, there were 82 patients, out of which 50 took insulin therapy and 32 took metformin therapy for type 2 diabetes. For our exploratory analysis, we compared 32 diabetic patients on metformin to 146 non-diabetic patients, not on metformin. Baseline characteristics were compared among the population. The time from the start of treatment until the date of progression of renal cell cancer and date of death or last follow-up were estimated for survival analysis. ^ In our primary analyses, there was a significant difference in the time to progression of patients receiving metformin therapy vs insulin therapy, which was also seen in our exploratory analyses. The median time to progression in primary analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 540 days (95% CI: 350-894) in patients who were receiving insulin therapy (p=0.024). The median time to progression in exploratory analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 279 days (95% CI: 202-372 days) in non-diabetic group (p-value <0.0001). ^ The median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 816 days (95%CI: 558-1405 days) in insulin group (p-value<0.91). For the exploratory analyses, the median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 766 days (95%CI: 649-965 days) in the non-diabetic group (p-value<0.78). Metformin was observed to increase the progression free survival in both the primary and exploratory analyses (HR=0.52 in metformin Vs insulin group and HR=0.36 in metformin Vs non-diabetic group, respectively). ^ Conclusion: In laboratory studies and a few clinical studies metformin has been proven to have dual benefits in patients suffering from cancer and type 2-diabetes via its action on the mammalian target of Rapamycin pathway and effect in decreasing blood sugar by increasing the sensitivity of the insulin receptors to insulin. Several studies in breast cancer patients have documented a beneficial effect (quantified by pathological remission of cancer) of metformin use in patients taking treatment for breast cancer therapy. Combination of metformin therapy in patients taking frontline therapy for renal cell cancer may provide a significant benefit in prolonging the overall survival in patients with metastatic renal cell cancer and diabetes. ^

Scaling up the production of biogenic magnetite nanoparticles

Although there are numerous examples of large-scale commercial microbial synthesis routes for organic bioproducts, few studies have addressed the obvious potential for microbial systems to produce inorganic functional biomaterials at scale. Here we address this by focusing on the production of nano-scale biomagnetite particles by the Fe(III)-reducing bacterium Geobacter sulfurreducens, which was scaled-up successfully from lab-scale to pilot plant-scale production, whilst maintaining the surface reactivity and magnetic properties which make this material well suited to commercial exploitation. At the largest scale tested, the bacterium was grown in a 50 L bioreactor, harvested and then inoculated into a buffer solution containing Fe(III)-oxyhydroxide and an electron donor and mediator, which promoted the formation of magnetite in under 24 hours. This procedure was capable of producing up to 120 g biomagnetite. The particle size distribution was maintained between 10 and 15 nm during scale-up of this second step from 10 ml to 10 L, with conserved magnetic properties and surface reactivity; the latter demonstrated by the reduction of Cr(VI). The process presented provides an environmentally benign route to magnetite production and serves as an alternative to harsher synthetic techniques, with the clear potential to be used to produce kg to tonne quantities.

Design and evaluation of acceleration strategies for speeding up the development of dialog applications

In this paper, we describe a complete development platform that features different innovative acceleration strategies, not included in any other current platform, that simplify and speed up the definition of the different elements required to design a spoken dialog service. The proposed accelerations are mainly based on using the information from the backend database schema and contents, as well as cumulative information produced throughout the different steps in the design. Thanks to these accelerations, the interaction between the designer and the platform is improved, and in most cases the design is reduced to simple confirmations of the “proposals” that the platform dynamically provides at each step. In addition, the platform provides several other accelerations such as configurable templates that can be used to define the different tasks in the service or the dialogs to obtain or show information to the user, automatic proposals for the best way to request slot contents from the user (i.e. using mixed-initiative forms or directed forms), an assistant that offers the set of more probable actions required to complete the definition of the different tasks in the application, or another assistant for solving specific modality details such as confirmations of user answers or how to present them the lists of retrieved results after querying the backend database. Additionally, the platform also allows the creation of speech grammars and prompts, database access functions, and the possibility of using mixed initiative and over-answering dialogs. In the paper we also describe in detail each assistant in the platform, emphasizing the different kind of methodologies followed to facilitate the design process at each one. Finally, we describe the results obtained in both a subjective and an objective evaluation with different designers that confirm the viability, usefulness, and functionality of the proposed accelerations. Thanks to the accelerations, the design time is reduced in more than 56% and the number of keystrokes by 84%.

Metodologías de modelado, monitorización y asistencia robótica en neurorrehabilitación funcional de extremidad superior

La Organización Mundial de la Salud (OMS) prevé que para el año 2020, el Daño Cerebral Adquirido (DCA) estará entre las 10 causas más comunes de discapacidad. Estas lesiones, dadas sus consecuencias físicas, sensoriales, cognitivas, emocionales y socioeconómicas, cambian dramáticamente la vida de los pacientes y sus familias. Las nuevas técnicas de intervención precoz y el desarrollo de la medicina intensiva en la atención al DCA han mejorado notablemente la probabilidad de supervivencia. Sin embargo, hoy por hoy, las lesiones cerebrales no tienen ningún tratamiento quirúrgico que tenga por objetivo restablecer la funcionalidad perdida, sino que las terapias rehabilitadoras se dirigen hacia la compensación de los déficits producidos. Uno de los objetivos principales de la neurorrehabilitación es, por tanto, dotar al paciente de la capacidad necesaria para ejecutar las Actividades de Vida Diaria (AVDs) necesarias para desarrollar una vida independiente, siendo fundamentales aquellas en las que la Extremidad Superior (ES) está directamente implicada, dada su gran importancia a la hora de la manipulación de objetos. Con la incorporación de nuevas soluciones tecnológicas al proceso de neurorrehabilitación se pretende alcanzar un nuevo paradigma centrado en ofrecer una práctica personalizada, monitorizada y ubicua con una valoración continua de la eficacia y de la eficiencia de los procedimientos y con capacidad de generar conocimientos que impulsen la ruptura del paradigma de actual. Los nuevos objetivos consistirán en minimizar el impacto de las enfermedades que afectan a la capacidad funcional de las personas, disminuir el tiempo de incapacidad y permitir una gestión más eficiente de los recursos. Estos objetivos clínicos, de gran impacto socio-económico, sólo pueden alcanzarse desde una apuesta decidida en nuevas tecnologías, metodologías y algoritmos capaces de ocasionar la ruptura tecnológica necesaria que permita superar las barreras que hasta el momento han impedido la penetración tecnológica en el campo de la rehabilitación de manera universal. De esta forma, los trabajos y resultados alcanzados en la Tesis son los siguientes: 1. Modelado de AVDs: como paso previo a la incorporación de ayudas tecnológicas al proceso rehabilitador, se hace necesaria una primera fase de modelado y formalización del conocimiento asociado a la ejecución de las actividades que se realizan como parte de la terapia. En particular, las tareas más complejas y a su vez con mayor repercusión terapéutica son las AVDs, cuya formalización permitirá disponer de modelos de movimiento sanos que actuarán de referencia para futuros desarrollos tecnológicos dirigidos a personas con DCA. Siguiendo una metodología basada en diagramas de estados UML se han modelado las AVDs 'servir agua de una jarra' y 'coger un botella' 2. Monitorización ubícua del movimiento de la ES: se ha diseñado, desarrollado y validado un sistema de adquisición de movimiento basado en tecnología inercial que mejora las limitaciones de los dispositivos comerciales actuales (coste muy elevado e incapacidad para trabajar en entornos no controlados); los altos coeficientes de correlación y los bajos niveles de error obtenidos en los corregistros llevados a cabo con el sistema comercial BTS SMART-D demuestran la alta precisión del sistema. También se ha realizado un trabajo de investigación exploratorio de un sistema de captura de movimiento de coste muy reducido basado en visión estereoscópica, habiéndose detectado los puntos clave donde se hace necesario incidir desde un punto de vista tecnológico para su incorporación en un entorno real 3. Resolución del Problema Cinemático Inverso (PCI): se ha diseñado, desarrollado y validado una solución al PCI cuando el manipulador se corresponde con una ES humana estudiándose 2 posibles alternativas, una basada en la utilización de un Perceptrón Multicapa (PMC) y otra basada en sistemas Artificial Neuro-Fuzzy Inference Systems (ANFIS). La validación, llevada a cabo utilizando información relativa a los modelos disponibles de AVDs, indica que una solución basada en un PMC con 3 neuronas en la capa de entrada, una capa oculta también de 3 neuronas y una capa de salida con tantas neuronas como Grados de Libertad (GdLs) tenga el modelo de la ES, proporciona resultados, tanto de precisión como de tiempo de cálculo, que la hacen idónea para trabajar en sistemas con requisitos de tiempo real 4. Control inteligente assisted-as-needed: se ha diseñado, desarrollado y validado un algoritmo de control assisted-as-needed para una ortesis robótica con capacidades de actuación anticipatoria de la que existe un prototipo implementado en la actualidad. Los resultados obtenidos demuestran cómo el sistema es capaz de adaptarse al perfil disfuncional del paciente activando la ayuda en instantes anteriores a la ocurrencia de movimientos incorrectos. Esta estrategia implica un aumento en la participación del paciente y, por tanto, en su actividad muscular, fomentándose los procesos la plasticidad cerebral responsables del reaprendizaje o readaptación motora 5. Simuladores robóticos para planificación: se propone la utilización de un simulador robótico assisted-as-needed como herramienta de planificación de sesiones de rehabilitación personalizadas y con un objetivo clínico marcado en las que interviene una ortesis robotizada. Los resultados obtenidos evidencian como, tras la ejecución de ciertos algoritmos sencillos, es posible seleccionar automáticamente una configuración para el algoritmo de control assisted-as-needed que consigue que la ortesis se adapte a los criterios establecidos desde un punto de vista clínico en función del paciente estudiado. Estos resultados invitan a profundizar en el desarrollo de algoritmos más avanzados de selección de parámetros a partir de baterías de simulaciones Estos trabajos han servido para corroborar las hipótesis de investigación planteadas al inicio de la misma, permitiendo, asimismo, la apertura de nuevas líneas de investigación. Summary The World Health Organization (WHO) predicts that by the year 2020, Acquired Brain Injury (ABI) will be among the ten most common ailments. These injuries dramatically change the life of the patients and their families due to their physical, sensory, cognitive, emotional and socio-economic consequences. New techniques of early intervention and the development of intensive ABI care have noticeably improved the survival rate. However, in spite of these advances, brain injuries still have no surgical or pharmacological treatment to re-establish the lost functions. Neurorehabilitation therapies address this problem by restoring, minimizing or compensating the functional alterations in a person disabled because of a nervous system injury. One of the main objectives of Neurorehabilitation is to provide patients with the capacity to perform specific Activities of the Daily Life (ADL) required for an independent life, especially those in which the Upper Limb (UL) is directly involved due to its great importance in manipulating objects within the patients' environment. The incorporation of new technological aids to the neurorehabilitation process tries to reach a new paradigm focused on offering a personalized, monitored and ubiquitous practise with continuous assessment of both the efficacy and the efficiency of the procedures and with the capacity of generating new knowledge. New targets will be to minimize the impact of the sicknesses affecting the functional capabilitiies of the subjects, to decrease the time of the physical handicap and to allow a more efficient resources handling. These targets, of a great socio-economic impact, can only be achieved by means of new technologies and algorithms able to provoke the technological break needed to beat the barriers that are stopping the universal penetration of the technology in the field of rehabilitation. In this way, this PhD Thesis has achieved the following results: 1. ADL Modeling: as a previous step to the incorporation of technological aids to the neurorehabilitation process, it is necessary a first modelling and formalization phase of the knowledge associated to the execution of the activities that are performed as a part of the therapy. In particular, the most complex and therapeutically relevant tasks are the ADLs, whose formalization will produce healthy motion models to be used as a reference for future technological developments. Following a methodology based on UML state-chart diagrams, the ADLs 'serving water from a jar' and 'picking up a bottle' have been modelled 2. Ubiquitous monitoring of the UL movement: it has been designed, developed and validated a motion acquisition system based on inertial technology that improves the limitations of the current devices (high monetary cost and inability of working within uncontrolled environments); the high correlation coefficients and the low error levels obtained throughout several co-registration sessions with the commercial sys- tem BTS SMART-D show the high precision of the system. Besides an exploration of a very low cost stereoscopic vision-based motion capture system has been carried out and the key points where it is necessary to insist from a technological point of view have been detected 3. Inverse Kinematics (IK) problem solving: a solution to the IK problem has been proposed for a manipulator that corresponds to a human UL. This solution has been faced by means of two different alternatives, one based on a Mulilayer Perceptron (MLP) and another based on Artificial Neuro-Fuzzy Inference Systems (ANFIS). The validation of these solutions, carried out using the information regarding the previously generated motion models, indicate that a MLP-based solution, with an architecture consisting in 3 neurons in the input layer, one hidden layer of 3 neurons and an output layer with as many neurons as the number of Degrees of Freedom (DoFs) that the UL model has, is the one that provides the best results both in terms of precission and in terms of processing time, making in idoneous to be integrated within a system with real time restrictions 4. Assisted-as-needed intelligent control: an assisted-as-needed control algorithm with anticipatory actuation capabilities has been designed, developed and validated for a robotic orthosis of which there is an already implemented prototype. Obtained results demonstrate that the control system is able to adapt to the dysfunctional profile of the patient by triggering the assistance right before an incorrect movement is going to take place. This strategy implies an increase in the participation of the patients and in his or her muscle activity, encouraging the neural plasticity processes in charge of the motor learning 5. Planification with a robotic simulator: in this work a robotic simulator is proposed as a planification tool for personalized rehabilitation sessions under a certain clinical criterium. Obtained results indicate that, after the execution of simple parameter selection algorithms, it is possible to automatically choose a specific configuration that makes the assisted-as-needed control algorithm to adapt both to the clinical criteria and to the patient. These results invite researchers to work in the development of more complex parameter selection algorithms departing from simulation batteries Obtained results have been useful to corroborate the hypotheses set out at the beginning of this PhD Thesis. Besides, they have allowed the creation of new research lines in all the studied application fields.

Image analysis by moment invariants using a set of step-like basis functions

Moment invariants have been thoroughly studied and repeatedly proposed as one of the most powerful tools for 2D shape identification. In this paper a set of such descriptors is proposed, being the basis functions discontinuous in a finite number of points. The goal of using discontinuous functions is to avoid the Gibbs phenomenon, and therefore to yield a better approximation capability for discontinuous signals, as images. Moreover, the proposed set of moments allows the definition of rotation invariants, being this the other main design concern. Translation and scale invariance are achieved by means of standard image normalization. Tests are conducted to evaluate the behavior of these descriptors in noisy environments, where images are corrupted with Gaussian noise up to different SNR values. Results are compared to those obtained using Zernike moments, showing that the proposed descriptor has the same performance in image retrieval tasks in noisy environments, but demanding much less computational power for every stage in the query chain.

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15–20 μg/ml of canine factor IX was detected in the plasma of mice injected with 5.6 × 1011 particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.

A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of β-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-Å resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type d-alanyl-d-alanine carboxypeptidase/transpeptidase (EC 3.4.16.4) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Fusigenic viral liposome for gene therapy in cardiovascular diseases.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy, we have developed a fusigenic liposome vector based on principles of viral cell fusion. The fusion proteins of hemagglutinating virus of Japan (HVJ; also Sendai virus) are complexed with liposomes that encapsulate oligodeoxynucleotide or plasmid DNA. Subsequent fusion of HVJ-liposomes with plasma membranes introduces the DNA directly into the cytoplasm. In addition, a DNA-binding nuclear protein is incorporated into the HVJ-liposome particle to enhance plasmid transgene expression. The fusigenic viral liposome vector has proven to be efficient for the intracellular introduction of oligodeoxynucleotide, as well as intact genes up to 100 kbp, both in vitro and in vivo. Many animal tissues have been found to be suitable targets for fusigenic viral liposome DNA transfer. In the cardiovascular system, we have documented successful cytostatic gene therapy in models of vascular proliferative disease using antisense oligodeoxynucleotides against cell cycle genes, double-stranded oligodeoxynucleotides as "decoys" to trap the transcription factor E2F, and expression of a transgene encoding the constitutive endothelial cell form of nitric oxide synthase. Similar strategies are also effective for the genetic engineering of vein grafts and for the treatment of a mouse model of immune-mediated glomerular disease.