The sensitivity of diffusion MRI to microstructural properties and experimental factors

Diffusion MRI is a non-invasive technique to study brain microstructure. Differences in the microstructural properties of tissue, including size and anisotropy, can be represented in the signal if the appropriate method of acquisition is used. However, to depict the underlying properties, special care must be taken when designing the acquisition protocol as any changes in the procedure might impact on quantitative measurements. This work reviews state-of-the-art methods for studying brain microstructure using diffusion MRI and their sensitivity to microstructural differences and various experimental factors. Microstructural properties of the tissue at a micrometer scale can be linked to the diffusion signal at a millimeter-scale using modeling. In this paper, we first give an introduction to diffusion MRI and different encoding schemes. Then, signal representation-based methods and multi-compartment models are explained briefly. The sensitivity of the diffusion MRI signal to the microstructural components and the effects of curvedness of axonal trajectories on the diffusion signal are reviewed. Factors that impact on the quality (accuracy and precision) of derived metrics are then reviewed, including the impact of random noise, and variations in the acquisition parameters (i.e., number of sampled signals, b-value and number of acquisition shells). Finally, yet importantly, typical approaches to deal with experimental factors are depicted, including unbiased measures and harmonization. We conclude the review with some future directions and recommendations on this topic

Diffusion and Perfusion MRI in Paediatric Posterior Fossa Tumours

Brain tumours in children frequently occur in the posterior fossa. Most undergo surgical resection, after which up to 25% develop cerebellar mutism syndrome (CMS), characterised by mutism, emotional lability and cerebellar motor signs; these typically improve over several months. This thesis examines the application of diffusion (dMRI) and arterial spin labelling (ASL) perfusion MRI in children with posterior fossa tumours. dMRI enables non-invasive in vivo investigation of brain microstructure and connectivity by a computational process known as tractography. The results of a unique survey of British neurosurgeons’ attitudes towards tractography are presented, demonstrating its widespread adoption and numerous limitations. State-of-the-art modelling of dMRI data combined with tractography is used to probe the anatomy of cerebellofrontal tracts in healthy children, revealing the first evidence of a topographic organization of projections to the frontal cortex at the superior cerebellar peduncle. Retrospective review of a large institutional series shows that CMS remains the most common complication of posterior fossa tumour resection, and that surgical approach does not influence surgical morbidity in this cohort. A prospective case-control study of children with posterior fossa tumours treated at Great Ormond Street Hospital is reported, in which children underwent longitudinal MR imaging at three timepoints. A region-of-interest based approach did not reveal any differences in dMRI metrics with respect to CMS status. However, the candidate also conducted an analysis of a separate retrospective cohort of medulloblastoma patients at Stanford University using an automated tractography pipeline. This demonstrated, in unprecedented spatiotemporal detail, a fine-grained evolution of changes in cerebellar white matter tracts in children with CMS. ASL studies in the prospective cohort showed that following tumour resection, increases in cortical cerebral blood flow were seen alongside reductions in blood arrival time, and these effects were modulated by clinical features of hydrocephalus and CMS. The results contained in this thesis are discussed in the context of the current understanding of CMS, and the novel anatomical insights presented provide a foundation for future research into the condition

Studying the folding of peptide dendrimers using molecular simulation methods

Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2009Dendrimers are a family of branched compounds that share a common layout where wedges emerge radially from a core by means of a regular branching pattern. Peptide dendrimers are a specific kind of dendrimers formed by alternating functional amino acids with branching diamino acids. There has been increasing interest in the synthesis of peptide-based dendritic architectures modelling specific aspects of biological function. Some results are already available, demonstrating these molecules ability to act as enzyme models and to mimic natural ligands. Unfortunately, most studies concerning peptide dendrimers lack structural information at the molecular level. The theoretical study published so far, reported peptide dendrimers presenting shapes close to spheres, though experimental studies on the same systems suggest the existence of more disordered states. Herein, we characterize five third-generation peptide dendrimers (B1, B1H, B1HH, B1HHH and C1) through multiple long molecular dynamics simulations (MD), and analyse their conformational details and folding preferences in solution. Special emphasis is placed on the analysis of conformational trends representative of the examined models. The conformational sampling results, obtained through MM/MD simulations, were scrutinized using several approaches. Namely, histogram analysis, phi-psi dihedral distributions, inter-residue distance matrices, shape analysis and principal coordinate analysis. The adequacy of each approach to discern the conformation space of peptide dendrimers is discussed. Using these analysis procedures we were able to observe two distinct types of behaviour (sphere-like and bowl-like structures), both asserting the enormous structural flexibility characterizing these molecules; and the myriad of conformational states available to them. Our conclusions can be interpreted together with the available experimental results, contributing to a synergistic understanding of the structure-function relation in peptide dendrimers, and casting the bases for novel knowledge-based applications.Os dendrímeros são uma família de compostos ramificados que partilham uma arquitectura comum, onde diferentes cadeias emergem radialmente de um mesmo núcleo (ou centro) através de um padrão de ramificação regular. Os dendrímeros peptídicos, são uma classe particular de dendrímeros, constituída por estruturas que incorporam de forma alternada e iterativa resíduos de amino-ácidos funcionais (resíduos de espaçamento) com resíduos de diamino-ácidos ramificados (resíduos de ramificação). Os resíduos de diamino-ácidos ramificados promovem a bifurcação das cadeias peptídicas e a aquisição da estrutura dendrítica. A possibilidade de sintetizar dendrímeros com composições que mimetizem as funções de moléculas biológicas, constitui o aliciante para a investigação neste campo cientifico. Em particular, a síntese planeada e controlada de estruturas dendríticas baseadas nos componentes apresentados pelas moléculas biológicas, como peptidos ou glícidos, constitui um desafio atractivo pelas potenciais aplicações que dai podem emergir. De facto, já foram reportados dendrímeros peptídicos que modelam aspectos específicos de funções biológicas, tais como: modelos enzimáticos para catálise dirigida (“enzimas artificiais”); mimetização de co-factores naturais (de que e exemplo a vitamina B12); transportadores de fármacos, pois quando acopladas aos ligandos adequados estas moléculas tem a capacidade de aderir à membrana celular. É também importante referir que várias destas moléculas têm sido estudadas enquanto modelos de folding das proteínas naturais, pois investigações experimentais indiciam que alguns dendrímeros peptídicos podem apresentar, em solução, uma estrutura compacta semelhante à das proteínas globulares. Contudo, a maioria dos estudos experimentais realizados até à data são omissos no que concerne a informação estrutural, e carecem do enquadramento adequado a nível molecular e atómico. O único estudo teórico publicado sobre dendrímeros peptídicos parece confirmar a ideia de que, em solução estas moléculas apresentam, de facto, formas semelhantes a esferas, isto apesar de existirem evidências experimentais que sugerem a existência de estados conformacionais mais desordenados, nesses mesmos sistemas. Considerando o grande interesse que estas moléculas tem vindo a despertar, a verdade é que pouco se sabe sobre o seu arranjo estrutural tridimensional, e sobre os processos que a ele conduzem (folding). Nesta tese tentamos preencher algumas destas lacunas. Para tal, procedemos à caracterização de cinco dendrímeros peptídicos de terceira geração (que designamos por B1, B1H, B1HH, B1HHH e C1) com diferentes constituintes peptídicos. Os sistemas que escolhemos como objecto de estudo, estão directamente relacionados com a coordenação da aquocobalamina (análogo da vitamina B12) a dendrímeros peptídicos, ainda que apenas três deles tenham sido sintetizados e caracterizados experimentalmente (B1, B1H e C1). Deste modo, pretendemos não só investigar as suas preferências conformacionais, mas também inferir possíveis relações entre a sua estrutura e a capacidade para desempenhar uma função análoga à das moléculas biológicas (transcobalamina). É importante salientar que de entre os dendrímeros que foram sintetizados experimentalmente, e que são também aqui estudados, os que apresentam maior capacidade de coordenação com a aquacobalamina, são os que possuem um menor número de resíduos com potencial de coordenação. Este aparente paradoxo é por si só interessante e pode estar interligada com aspectos mais estruturais. Como temos por objectivo compreender as alterações e a variabilidade subjacentes às estruturas tridimensionais dos diferentes dendrímeros, empregamos metodologias adequadas ao detalhe da escala que pretendemos investigar. Nomeadamente, métodos computacionais de simulação molecular (MM/MD). Optámos portanto por simular cada um destes cinco sistemas através de múltiplas e longas simulações de dinâmica molecular, utilizando a água enquanto solvente explícito. Com efeito, no trabalho que conduziu a esta tese, realizamos simulações que contabilizam aproximadamente 1 μs-1 para cada um dos dendrímeros em estudo. No que respeita a estes sistemas, isto é muito superior ao tempo simulado em estudos anteriores. Nas últimas décadas a investigacao científica tem beneficiado imenso do avanço das técnicas de simulação computacional, que providenciam resultados e formas de escrutinar sistemas, que são de outra forma normalmente inacessíveis. A dinâmica molecular, especificamente, permite “seguir” a evolução temporal dos átomos que constituem um sistema, através da integração das equações de Newton para o movimento de corpos. É inclusive um dos métodos computacionais de eleição para estudar fenómenos biomoleculares. Os resultados obtidos com esta técnica de amostragem conformacional permitiram-nos analisar e identificar de forma adequada, os detalhes estruturais de cada um dos dendrímeros peptídicos. Colocamos especial ênfase nos arranjos estruturais mais estáveis. As conformações tridimensionais obtidas a partir das trajectórias resultantes das simulações, foram agrupadas de forma a obtermos os ensembles conformacionais característicos de cada dendrímero. Sobre estes conjuntos de conformações realizamos várias análises. Começamos por investigar algumas das propriedades que caracterizam estes sistemas, como o raio de giração, o número total de ligações de hidrogénio, a distância máxima entre os dois átomos mais afastados de cada estrutura, a superfície acessível ao solvente, entre outros. O raio de giração revelou ser a propriedade que individualmente, melhor espelha as variações intrínsecas a estes sistemas. Adicionalmente, procedemos também a caracterização da distribuição dos valores de phi-psi característicos dos diedros de cada um dos dendrímeros. Complementamos esta análise com o estudo das matrizes que reflectem as distãncias mínimas entre os resíduos de todas as conformações. Posteriormente aplicamos metodologias de análise conformacional que envolvem a determinação da energia livre, associada a diferentes coordenadas reaccionais (ou de folding) para cada estrutura nos diferentes ensembles, obtendo assim as correspondentes superfícies energéticas (folding landscapes). Utilizamos esta abordagem por forma a obter folding landscapes bi- e tridimensionais. Em especifíco, utilizamos como coordenadas de folding os valores do raio de giração, do root mean square deviation (RMSD), dos componentes principais do tensor do raio de giração diagonalizado, e os valores para a posição relativa das diferentes conformações, num espaço concordante com a matriz de RMSD, utilizando para tal o método de análise das coordenadas principais (PCoorA). Utilizando o tensor do raio de giração, foi possível investigar a forma dos arranjos estruturais de cada dendrímero peptídico, tendo inclusive sido definido um espaço tridimensional baseado nos componentes principais do tensor diagonalizado (espaço de giração). A capacidade de cada uma destas abordagens para discriminar de forma adequada o espaço das conformações dos dendrímeros peptídicos é discutida ao longo da tese. Dos diversos procedimentos de análise conformacional empregues, resulta uma clara indicação de que, em solução, os dendrímeros peptídicos podem apresentar dois comportamentos preferenciais distintos: estruturas compactas que privilegiam as interacções entre os diferentes resíduos, semelhantes a esferas (sphere-like); e estruturas “abertas” com as diferentes ramificações espaçadas, em que as interacções entre resíduos não adjacentes são minimizadas, semelhantes a taças (bowl-like). Ambas estas configurações atómicas consubstanciam a enorme flexibilidade estrutural que parece caracterizar estas moléculas, dando provas da miríade de estados conformacionais que lhes estão acessíveis. Foi ainda possível verificar a existência de evidências que suportam a ideia de que estas moléculas possuem uma grande robustez estrutural. Isto é, pequenas alterações na composição dos resíduos de amino-ácidos que as constituem não parecem desencadear alterações conformacionais significativas nos arranjos estruturais preferenciais. Através da comparação entre o coeficiente de difusao experimental disponível para um dos dendrímeros, e o coeficiente de difusão calculado com base nas trajectórias obtidas por simulação, foi possível verificar que os modelos utilizados, reflectem de forma adequada os sistemas experimentais. Concluiu-se também que o campo de força (force field) GROMOS 53A6 possui a capacidade de transferabilidade apropriada para lidar com estas moléculas. Os pontos fortes e fracos dos nossos modelos são discutidos ao longo da tese. Durante este trabalho foi ainda desenvolvida e implementada uma metodologia que permite o cálculo eficiente do RMSD entre estruturas dendríticas. As conclusões apresentadas nesta tese podem ser interpretadas juntamente com os resultados experimentais disponíveis, de forma a contribuir para uma compreensão sinérgica da relação entre a estrutura e a função dos dendrímeros peptídicos, lançando as fundações para aplicações inovadoras

The influence of soil structure on microbial processes in microfluidic models

The way microbes behave in nature can vary widely depending on the spatial characteristics of the habitats they are located in. The spatial structure of the microbial environment can determine whether and to which extent processes such as organic matter degradation, and synergistic or antagonistic microbial processes occur. Investigating how the different spatial characteristics of microhabitats influence microbes has been challenging due to methodological limitations. In the case of soil sciences, attempts to describe the inner structure of the soil pore space, and to connect it to microbial processes, such as to determine the access of nutrient limited soil microorganisms to soil organic matter pools, has been one of the main goals of the field in the last years. The present work aimed at answering the question of how spatial complexity affects microbial dispersal, growth, and the degradation of a dissolved organic substrate. Using microfluidic devices, designed to mimic the inner soil pore physical structures, we first followed the dispersal and growth of soil microbes in the devices, using soil inocula or burying the microfluidic devices in the top layer of a soil (Paper I). We found that inter-kingdom interactions can play an important role for the dispersal of water-dwelling organisms and that these physically modified their environment. To reveal the effect of the different structures on microbes in more detail we tested the influence of increasing spatial complexity in a porespace on the growth and substrate degradation of bacterial and fungal laboratory strains. The parameters we used to manipulate the pore space’s complexity were two: via the turning angle and turning order of pore channels (Paper II), and via the fractal order of a pore maze (Paper III). When we tested the effect of an increase in turning angle sharpness on microbial growth, we found that as angles became sharper, bacterial and fungal growth decreased, but fungi were more affected than bacteria. We also found that their substrate degradation was only affected when bacteria and fungi grew together, being lower as the angles were sharper. Our next series of experiments, testing the effect of maze fractal complexity, however, showed a different picture. The increase in maze complexity reduced fungal growh, similar to the previous experiments, but increased bacterial growth and substrate consumption, at least until a certain depth into the mazes, contrary to our initial hypothesis. To increase the relevance of our studies, we performed experiments in both microfluidic device designs inoculated with a soil microbial extract and followed the substrate degradation patterns over time (Paper IV). We found that as complexity increased, both in terms of angle sharpness and fractal order, substrate consumption also increased. Our results, specially in mazes, might be caused by a reduced competition among bacterial communities and individuals in complex habitats, allowing co-existence of different metabolic strategies and the onset of bacterial biofilm formation leading to a higher degradation efficiency, but further studies are required to confirm this. Our results show that the spatial characteristic of microhabitats is an important factor providing microbes with conditions for a wide variety of ecological interactions that determine their growth and their organic matter turnover

Tumor vasculature and microenvironment during progression and treatment : insights from optical microscopy

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2010.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.In addition to cancer cells, solid tumors consist of a variety of cell types and tissues defining a complex microenvironment that influences disease progression and response to therapy. To fully characterize and probe the tumor microenvironment, new tools are needed to quantitatively assess microanatomical and physiological changes during tumor growth and treatment. Particularly important, is the metabolic microenvironment defined in tumors by hypoxia (low p02) and acidity (low pH). These parameters have been shown to influence response to radiation therapy and chemotherapy. However, very little is known about spatio-temporal changes in p02 and pH during tumor progression and therapy. By modifying the technique of intravital multiphoton microscopy (MPM) to perform phosphorescence quenching microscopy, I developed a non-invasive method to quantify oxygen tension (p02) in living tissue at high three-dimensional resolution. To probe functional changes in the metabolic microenvironment, I measured in vivo P02 during tumor growth and antiangiogenic (vascular targeted) treatment in preclinical tumor models. Nanotechnology is rapidly emerging as an important source of biocompatible tools that may shape the future of medical practice. Fluorescent semiconductor nanocrystals (NCs), also known as quantum dots, are a powerful tool for biological imaging, cellular targeting and molecular sensing.(cont.) I adapted novel fluorescence resonance energy transfer (FRET) -based nanocrystal (NC) biosensors for use with MPM to qualitatively measure in vivo extracellular pH in tumors at high-resolution. While intravital multiphoton microscopy demonstrates utility and adaptability in the study of cancer and response to therapy, the requisite high numerical aperture and exogenous contrast agents result in a limited capacity to investigate substantial tissue volumes or probe dynamic changes repeatedly over prolonged periods. By applying optical frequency domain imaging (OFDI) as an intravital microscopic tool, the technical limitations of multiphoton microscopy can be circumvented providing unprecedented access to previously unexplored, critically important aspects of tumor biology. Using entirely intrinsic mechanisms of contrast within murine tumor models, OFDI is able to simultaneously, rapidly, and repeatedly probe the microvasculature, lymphatic vessels, and tissue microstructure and composition over large volumes. Using OFDI-based techniques, measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy were demonstrated, thereby highlighting the potential of OFDI to facilitate the exploration of pathophysiological processes and the evaluation of treatment strategies.by Ryan M. Lanning.Ph.D

Biofabrication Approaches With Hyaluronic Acid Hydrogels For Cartilage Repair

Current therapies to repair damaged articular cartilage fail to consistently or fully restore the biomechanical function of cartilage. Although cell-based clinical techniques have emerged for the treatment of focal defects in articulating joints, these approaches typically lead to inferior tissue formation when compared to native, healthy cartilage. Alternatively, subchondral microfracture is a surgical procedure that aims to recruit endogenous mesenchymal stromal cells (MSCs) from the underlying bone marrow to facilitate neocartilage formation in focal defects. Similarly, microfracture typically results in the formation of repair cartilage incapable of withstanding the loading environment of the articulating joint over time. New biomaterial-based strategies are therefore in significant demand to improve cartilage tissue formation and maturation within focal defects. Hyaluronic acid (HA) is a glycosaminoglycan that is found in native cartilage and that shows promise as a biomaterial for cartilage tissue engineering due to its innate bioactivity and ability to form hydrogels, water-swollen polymer networks that may be engineered to mimic the native extracellular matrix (ECM). Moreover, hydrogels may be employed as materials for biofabrication, which involves the use of automated additive manufacturing processes such as 3D printing to fabricate living, biological constructs. This dissertation describes the design and implementation of HA hydrogels for the biofabrication of articular cartilage towards improving existing therapies for damaged cartilage. Multiple biofabrication approaches, including extrusion bioprinting, melt-electrowriting, and digital light processing are investigated to engineer scaffolds with rationally designed geometries, mechanical properties, porosities, and biodegradability. Conserved across all these approaches is the use of thiol-ene based photochemistry to control the formation and resultant material properties of HA hydrogels modified with norbornene functional groups. Taken together, the employment of these biofabrication approaches for cartilage repair has significantly informed the design and implementation of future therapies for articular cartilage damage

44th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 44th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Denver, Colorado, July 28 - August 1, 2002

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function

Hybrid bio-robotics: from the nanoscale to the macroscale

[eng] Hybrid bio-robotics is a discipline that aims at integrating biological entities with synthetic materials to incorporate features from biological systems that have been optimized through millions of years of evolution and are difficult to replicate in current robotic systems. We can find examples of this integration at the nanoscale, in the field of catalytic nano- and micromotors, which are particles able to self-propel due to catalytic reactions happening in their surface. By using enzymes, these nanomotors can achieve motion in a biocompatible manner, finding their main applications in active drug delivery. At the microscale, we can find single-cell bio-swimmers that use the motion capabilities of organisms like bacteria or spermatozoa to transport microparticles or microtubes for targeted therapeutics or bio-film removal. At the macroscale, cardiac or skeletal muscle tissue are used to power small robotic devices that can perform simple actions like crawling, swimming, or gripping, due to the contractions of the muscle cells. This dissertation covers several aspects of these kinds of devices from the nanoscale to the macro-scale, focusing on enzymatically propelled nano- and micromotors and skeletal muscle tissue bio-actuators and bio-robots. On the field of enzymatic nanomotors, there is a need for a better description of their dynamics that, consequently, might help understand their motion mechanisms. Here, we focus on several examples of nano- and micromotors that show complex dynamics and we propose different strategies to analyze their motion. We develop a theoretical framework for the particular case of enzymatic motors with exponentially decreasing speed, which break the assumptions of constant speed of current methods of analysis and need different strategies to characterize their motion. Finally, we consider the case of enzymatic nanomotors moving in complex biological matrices, such as hyaluronic acid, and we study their interactions and the effects of the catalytic reaction using dynamic light scattering, showing that nanomotors with negative surface charge and urease-powered motion present enhanced parameters of diffusion in hyaluronic acid. Moving towards muscle-based robotics, we investigate the application of 3D bioprinting for the bioengineering of skeletal muscle tissue. We demonstrate that this technique can yield well-aligned and functional muscle fibers that can be stimulated with electric pulses. Moreover, we develop and apply a novel co-axial approach to obtain thin and individual muscle fibers that resemble the bundle-like organization of native skeletal muscle tissue. We further exploit the versatility of this technique to print several types of materials in the same process and we fabricate bio-actuators based on skeletal muscle tissue with two soft posts. Due to the deflection of these cantilevers when the tissue contracts upon stimulation, we can measure the generated forces, therefore obtaining a force measurement platform that could be useful for muscle development studies or drug testing. With these applications in mind, we study the adaptability of muscle tissue after applying various exercise protocols based on different stimulation frequencies and different post stiffness, finding an increase of the force generation, especially at medium frequencies, that resembles the response of native tissue. Moreover, we adapt the force measurement platform to be used with human-derived myoblasts and we bioengineer two models of young and aged muscle tissue that could be used for drug testing purposes. As a proof of concept, we analyze the effects of a cosmetic peptide ingredient under development, focusing on the kinematics of high stimulation contractions. Finally, we present the fabrication of a muscle-based bio-robot able to swim by inertial strokes in a liquid interface and a nanocomposite-laden bio-robot that can crawl on a surface. The first bio-robot is thoroughly characterized through mechanical simulations, allowing us to optimize the skeleton, based on a serpentine or spring-like structure. Moreover, we compare the motion of symmetric and asymmetric designs, demonstrating that, although symmetric bio-robots can achieve some motion due to spontaneous symmetry breaking during its self-assembly, asymmetric bio-robots are faster and more consistent in their directionality. The nanocomposite-laden crawling bio-robot consisted of embedded piezoelectric boron nitride nanotubes that improved the differentiation of the muscle tissue due to a feedback loop of piezoelectric effect activated by the same spontaneous contractions of the tissue. We find that bio-robots with those nanocomposites achieve faster motion and stronger force outputs, demonstrating the beneficial effects in their differentiation. This research presented in this thesis contributes to the development of the field of bio-hybrid robotic devices. On enzymatically propelled nano- and micromotors, the novel theoretical framework and the results regarding the interaction of nanomotors with complex media might offer useful fundamental knowledge for future biomedical applications of these systems. The bioengineering approaches developed to fabricate murine- or human-based bio-actuators might find applications in drug screening or to model heterogeneous muscle diseases in biomedicine using the patient’s own cells. Finally, the fabrication of bio-hybrid swimmers and nanocomposite crawlers will help understand and improve the swimming motion of these devices, as well as pave the way towards the use of nanocomposite to enhance the performance of future actuators.[spa] La bio-robótica híbrida es una disciplina cuyo objetivo es la integración de entidades biológicas con materiales sintéticos para superar los desafíos existentes en el campo de la robótica blanda, incorporando características de los sistemas biológicos que han sido optimizadas durante millones de años de evolución natural y no son fáciles de reproducir artificialmente. Esta tesis cubre varios aspectos de este tipo de dispositivos desde la nanoescala a la macroescala, enfocándose en nano- y micromotores propulsados enzimáticamente y bio-actuadores y bio-robots basados en tejido muscular esquelético. En el campo de nanomotores enzimáticos, existe la necesidad de encontrar mejores modelos que puedan describir la dinámica de su movimiento para llegar a entender sus mecanismos de propulsión subyacentes. Aquí, nos enfocamos en diversos ejemplos de nano- y micromotores que muestran dinámicas de movimiento complejas y proponemos diferentes estrategias que se pueden utilizar para analizar y caracterizar este movimiento. Moviéndonos hacia robots basados en células musculares, investigamos la aplicación de la técnica de bioimpresión en 3D para la biofabricación de músculo esquelético. Demostramos que esta técnica puede producir fibras musculares funcionales y bien alineadas que puede ser estimuladas y contraerse con pulsos eléctricos. Investigamos la versatilidad de esta técnica para imprimir varios tipos de materiales en el mismo proceso y fabricamos bio-actuadores basados en músculo esquelético. Debido a los movimientos de unos postes gracias a las contracciones musculares, podemos obtener medidas de la fuerza ejercida, obteniendo una plataforma de medición de fuerzas que podría ser de utilidad para estudios sobre el desarrollo del músculo o para testeo de fármacos. Finalmente, presentamos la fabricación de un bio-robot basado en músculo esquelético capaz de nadar en la superficie de un líquido y un bio-robot con nanocompuestos incrustados que puede arrastrarse por una superficie sólida. El primer de ellos es minuciosamente caracterizado a través de simulaciones mecánicas, permitiéndonos optimizar su esqueleto, basado en una estructura tipo serpentina o muelle. El segundo bio-robot contiene nanotubos piezoeléctricos incrustados en su tejido, los cuales ayudan en la diferenciación del músculo debido a una retroalimentación basada en su efecto piezoeléctrico y activada por las contracciones espontáneas del tejido. Mostramos que estos bio-robots pueden generar un movimiento más rápido y una mayor generación de fuerza, demostrando los efectos beneficiales en la diferenciación del tejido