A lunar base reference mission for the phased implementation of bioregenerative life support system components

Previous design efforts of a cost effective and reliable regenerative life support system (RLSS) provided the foundation for the characterization of organisms or 'biological processors' in engineering terms and a methodology was developed for their integration into an engineered ecological LSS in order to minimize the mass flow imbalances between consumers and producers. These techniques for the design and the evaluation of bioregenerative LSS have now been integrated into a lunar base reference mission, emphasizing the phased implementation of components of such a BLSS. In parallel, a designers handbook was compiled from knowledge and experience gained during past design projects to aid in the design and planning of future space missions requiring advanced RLSS technologies. The lunar base reference mission addresses in particular the phased implementation and integration of BLS parts and includes the resulting infrastructure burdens and needs such as mass, power, volume, and structural requirements of the LSS. Also, operational aspects such as manpower requirements and the possible need and application of 'robotics' were addressed

Aplicaciones de los autómatas transductores finitos con pesos (WFST) en la corrección simbólica en interfaces persona-máquina

[EN] In this thesis a real application related to the string correction process from an OCR classifier in a form digitizing task is presented. These strings come from a classifier with a given error ratio that implies that some characters in the string have been potentially misclassified, producing erroneous words. This raises the need to introduce some kind of postprocess to improve the strings. The implementation of such postprocess takes into account all the available evidence in a given moment. In the case proposed here, these evidences are the charactersrecognized by the classifier with their posterior probabilities, the confusion matrix between symbols and the language model finally accepted. Each evidence is modelled independently by means of a WFST and then combined by means of the composition operation into a single integrated automata. From this automata, the path that maximizes the probability is selected. This path is the string, that belongs to the language model, that is the nearest string to the OCR hypothesis according to the confusion matrix. The final system offers two different results: on the one hand the corrected string, on the other hand the transformation cost produced during the string correction. Additionally, it is proposed a general method of error estimation using the input string transformation cost that establishes a threshold in terms of the cost and the proposed end-user parameter: the acceptable final error. This thesis presents a method for estimating adaptive rejection threshold estimation that allows for a certain percentage of error in a lot of strings from one language (sample) that presents several advantages. On the one hand, it is independent from transformation cost postprocessing distribution of such samples. On the other hand, it allows the user to set the threshold for a familiar and advantageous manner, as is setting the desired rate of sampling error. For this, first, and for a given language, a model that estimates the probability of error associated with the acceptation of postprocessed strings with a given transformation cost is defined. Then, the procedure that performs the rejection threshold estimation adaptively in order to achieve predefined rate error for a test batch is presented. In addition, an approach to obtain the above model is proposed when there are no real and supervised OCR hypothesis in the learning stage. The chapter is accompanied by experiments whose results demonstrate the utility of the proposed method. Next, linking in somehow with the search for an increased productivity in a possible string validation task, of previously strings rejected by the system through the foregoing error estimation method, a method of multimodal and interactive human-computer interaction that composes the above information with the prefix introduced by the user, while the validation process occurs, making use, for this, of WFST and the automata composition operation. The search for the most likely string for each new interaction offered by the user, in the composed automata, presented here, shows a clear increase in productivity by requiring fewer keystrokes in obtaining the correct string. Finally, a tolerant fault multimodal and interactive interface, using also WFST, is shown by making the composition of different information sources together with an error model related with the possible confusion caused due to the arrangement of keys on a keyboard. The application shown in this case is related to the introduction of a destination into a GPS device where is considered both the information related to the next destinations to a specific place, such as the information related to the entered prefix and errors that may occur due to the arrangement of keys on the input device considered.[ES] En esta tesis se presenta inicialmente una aplicación real de corrección de cadenas procedentes de un clasificador OCR en una tarea de digitalización de formularios. Estas cadenas, proceden de un clasificador con cierta probabilidad de error, lo que implica la posibilidad de que alguno de los caracteres pertenecientes a una palabra sea erróneo, produciendo finalmente palabras incorrectas. Esto plantea la necesidad de introducir algún tipo de postproceso que mejore dichas cadenas. Para implementar dicho postproceso, se tienen en cuenta todas las evidencias disponibles en un momento dado. En el caso propuesto aquí serán los caracteres reconocidos por el propio clasificador con su probabilidad a posteriori, la matriz de confusión entre símbolos y el modelo de lenguaje finalmente aceptado. Cada una de estas evidencias se modela de manera independiente en forma de un WFST. Una vez modeladas se fusionan mediante la operación de composición de autómatas en un único autómata integrado. A partir de este autómata, se selecciona el camino que maximiza la probabilidad y que corresponde con la cadena perteneciente al lenguaje más cercana a la hipótesis OCR según la matriz de confusión entre símbolos. El sistema final ofrecerá dos resultados diferentes: por una parte la cadena corregida y por otra el coste de transformación de dicha corrección. Por otra parte, se plantea un método general de estimación del error frente a un coste de transformación de las cadenas de entrada que permite establecer un umbral dinámico en función de dicho coste y un parámetro propuesto por el usuario final: el error final asumible. Para ello en esta tesis se presenta un método adaptativo de estimación del umbral de rechazo que permite estimarlo para obtener un determinado porcentaje de error en un lote de cadenas de un lenguaje (muestra) que presenta diversas ventajas. Por un lado, es independiente de la distribución de los costes de transformación de dichas muestras. Por otro lado, permite al usuario establecer el umbral de una manera familiar y ventajosa, como es fijando la tasa de error deseada de la muestra. Para todo ello, en primer lugar, y para un lenguaje dado, se define un modelo que estima la probabilidad de error asociada a aceptar cadenas con un coste de transformación determinado. A continuación, se expone el procedimiento que lleva a cabo la estimación del umbral de rechazo de manera adaptativa con el objetivo de alcanzar la tasa de error predefinida para un lote de test. Además, se propone una aproximación para la obtención del modelo anterior cuando no se dispone de hipótesis OCR reales y supervisadas en la etapa de aprendizaje. Seguidamente y enlazando en cierta forma con la búsqueda de un incremento de productividad en una posible validación de las cadenas, previamente rechazadas por el sistema a través del método de estimación del error anteriormente expuesto, se presenta un método de interacción persona-máquina multimodal e interactivo que fusiona la información anterior junto al prefijo introducido, por el propio usuario, durante dicho proceso de validación, haciendo uso para ello de los WFST y la operación de composición de autómatas. Para finalizar, se muestra otra interfaz multimodal e interactiva tolerante a fallos, mediante la fusión de diferentes fuentes de información junto a un modelo de error relacionado con las posibles confusiones producidas debido a la disposición de las teclas de un teclado. Para ello, se hace uso también de WFST para su modelado. La aplicación mostrada en este caso está relacionada con la introducción de un destino en un dispositivo GPS y en ella se considera, tanto la información de los destinos próximos a un lugar concreto, como la información relativa al prefijo introducido y los errores que pueden aparecer debido a la propia disposición de las teclas en el dispositivo de entrada.[CA] En aquesta tesi es presenta inicialment una aplicació real de correcció de cadenes procedents d'un classificador OCR en una tasca de digitalització de formularis. Aquestes cadenes, procedeixen d'un classificador amb una determinada probabilitat d'error, la qual cosa implica la possibilitat de que algun dels caràcters que pertanyen a una paraula siga erroni, produint finalment paraules incorrectes. Això planteja la necessitat d'introduir algun tipus de postprocés que millore aquestes cadenes. Per implementar aquest postprocés, es tenen en compte totes les evidències disponibles en un moment donat. En el cas proposat ací, seran els caràcters reconeguts pel propi classificador amb la seua probabilitat a posteriori, la matriu de confusió entre símbols i el model de llenguatge finalment acceptat. Cadascuna d'aquestes evidències es modela de manera independent en forma d'un WFST. Una vegada modelades es fusionen mitjançant l'operació de composició d'autòmats en un únic autòmat integrat. A partir d'aquest autòmat, es selecciona el camí que fa màxima la probabilitat i que es correspon amb la cadena més propera a la hipòtesi OCR que pertany al llenguatge segons la matriu de confusió entre símbols. El sistema final oferirà dos resultats diferents: d'una banda la cadena corregida, i d'una altra, el cost de transformació d'aquesta correcció. D'una altra banda, es planteja un mètode general d'estimació de l'error front al cost de transformació de les cadenes d'entrada que permet establir un llindar dinàmic en funció d'aquest cost i un paràmetre proposat per l'usuari final: l'error final assumible. Per això en aquesta tesi es presenta un mètode adaptatiu d'estimació de rebuig, amb la finalitat d'obtindre un determinat percentatge d'error en un lot de cadenes d'un llenguatge (mostra) que presenta diversos avantatges. D'una banda és independent de la distribució dels costos de transformació de les mostres esmentades. D'altra banda, permet l'usuari establir el llindar d'una manera familiar i avantatjosa, com és fixant la tasa d'error desitjada per la mostra. Per tot això, en primer lloc, i donat un llenguatge, es defineix un model que estima la probabilitat d'error associada a acceptar cadenes amb un cost de transformació determinat. A continuació, s'exposa el procediment que du a terme l'estimació del llindar de rebuig de manera adaptativa amb l'objectiu de arribar a la tasa d'error predefinida per a un lot de test. A més a més, es proposa una aproximació per a obtindre el model anterior quant no es disposa d'hipòtesi OCR reals i supervisades a l'etapa d'aprenentatge. Seguidament, i enllaçant amb la recerca d'un increment en la productivitat en una possible validació de cadenes prèviament rebutjades pel sistema a través del mètode d'estimació de l'error anteriorment exposat, es presenta un mètode d'interacció persona-màquina multimodal i interactiu que fusiona la informació anterior, juntament amb el prefix introduït pel propi usuari durant l'esmentat procés de validació, fent ús dels WFST i l'operació de composició d'autòmats. La recerca de la cadena més probable, en cada nova interacció oferida per l'usuari ens mostra un clar increment de la productivitat, al requerir un nombre menor de pulsacions de teclat per obtindre la cadena correcta. Per finalitzar, es mostra una altra interfície multimodal i interactiva tolerant a errades, mitjançant la fusió de diferents fonts d'informació juntament a un model d'error relacionat amb les possibles confusions produïdes a causa de la disposició de les lletres d'un teclat. En aquest cas es fa ús també dels WFST en el seu modelat. L'aplicació mostrada en aquest cas està relacionada amb la introducció d'una destinació en un dispositiu GPS i en aquesta es considera tant la informació pròxima a un lloc concret, com la informació relativa al prefix introduït, junt als errors que poden aparèixer a causa de la pròpia disposNavarro Cerdán, JR. (2016). Aplicaciones de los autómatas transductores finitos con pesos (WFST) en la corrección simbólica en interfaces persona-máquina [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62688TESI

NBS monograph

From Introduction: "This report is the first of a series intended to provide a selective overview of research and development efforts and requirements in the somewhat overlapping fields of the computer and information sciences and technologies. The projected series of reports will attempt to outline the probable range of R & D activities in the computer and information sciences and technologies through selective reviews of the literature and to develop a reasonable consensus with respect to the opinions of workers in these and potentially related fields as to areas of continuing R & D concern for research program planning or review in these areas.

Focusing on metabolomic dysregulation and modulation of retinal metabolism to develop novel therapeutic strategies for diabetic retinopathy

Tese de doutoramento, Medicina (Oftalmologia), Universidade de Lisboa, Faculdade de Medicina, 2015Diabetic retinopathy (DR) is a neurovascular complication of diabetes mellitus and a leading cause of blindness in adults below the age of 65, in industrialized nations. Currently there are 126.6 million people with DR worldwide (34.6% of the total diabetic population) and it is estimated that this number will increase to 191.0 million by 2030. Generally, DR is divided into two stages: non-proliferative diabetic retinopathy (NPDR), an earlier phase characterized by appearance of microaneurysms, dot and blot hemorrhages, capillary occlusions and nerve fiber layer infarcts and; and proliferative diabetic retinopathy (PDR), the late-stage disease, which is diagnosed when pathological neovascular changes are identified on the retinal surface and/or vitreous. Diabetic macular edema (DME) can develop at any stage and reflects a pathological increase in retinal vascular permeability. Despite its high prevalence, current availability of preventive and therapeutic strategies is far from ideal. In fact, there are no reliable biomarkers to predict risk of developing DR and no effective, targeted and early-acting therapies to sustainably and safely prevent disease progression into its vision threatening stages: PDR and DME. The two main therapeutic options currently available for DR are laser photocoagulation and intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents; these constitute “non-selective” and destructive (especially for laser therapy) approaches that are not only unable to effectively and sustainably arrest retinal disease progression in every case, but can also potentially induce a myriad of undesired off-target effects at the retinal level and even systemically, in the case of VEGF antagonists. Moreover, they act late in the disease course. The lack of reliable rodent models - there is no diabetic mouse model that spontaneously recapitulates the late stages of DR - has greatly hindered research progress and development of novel and effective drugs for PDR, further contributing to the present therapeutic scenario. In this dissertation I will introduce and present my experimental work in the context of the following concepts, which can potentially lead to development of targeted, earlier acting, less destructive and more effective future therapies for DR: (1) DR has long been regarded as a vascular disease and present-day DR management guidelines are still based on this assumption. However, a growing body of evidence shows that retinal neuronal function becomes impaired before vascular changes can be detected; these findings along with those showing that adequate retinal functioning depends on stable intercellular interactions within neurovascular units, suggest that disrupting retinal neurovascular crosstalk may play a critical role in promoting disease development and progression. (2) Metabolomic studies have been surprisingly neglected in the investigation of DR’s pathophysiology, and this is clearly reflected by the fact that the metabolome of human DR remains unknown. Furthermore, the neuroretina is one of the most metabolically demanding tissues in the body per unit weight, and diabetes is triggered by a metabolic defect that profoundly impairs cellular energy production. These features constitute a potentially disastrous combination in regard to retinal functioning and suggest that studying retinal energy metabolism in DR is critical. (3) Metabolic cycles of photoreceptors, interneurons and glial cells are still under debate and, even though it is known that intercellular communications within the neurovascular unit (NVU) are mediated by metabolites whose production becomes dysregulated under pathological conditions, the precise mechanisms underlying retinal neurovascular coupling are not fully identified. Gaining further insight into these interactions is pivotal because retinal NVUs are responsible for regulating blood flow for functionally dynamic retinal neuronal networks and, thus, for their proper functioning. Besides the points stated above, additional clinical clues were considered to guide the research plan presented in this dissertation. One the strongest came from a subset of long-term diabetic patients who appear to be protected from developing late-stage DR, by an unknown mechanism. Studying these patients provides an excellent opportunity to identify protective factors and to further understand the mechanisms involved in progression of DR. In order to better understand how metabolic dysregulation impacts development of DR, how neurovascular interactions become compromised in the diabetic retina, and to develop strategies to potentially restore homeostasis within the NVU, we decided to use metabolomic analyses. A highly sensitive metabolomics mass-spectrometry based approach was used in ocular and serum samples to identify the most prominent metabolic perturbations, to acquire a global overview of the metabolomic landscape in late-stage DR and to identify potentially protective circulating factors. At the ocular level, late-stage diabetic retinopathy was associated with severe dysregulation in amino acid levels; this was especially prominent in those generated during arginine metabolism, suggesting a preferential activity in the arginase pathway over the alternative Nitric Oxide Synthase (NOS) pathway; Studies in the Oxygen-Induced-Retinopathy (OIR) mouse, a non-diabetic model that develops features of ischemic retinopathy, revealed a very similar metabolic landscape and, in vivo global isotope analysis confirmed the presence of asymmetrical arginine metabolism by showing: (1) over-activity in the arginase pathway leading to enhanced proline production; and (2) reduced activity in the alternative NOS pathway, with potentially reduced NO production. Even though NO’s role in DR and other retinopathies is not clearly understood, NO is known to be an important modulator of cellular interactions within the NVU and its lower availability in specific locations and/or time-points in pathological conditions may significantly contribute to the disruption of retinal neurovascular crosstalk. The work presented in this dissertation has also described novel functions for interneurons (amacrine and horizontal cells) and photoreceptors within the NVU, by showing that these cells play an active role in regulating their primary vasculature and thus, their blood supply. Furthermore, it has also shown that dysfunction of retinal neurons in this capacity can directly alter their own blood supply, therefore providing additional clues for disrupted retinal neurovascular crosstalk. Metabolomic analyses comparing serum samples from diabetic patients with or without PDR (long-term diabetic patients “protected” from late-stage DR versus those who were non-protected) revealed that “protected” patients had higher circulating levels of a purine metabolite, inosine. To assess its therapeutic potential in conditions of retinal ischemia, inosine was delivered to the eye of the OIR mouse where it enhanced effective revascularization of ischemic retinal areas, thus significantly reducing pathological neovascularization. These effects were associated with a favorable modulation of the local pro-inflammatory response that could result from an improved overall retinal metabolic status. These beneficial effects on retinal metabolism induced by inosine injections were observed as: (1) a reduction in basal mitochondrial respiration in vaso-obliterated areas (i.e., ischemic areas), which can potentially increase retinal neuronal tolerance to hypoxia by reducing the metabolic mismatch created by scarce metabolic supply and high neuronal demand; and (2) a reduction in proline production, suggesting antagonism of the arginase pathway (which is hyperactive in oxygen-induced-retinopathy and potentially in human PDR). In summary, the work presented in this dissertation employed a metabolomic-focused approach with a strong focus on neurovascular crosstalk to answer two intriguing questions regarding DR: (1) What is the characteristic ocular metabolic landscape of severe DR? (2) Is the “protection” against severe DR (observed in some long-term diabetic patients) associated with differences in circulating metabolic factors? The answers to these questions, presented below, could serve as the basis of future targeted, more effective and earlier acting therapeutics that would revolutionize DR patient management. Identification of the most prominently affected metabolic pathways in eyes with severe DR has identified specific pathways of amino acid metabolism as potential targets for development of new drugs for DR. We have also identified a circulating protective factor, inosine, in “protected” patients and further investigated its ability to (1) prevent development of retinal ischemia and pathological neovascularization; (2) adjust retinal metabolism to the limited energy supplies in ischemic areas; and (3) counteract development of prominent metabolic dysregulation by potentially inhibiting the pathology-promoting arginase pathway. We believe that inosine can potentially become an effective, early-acting therapeutic agent to prevent progression of DR. In addition, these metabolites could potentially be used as reliable biomarkers for monitoring response to therapy and for predicting risk of developing or progressing DR. Finally, the work presented in this dissertation supports the concept that early intervention for treating DR will restore balance and stabilize cellular interactions within the NVU, thereby reversing the chronic stressors (e.g., extreme conditions of metabolic insufficiency in retinal ischemic areas) that ultimately drive development and progression of retinal pathology.Fundação para a Ciência e a Tecnologia (FCT

Blocking Immunosuppressive Factors and Taking Advantage of the Nutrient Supply Within the Tumor Microenvironment: Pathways to Achieve Improved Cancer Immunotherapeutic Efficacy for Patients With Metastatic Melanoma

The incidence of melanoma is increasing. Immunotherapy commonly fails due to the immunosuppressive tumor microenvironment (TME). The aim of my dissertation is to develop strategies that dampen the TME’s immunosuppressive capacity and improve the antitumor performance of vaccine-induced melanoma-associated antigen (MAA)-specific CD8+T cells. I pursued this goal using three approaches. First, interactions between co-inhibitors on CD8+ tumor-infiltrating lymphocytes (TILs) with immunoinhibitory ligands within the TME impair T cells’ effector functions. I assessed whether blocking immunoinhibitory signaling during T cell priming augments their antitumor activity. I designed a melanoma vaccine expressing MAAs within herpes simplex virus (HSV) glycoprotein D (gD), which blocks the inhibitory BTLA/CD160-HVEM pathway. Compared to a non-gD vaccine, the gD-adjuvanted vaccine enhances CD8+T cells to low avidity epitopes and prolongs survival of tumor-bearing mice. gD renders MAA-specific CD8+TILs more resistant to functional impairment within TME, which increases their ability to limit tumor progression. Second, the stroma of solid tumors is crucial for tumorigenesis and suppresses the CD8+TILs’ effector functions. To determine whether destroying tumor stroma could improve the MAA-specific CD8+T cells’ tumoricidal capacity, I designed a vaccine targeting the fibroblast activation protein (FAP), which is expressed at high levels on tumor-stromal fibroblasts. Combining the vaccines to FAP and MAAs significantly improves the survival of tumor-bearing mice. This is caused by destruction of FAP+ cells, which reduces frequencies and inhibitory functions of immunosuppressive cells. It also decreases the MAA-specific CD8+TILs’ metabolic stress and delays their progression towards functional exhaustion. Finally, the TME commonly lacks nutrients and oxygen needed for the CD8+TILs’ energy production. My data demonstrate that these metabolic challenges profoundly contribute to the CD8+TILs\u27 functional impairment. Using 13C-stable isotope tracing in vivo, I show that metabolically stressed CD8+TILs in late stage tumors increasingly depend on fatty acids (FAs) catabolism for energy production. Promoting FA catabolism by CD8+TILs improves their effector functions and capacity to delay tumor growth. Overall, my studies show that blocking inhibitory factors while taking advantage of the available nutrients within the TME could improve the performance of vaccine- or adoptive transfer-induced CD8+TILs. These strategies provide new avenues for cancer immunotherapy that may benefit cancer patients

Loss-of-function of leptin receptor impairs metabolism in human cardiomyocytes

Background and aims: Leptin resistance or leptin signalling deficiency are associated with increased risk of diabetic cardiomyopathy and heart failure, which is a leading cause of obesity- and diabetes type 2 (T2DM)-related morbidity and mortality. Various metabolic disturbances are involved in this pathogenesis, such as elevated glucose and fatty acid levels, insulin resistance and altered myocardial substrate utilization. Rodent models provided useful insights into the underlying molecular mechanisms of obese- and T2DM-associated cardiometabolic diseases, however, they cannot fully recapitulate the disease phenotype of obese or T2DM patients. The aims of this study were to study the effect of leptin receptor (LEPR) mutations on the leptin-mediated signalling pathways in human cardiomyocytes, and to investigate glucose and fatty acid metabolism in the heart under (patho)physiological conditions. Methods and results: To study the role of LEPR in human cardiomyocytes (CMs), human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used as a model. In the first part of this study, LEPR expression and function was investigated in wild type (WT)-iPSC-CMs by PCR and Western Blot. LEPR protein expression was almost not detectable in iPSCs and during early cardiac differentiation stages, however mRNA LEPR expression was comparable in the different steps of cardiac development. Importantly, LEPR protein expression was observed in WT-iPSC-CMs at the maturation stages, indicating that LEPR plays an important role in matured CMs. Thanks to CRISPR/Cas9 technology, LEPR mutations were introduced into iPSCs. Among the several clones obtained, 1B2 LEPRΔ/Δ-iPSC line was fully characterized and showed normal capacity to differentiate into spontaneously beating CMs. Although the B27 medium represents a well-established medium to cultivate iPSC-CMs, it has limitations for studying CM metabolism due to its high concentration of insulin and glucose, but low concentration of fatty acids. Physiological medium condition (F2) including physiological range of glucose, insulin and fatty acids was found to be fundamental to study LEPR signalling pathway in iPSC-CMs. Western blot analysis showed functional LEPR downstream pathway activation in WT-iPSC-CMs, while the absence of LEPR function was demonstrated in LEPRΔ/Δ-iPSC-CMs cultured in F2 medium. Moreover, improved medium condition, offered by the F2 medium, ameliorates insulin sensitivity as result of increased insulin-dependent AKT phosphorylation in WT-iPSC-CMs, while loss of LEPR function was associated with downregulation of insulin pathway activation. Additionally, leptin direct effect was observed on the regulation of glucose metabolism in WT-iPSC-CMs by reducing glycolytic fluxes, which was not observed in LEPRΔ/Δ-iPSC-CMs, as measured by 13C-isotope-assisted glucose metabolic flux. These data indicate that the signalling interaction between insulin and leptin is important in regulation of glucose metabolism and is abolished in LEPRΔ/Δ-iPSC-CMs. The matured WT-iPSC-CMs in F2 medium display adult CM-like metabolic phenotype such as enhanced mitochondrial respiration and glycolytic function, as measured by Seahorse analyser, compared to the same group cultured in the B27 medium. The mutation generated in LEPRΔ/Δ-iPSC-CMs caused an “energy starvation” status which led to increased AMPK phosphorylation compared to the WT group in B27 medium, which was associated with lower mitochondrial oxygen consumption rate (OCR) linked basal respiration and ATP production. In the next part of this study, the long-term leptin treatment of iPSC-CMs under physiological medium conditions in the presence of physiological range of insulin, glucose, and fatty acids (F2+) influenced LEPR downstream pathway activation such as JAK2 and AMPK suggesting a leptin-dependent role in fatty acid uptake and oxidation in WT-iPSC-CMs. On the contrary, leptin did not affect JAK2 and AMPK activation in LEPRΔ/Δ-iPSC-CMs. Culturing of (WT)-iPSC-CMs in F2+ medium demonstrated no significant difference in mitochondrial oxygen consumption, while slightly lower glycolysis and glycolytic capacity was observed. However, a leptin effect on fatty acid and glucose metabolism was observed in LEPR∆/∆-iPSC-CMs, which is independent from LEPR downstream regulation. To study the effect of high leptin levels, a medium mimicking some of the diabetic hallmarks, such as high glucose, high insulin, and high leptin levels, was used. Metabolic flexibility was observed in WT-iPSC-CMs in F3+ medium as showed by no difference in mitochondrial function in WT-iPSC-CMs in the presence or absence of high leptin. In contrast, LEPRΔ/Δ-iPSC-CMs in F3+ medium demostrated higher OCR compared to F2 medium, which is accompanied by lower glycolysis and glycolytic capacity, indicating the incapability of LEPRΔ/Δ-iPSC-CMs to use glucose as energy source, as measured by Seahorse analysis. Conclusion and outlook: Taken together, this study demonstrates the importance of leptin and LEPR at the late stage of CM maturation and the fundamental role of metabolic medium condition including physiological range of glucose and fatty acid to study the role of leptin in iPSC-CMs. In addition, LEPRΔ/Δ-iPSC-CMs in diabetic condition (F3+) represent a suitable model to investigate leptin-dependent cardiac metabolism, resulting in increased mitochondrial oxygen consumption and decreased glycolytic function, resembling the condition known in obesity-related T2DM patients. Further studies should focus on the regulation of the metabolic switch between glucose and fatty acid utilization in the absence of a functional LEPR. Understanding the contribution of leptin/LEPR signalling in human CM metabolism will shed light on novel therapeutic approaches to treat diabetic cardiomyopathy

The anisotropic small strain stiffness of Cambridge Gault Clay.

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN024001 / BLDSC - British Library Document Supply CentreGBUnited Kingdo