Proyectos

Identificación de bacterias de ralves de la región de O'Higgins para el estudio de posibles funciones biotecnológicas.

Identificación de bacterias de ralves de la región de O'Higgins para el estudio de posibles funciones biotecnológicas.

La Piscirickettsiosis es una grave enfermedad bacteriana que resulta en altos niveles de mortalidad en diferentes especies de salmónidos. Las pérdidas totales, incluidos los costos de vacunación y los tratamientos antimicrobianos se han estimado en alrededor de $ 450 millones de dólares por año en Chile. Desafortunadamente, las vacunas contra este patógeno han mostrado ser poco efectivas para prevenir esta enfermedad, lo que se ha asociado a fenómenos de coinfección de Caligus, variabilidad del patógeno y del hospedero. Recientes estudios muestran, además, que los mecanismos de respuesta inmune innata son los responsables de otorgar protección a los peces frente a un desafío con esta bacteria. De esta manera, la falta de tratamientos eficaces contra Piscirickettsia salmonis fundamentan la necesidad de buscar nuevas alternativas de tratamientos profilácticos, las que deberían estar orientados a reforzar el sistema inmune innato del hospedador. En base a estas necesidades nosotros proponemos evaluar proteínas nanoestructuradas, también conocidas como cuerpos de inclusión como inmunoestimulantes para el control de P. salmonis en salmón del Atlántico (Salmo salar). Estas proteínas nanoestructuradas pueden expresar cualquier proteína con una función de interés, la cual es liberada gradualmente en el tiempo. Además, son muy estables y resisten duras condiciones fisicoquímicas manteniendo su funcionalidad por lo que no es necesario su encapsulación. Debido a que la producción de estas proteínas es altamente escalable en biorreactores bacterianos surgen entonces como una herramienta de biotecnología viable de llegar a ser una solución comercial. Recientemente he evaluado con éxito el uso de proteínas nanoestructuradas de citoquinas como inmunoestimulantes en trucha arcoíris (Onchorhynchuss mykiss) y el pez cebra (Danio rerio). La prueba de concepto de esta tecnología mostró que los cuerpos de inclusión pueden estimular una respuesta inmune en macrófagos de trucha arcoíris. Además, las proteínas nanoestructuradas administrados por vía intraperitoneal protegen al pez cebra frente a una infección bacteriana letal y al ser administrados oralmente son absorbidos por células presentes en la mucosa intestinal. En base a esto, proponemos como proyecto desarrollar proteínas nanoestructuradas como inmunoestimulantes contra Piscirickettsia, para ello contaremos con la asesoría técnica de tres laboratorios de la PUCV. Como primer objetivo realizaremos la clonación de los genes que expresan las proteínas de interés y la producción de los plásmidos para ser expresados en bacterias. Este objetivo se llevará a cabo en el Laboratorio de Genética y Genómica Aplicada. En el segundo objetivo, produciremos las proteínas recombinantes con la asesoría técnica y en las instalaciones del Laboratorio de Cultivo de Células Microbianas. Este grupo es experto en la producción de proteínas recombinantes en biorreactores de bacterias. Como tercer objetivo cuantificaremos y caracterizaremos las proteínas producidas con la asesoría y en el laboratorio del Grupo de Marcadores Inmunológicos de Organismos Acuáticos. Este laboratorio es especialista en la purificación, cuantificación y caracterización de proteínas. Además, contaremos con la asesoría del Laboratorio de Genética y Genómica Aplicada en cuanto a cómo dar continuidad al presente proyecto e introducir el producto en la industria. Nosotros esperamos como resultado de este proyecto desarrollar proteínas nanoestructuradas que sean administradas a peces como inmunoestimulantes contra Piscirickettsia. El desarrollo de un método profiláctico efectivo contra P. salmonis permitirá disminuir las pérdidas económicas asociadas a la Piscirickettsiosis y disminuir el uso excesivo de antibióticos en la industria de cultivo de salmones en Chile.

La Fundación financió las excavaciones arqueológicas y paleontológicas del yacimiento Tagua Tagua 3

La Fundación financió las excavaciones arqueológicas y paleontológicas del yacimiento Tagua Tagua 3

Stochasticity in optimization and game theory is a very important aspect to model more accurately real-world problems in many different areas (see for instance [6]). In optimization problems as well as in one-level games, namely, Nash equilibrium problems, stochasticity aspects have received quite a lot of attention for a while and have also been well studied [22]. However, for the branch of bilevel games quite few studies have included in their analysis stochastic aspects in their models. A bilevel game is basically to split a finite set of players into two levels: the leaders or upper-level players, and the followers or lower-level players. In the model, the followers react in a passive way to the leaders' actions, while the leaders compete in the upper level trying to actively anticipate the followers' reaction. Moreover, in each level, the interaction is non-cooperative as in Nash equilibrium problems. Bilevel games have been recognized as one of the most complex and at the same time very useful models in the literature [17]. Bilevel games, and more precisely the problem of the leaders in a bilevel game, face an ambiguity/uncertainty whenever the followers' reaction is not necessarily uniquely determined for each leaders' decision. To deal with this ambiguity two main approaches are well-known the optimistic and the pessimistic. The weakness of these two approaches is that both are quite extreme and the optimistic one lacks of real modeling foundations, putting the leaders in a quite naive position. Recently, in [9] a general stochastic approach has been proposed to solve this ambiguity, which is seen as an uncertainty of the problem, providing also a specific approach that seems to be more reasonable than the optimistic one, from a modeling point of view. The stochasticity in the stochastic approach is an endogenous one since it corresponds to a decision-dependent uncertainty [1, 23]. But, of course, stochasticity might also come from an exogenous side, that is, when some of the parameters defining the game, such as future demand and prices, 1 forecasts of winds and clouds, are uncertain and possibly follow some probability distribution. This has been considered in [34, 12, 13, 16]. In the second part of the project, which is the applied part, we are interested in using the developed theoretical framework of bilevel games with stochastic aspects to a problem of contaminated water resource management, which has high levels of stochasticity. The scarcity of water resource and its efficient use has been recognized as an extremely important problem in Chile and the whole world, for agricultural, industrial, and human use. Moreover, after any use, there is an outflow of water which has generally more contaminants than the inflow. Depending on the type and quantity of contaminants the outflow of water could be reused, but sometimes giving less profit to the entity. The general situation is full of uncertainties, since the entities do not share their information. Moreover, the main source of information for us will be measurements on the quality of water at different strategic points and punctual events of contamination registered by inspection, which is simply a qualitative data. Therefore, we propose first to apply predictive models and machine learning to do inverse engineering in order to understand the game played by the different actors. Then we want to study the underlying one-level game and study the design of mechanisms (bilevel game) so that we can move the equilibrium to a desired goal. In a somehow similar spirit, in [30, 31, 10] game theory techniques have been used to analyze the behavior of companies sharing contaminated water in the context of eco-industrial parks, while in [36] also a bilevel model is used for a water resource optimal allocation problem.

La Piscirickettsiosis es una grave enfermedad bacteriana que resulta en altos niveles de mortalidad en diferentes especies de salmónidos. Las pérdidas totales, incluidos los costos de vacunación y los tratamientos antimicrobianos se han estimado en alrededor de $ 450 millones de dólares por año. Desafortunadamente, las vacunas contra este patógeno han mostrado ser poco efectivas para prevenir esta enfermedad, lo que se ha asociado según estudios de nuestro laboratorio a fenómenos de coinfección de Caligus, variabilidad del patógeno y del hospedero. Recientes estudios muestran, además, que los mecanismos de respuesta inmune innata son los responsables de otorgar protección a los peces frente a un desafío con esta bacteria. De esta manera, la falta de tratamientos eficaces contra Piscirickettsia salmonis fundamentan la necesidad de buscar nuevas alternativas de tratamientos profilácticos, las que deberían estar orientados a reforzar el sistema inmune innato del hospedador. En base a estas necesidades nosotros proponemos evaluar cuerpos de inclusión como inmunoestimulantes de administración oral para el control de P. salmonis en salmón del Atlántico (Salmo salar). Los cuerpos de inclusión son agregados de proteínas recombinantes nanoestructuradas funcionales expresados usualmente en el citoplasma de bacterias recombinantes. Los cuerpos de inclusión pueden expresar cualquier proteína con una función de interés, la cual es liberada gradualmente en el tiempo. Además, son muy estables y resisten duras condiciones fisicoquímicas manteniendo su funcionalidad por lo que no es necesario su encapsulación. Debido a que la producción de los cuerpos de inclusión es altamente escalable en biorreactores bacterianos surgen entonces como una herramienta de biotecnología susceptible de llegar a ser una solución comercial. Recientemente he evaluado con éxito el uso de cuerpos de inclusión de citoquinas como inmunoestimulantes en trucha arcoíris (Onchorhynchuss mykiss) y el pez cebra (Danio rerio). La prueba de concepto de esta tecnología mostró que los cuerpos de inclusión pueden estimular una respuesta inmune en macrófagos de trucha arcoíris. Además, los cuerpos de inclusión administrados por vía intraperitoneal protegen al pez cebra frente a una infección bacteriana letal y al ser administrados oralmente son absorbidos por células presentes en la mucosa intestinal. Postulamos como hipótesis que los cuerpos de inclusión administrados por vía oral otorgan protección al salmón del atlántico contra P. salmonis. Como primer objetivo de este estudio proponemos realizar la producción y caracterización de los cuerpos de inclusión de dos proteínas relacionadas al sistema inmune innato de salmón del Atlántico. En el segundo objetivo, evaluaremos la capacidad de inmunomodulación del sistema inmune por parte de los cuerpos de inclusión al ser administrados en diferentes dosis a través de la intubación de los peces. Para ello usaremos RT-qPCR donde analizaremos la capacidad moduladora de los cuerpos de inclusión a través de la expresión génica de citoquinas proinflamatorias (TNFα, IL-1β, IFNγ), antiinflamatorias (IL-10), enzima proinflamatoria (COX-2), y receptor tipo Toll (TLR9). También analizaremos TNFα e IL-10 a través de ELISA. Igualmente, se analizará si los cuerpos de inclusión marcados con un fluoróforo son endocitados por células intestinales y si son trasladados al bazo a través de citometría de flujo. En el tercer objetivo, analizaremos la eficacia de los cuerpos de inclusión para el control de P. salmonis en salmón del Atlántico administrados a través del alimento en un desafío experimental. Se evaluará a través de técnicas moleculares (RT-qPCR y ELISA) citoquinas proinflamatorias, antiinflamatorias, enzima proinflamatoria, y receptor tipo Toll para analizar la capacidad inmunomoduladora de los cuerpos de inclusión durante un desafío bacteriano. Asimismo, evaluaremos sobrevivencia, carga de P. salmonis y necropsia de los tejidos. Nosotros esperamos como resultado de esta investigación que los cuerpos de inclusión aumenten la sobrevivencia de salmón del Atlántico frente a un desafío de P. salmonis. El desarrollo de un método profiláctico efectivo contra P. salmonis permitirá disminuir las pérdidas económicas asociadas a la Piscirickettsiosis y disminuir el uso excesivo de antibióticos en la industria de cultivo de salmones en Chile.

Introduction: Ultrasound (US) exams are extensively used in Chile and around the world. This non-invasive imaging technique has many advantages when compared to magnetic resonance, computed tomography, and others because it has a low cost, it does not need ionizing radiation, and it is portable equipment. However, this technique has many challenges; the most known is the balance between resolution and penetration depth. Recently, in 2011, a new technique of US has been described: the ultrasound localization microscopy (ULM). However, it was only in 2015 that this technique gained knowledge with the publication of Errico et al. (2015) who described the ultrafast ultrasound localization microscopy applied in vivo in rats’ brain. ULM eliminates the challenge of the balance between resolution and penetration; but a new challenge emerges: the balance between localization precision of microbubble, microbubble concentration, and acquisition time. The microbubbles (MB) are the contrast agent for the US technique. They have 1-5 µm in diameter and act as a blinking source. These MB are injected into the bloodstream and flow into the circulatory system. ULM is also known as super-resolution imaging; it can produce vascular images with a resolution around 10 µm, 10 times better than the conventional US image. This unprecedented resolution has numerous potential applications. In particular, ULM would have a high impact in oncology because the vascular structure of early tumors, that are in the range of 5 µm to 80 µm, provides information that can help in the early diagnosis and monitor therapy responses. The huge potentiality of ULM has produced a lot of excitement and expectation worldwide, and it became a hot topic in the medical ultrasound community. Unfortunately, this technique is not yet clinically approved because it is still in development stages and presents many challenges that must be solved before translating it into clinics. The mainly limitations to overcome before translating ULM into clinics are the following: contrast-to-tissue ratio (CTR), signal-to-noise ratio (SNR), acquisition time, microbubble concentration, motion, lack of a gold standard, data overdose, exploitation of ultrafast scanner uncommon in the clinic and so on. Therefore, the aim of this study is to optimize the technique to localize microbubbles, and to explore the physics of microbubble to provide a change in the paradigm of the processes to produce ULM by combining the superresolution processing with a controlled exterior force impulse. To achieve this aim, first a numerical study will be performed to simulate microbubbles into small vessels and find a better way to localize them. The robustness of the algorithm will be increased to consider the non-linear interactions between MB and US and to consider the parabolic velocity profile of the vessel/tube. Up to date, the studies on microbubbles localization in ULM are after the image acquisition. There are no tissue/flow simulations of the behavior of microbubbles into the vessels with this technique. To perform the simulation, we will use a computer with excellent storage capacity and great velocity of processing together with the MATLAB algorithm: The Full Wave Solver. Second, an experimental study will be performed to generate super resolution images in a conventional phantom. The state of art will be applied with a phantom made by microtubes and microbubbles and then, the improvement of the aim 1 will be considered into this experiment. The complexity of the phantom will be increased from a medium with only water, then gelatine and finally, we will add some respiratory simulated movement. To the experimental setup we will use the Verasonics Vantage 128 research ultrasound scanner with different types of ultrasound transducers, microbubbles, microtubes, gelatine and the respiratory movement will be simulated with a vibration testing system (Shaker VTS-100). Finally, the physics of microbubble will be explored to provide a change in the paradigm of the processes to produce ULM and to detect the MB in a more direct way, without the need to perform a filter, like the signal value decomposition (SVD). We want to apply the an external know push pulse that will produce differences in the shear waves between the microbubbles and the tissue around and with simulations we will be able to know the response of microbubbles and it may help us to separate them from the tissue. Expected results: As result of this study, we expect to develop a numerical simulation to the ULM method, by considering interactions of the US with the tissue and fluid dynamics of the blood into the vessel and significantly optimize the techniques of MB detection. Besides that, this project will help to improve the first fully programmable ultrasound scanner system in Chile. Potentially, this would open new research areas at the country level, such as ultrasound imaging, ultrasound super-resolution imaging and soft tissue characterization.

The importance of water for life is indisputable. Nevertheless, water quality and availability are affected by increases in consumption and climate change. Indeed, several areas of Chile are suffering acute water scarcity. Consequently, there is a critical need to develop efficient technologies for wastewater recovery. However, considerations must be given to the fact that some wastewaters have toxic recalcitrant pollutants requiring complex treatments, such as landfill leachate (LL). The general goal of the project is to evaluate the viability of experimentally improving LL quality by conjointly using the solar photo-Fenton process and ultrasound (US), thereby enhancing photocatalysis, ultimately reducing wastewater toxicity. The specific goals are to (i) measure the H2O2 and UV irradiation produced by US in LL (laboratory scale); (ii) evaluate the hydroxyl radicals generated during treatment processes (laboratory scale); (iii) maximize organic-pollutant removal in LL by defining the optimal operating conditions for the photo-Fenton/US process (laboratory scale); (iv) maximize organic-pollutant removal in LL by defining optimal US power/frequency in the sonolytic process (pilot-plant scale); and (v) evaluate toxicity elimination and energy consumption in LL treatments with solar-photocatalytic and US processes (laboratory and pilot-plant scales). The proposed investigation will use a scientific methodology, developing reproducible methods to observe the effects of diverse parameters, all with a focus on maximizing contaminants removal. To characterize LL, several parameters will be evaluated, including chemical oxygen demand, biological oxygen demand, total organic carbon, total dissolved nitrogen, pH, metals, ammonia, colour, biodegradability, toxicity, total suspended solids, conductivity and humic acid. To determine the amount of H2O2 generated by US in a simulated LL, a set of experiments will be run to produce the sonochemical process, applying different US frequencies (100 kHz, 200 kHz and 300 kHz) and powers (100 W, 170 W and 250 W), thus obtaining the kinetic reaction to H2O2 production. The amount of UV irradiation formed due to sonoluminescence will be quantified in the same beaker in the simulated LL. Sonoluminescence intensity during the runs will be measured using a spectro-radiometer. To evaluate the hydroxyl radicals (·OH) generated in the simulated LL during treatment processes, a method based on the oxidation of 2-proponol will be used. To determine the optimal operating conditions for the photo-Fenton/US process to maximize the removal of organic pollutants present in the simulated LL, a set of experiments will be carried out in the same photoreactor (1 L), applying different reagent concentrations, treatment times, and pH levels. To establish optimal US power and frequency in the sonolytic process to maximize the removal of organic pollutants present in a real LL (after its pretreatment), a set of runs will be carried out at pilot-plant scale in a solar photoreactor compound parabolic concentrator (CPC; 12 L useful volume). To evaluate toxicity elimination from the real LL an Aliivibrio fischeri test, respirometer assay, and phytotoxicity assay will be used, followed by determining median effective concentrations (EC50) according to the Probit model. Since a main disadvantage of the proposed treatments is high-energy consumption, specific energy consumption (SEC) and electrical energy per order (EEO) will be determined for all processes. All experiments will be done in triplicate, and filtration and coagulation/flocculation processes as a pretreatment will be used prior to all runs. The expected results of the proposed project are to (i) obtain new knowledge related to joint photo- Fenton and ultrasound wastewater treatments, (ii) demonstrate treatment synergies, and (iii) validate the use of advanced oxidation processes for improving LL. Project results will be reported in papers, through thesis work, and at scientific congresses, strengthening national and international research networks.