Engineered control of protein activity in living cells

Cell behavior results from the precise orchestration of molecular activity in time and space. The need to understand dynamics of proteins in the context of living systems has recently led to the development of a remarkable suite of protein ‘switches’, engineered domains and other approaches that cause proteins to respond to small molecules or light, enabling us to control the spatiotemporal dynamics of protein-protein interactions, posttranslational modifications, conformational change, and subcellular localization. However, existing methods suffer from many disadvantages including increased basal activity before protein activation, slow kinetics, difficulty in delivery and expression, and inefficient activation. This dissertation describes two strategies to manipulate protein activity to interrogate the role of the protein of interest in cell motility. In the first study, I developed a ligand-controlled switch to manipulate activities of various kinases dynamically. In the second study, I developed a novel and generalizable approach to control protein activity by splitting target proteins and regulating their reassembly using a ligand or light. Both methods were used to investigate the dynamics of proteins including kinases and guanine nucleotide exchange factors in cell motility.Doctor of Philosoph

Characterization and modelling of organic devices for simultaneous stimulation and recording of cellular electrical activity with Reference-Less Electrolyte-Gated Organic Field-Effect Transistors

The study of neuronal and neurodegenerative diseases requires the development of new tools and technologies to create functional neuroelectronics allowing both stimulation and recording of cellular electrical activity. In the last decade organic electronics is digging its way in the field of bioelectronics and researchers started to develop neural interfaces based on organic semiconductors. The interest in such technologies arise from the intrinsic properties of organic materials such as low cost, transparency, softness and flexibility, as well the biocompatibility and the suitability in realizing all organic printed systems. In particular, organic field-effect transistor (OFET) -based biosensors integrate the sensing and signal amplification in a single device, paving the way to new implantable neural interfaces for in vivo applications. To master the sensing and amplification properties of the OFET-based sensors, it is mandatory to gain an intimate knowledge of the single transistors (without any analytes or cells) that cannot be limited to basic characterizations or to general models. Moreover, organic transistors are characterized by different working principles and properties as respect to their inorganic counterpart. We performed pulsed and transient characterization on different OFETs (both p-type and n-type) showing that, even though the transistors can switch on and off very fast, the accumulation and/or the depletion of the conductive channel continues for times as long as ten seconds. Such phenomenon must be carefully considered in the realization of a biosensor and in its applications, since the DC operative point of the device can drift during the recording of the cellular signals, thus altering the collected data. We further investigate such phenomenon by performing characterizations at different temperatures and by applying the deep level transient spectroscopy technique. We showed that the slow channel accumulation (and depletion) is due to the semiconductor density-of-states that must be occupied in order to bring the Fermi energy level close to the conduction band. This is a phenomenon that can takes several seconds and we described it by introducing a time-depend mobility. We also proposed a technique to estimate the behavior, in time, of the position of the Fermi energy level as respect to the conduction band. To understand the electrochemical transduction processes between living cell and organic biosensor, we realized two-electrodes structure (STACKs) where a drop of saline solution is put directly in contact with the organic semiconductor. On these devices, we performed electrochemical impedance spectroscopy at different DC polarizations and we developed an equivalent circuit model for the metal-organic semiconductor-solution structures that are typically used as transducers in biosensor devices. Our approach was extending the standard range of the bias voltages applied for devices that operate in water. This particular characterization protocol allowed to distinguish and investigate the different mechanisms that occur at the different layers and interfaces: adsorption of ions in the semiconductor; accumulation and charge exchange of carriers at the semiconductor/electrolyte interface; percolation of the ionic species through the organic semiconductor; ion diffusion across the electrolyte; ion adsorption and charge exchange at the platinum interface. We highlighted the presence of ion percolation through the organic semiconductor layer, which is described in the equivalent circuit model by means of a de Levie impedance. The presence of percolation has been demonstrated by environmental scanning electron microscopy and profilometry analysis. Although percolation is much more evident at high negative bias values, it is still present even at low bias conditions. In addition, we analyze two case studies of devices featuring NaCl (concentration of 0.1M) and MilliQ water as solution, showing that both cases can be considered as a particular case of the general model presented in this manuscript. The very good agreement between the model and the experimental data makes the model a valid tool for studying the transducing mechanisms between organic films and the physiological environment. Hence this model could be a useful tool not only for the characterization and failure analysis of electronic devices, such as water-gated transistors, electrophysiological interfaces, fuel cells, and others electrochemical systems, but also this model might be used in other applications, in which a solution is in intimate contact with another material to determine and quantify, if undesired mechanisms such as percolation and/or redox corrosive processes occur. Lastly, the knowledge gain on OFETs and STACKs were put together to realize electrolyte-gated field effect transistors (EGOFETs). We then developed a model to describes EGOFETs as neural interfaces. We showed that our model can be successfully applied to understand the behaviour of a more general class of devices, including both organic and inorganic transistors. We introduced the reference-less (RL-) EGOFET and we showed that it might be successfully used as a low cost and flexible neural interface for extracellular recording in vivo without the need of a reference electrode, making the implant less invasive and easier to use. The working principle underlying RL-EGOFETs involves self-polarization and back-gate stimulation, which we show experimentally to be feasible by means of a custom low-voltage high-speed acquisition board that was designed to emulate a real-time neuron response. Our results open the door to using and optimizing EGOFETs and RL-EGOFETs for neural interfaces

Measuring and managing foot muscle weakness

Foot muscle weakness is caused by disease, injury, inactivity and ageing, with disabling consequences. Exercise improves muscle weakness however, adherence to correct technique is challenging. Biofeedback may improve performance. Chapter One reviews the literature on small foot muscles, muscle function, measurement, causes and consequences of foot muscle weakness, and the role of exercise. Chapter Two is a systematic review on the relationship between foot pain, muscle strength and size. Eight studies were identified evaluating the relationship between foot pain and foot muscle strength or size, with a significant association between foot pain and muscle weakness when pain is of high intensity and weakness measured by toe flexion force. Chapter Three is a reliability study assessing size of abductor hallucis and medial belly flexor hallucis brevis muscles by ultrasound in 21 adults and identify their relationship with toe strength, foot morphology, balance. Intra-rater reliability was excellent. Significant associations were found between cross-sectional area of abductor hallucis with great toe flexion force, arch height sit and stand, truncated and full foot length, balance. Significant associations found between cross-sectional area of medial belly flexor hallucis brevis with Foot Posture Index, truncated and full foot length. After controlling for body size, cross-sectional area of abductor hallucis remained a significant correlate of great toe flexor strength. Chapter Four describes the development of the Archie biofeedback device. Device feasibility is evaluated in Chapter Five by repeat testing of 30 adults performing four foot exercises using Archie, with 89% of exercise and foot location variables collected consistently. Biofeedback significantly improved foot location for all exercises and 97% of participants reported biofeedback helped exercise performance. Archie appears to be a safe and feasible biofeedback device to assist participants perform exercise

Developing a new generation of neuro-prosthetic interfaces: structure-function correlates of viable retina-CNT biohybrids

PhD ThesisOne of the many challenges in the development of neural prosthetic devices is the choice of electrode material. Electrodes must be biocompatible, and at the same time, they must be able to sustain repetitive current injections in a highly corrosive physiological environment. We investigated the suitability of carbon nanotube (CNT) electrodes for retinal prosthetics by studying prolonged exposure to retinal tissue and repetitive electrical stimulation of retinal ganglion cells (RGCs). Experiments were performed on retinal wholemounts isolated from the Cone rod homeobox (CRX) knockout mouse, a model of Leber congenital amaurosis. Retinas were interfaced at the vitreo-retinal juncture with CNT assemblies and maintained in physiological conditions for up to three days to investigate any anatomical (immunohistochemistry and electron microscopy) and electrophysiological changes (multielectrode array stimulation and recordings; electrodes were made of CNTs or commercial titanium nitride). Anatomical characterisation of the inner retina, including RGCs, astrocytes and Müller cells as well as cellular matrix and inner retinal vasculature, provide strong evidence of a gradual remodelling of the retina to incorporate CNT assemblies, with very little indication of an immune response. Prolonged electrophysiological recordings, performed over the course of three days, demonstrate a gradual increase in signal amplitudes, lowering of stimulation thresholds and an increase in cellular recruitment for RGCs interfaced with CNT electrodes, but not with titanium nitride electrodes. These results provide for the first time electrophysiological, ultrastructural and cellular evidence of the time-dependent formation of strong and viable bio-hybrids between the RGC layer and CNT arrays in intact retinas. We conclude that CNTs are a promising material for inclusion in retinal prosthetic devices

Advanced Direct Drive Shock Ignition Studies

The shock ignition approach to inertial confinement fusion offers a potential route to ignition and high gain. It proposes a low velocity fuel assembly on a low adiabat, and ignition through the launching of a late timed strong shock. Accurate descriptions of the coupling of laser energy into the capsule are required to model implosions, including driving the highly compressible fuel and the interaction of the shock launching spike with the coronal ablation plasma. Two well diagnosed experiments were performed on the Omega-60 laser facility that isolated key physics issues for the two steps of shock ignition. The first used a novel conical target to access for the first time the laser-plasma conditions relevant for full-scale shock ignition, in order to characterise the laser-plasma interactions and subsequent supra-thermal hot electrons. The dominant instability was identified as convective stimulated Raman scattering, producing hot electrons of ~40 keV with a laser energy conversion efficiency of 1-3%. This is unique and an essential measurement, as inclusion of hot electron generation and propagation in shock ignition simulations is crucial for constructing implosion designs that might be capable of reaching ignition. The second experiment investigated the implosion dynamics of warm deuterium filled capsules using shaped laser pulses that maintained a reduced shell adiabat and associated high fuel compressibility. A laser drive multiplier was tuned with trajectory measurements from a gated self-emission imager, a significant advancement in the ability to more accurately simulate reduced adiabat designs that are relevant for both shock ignition fuel and conventional central hot spot implosions. Despite significant low mode asymmetries that were identified during the in-flight fuel compression and within the late formed hot spot, the shell trajectory, hot spot morphology and peak neutron emission were well reproduced from one-dimensional simulations. More experiments coupled with predictive modelling are a necessity to determine whether inertial confinement fusion can be a future energy source

Bio-Micro-Systems for Diagnostic Applications, Disease Prevention and Creating Tools for Biological Research

This thesis, divided into two parts, describes the development of 5 novel Bio-Micro-System devices. The term Bio-Micro-System has been used here to describe BioMEMS and 3D printed devices, with the dimensions of key components ranging from micrometers to a millimeter. Part A is focused on ‘Medical’ Micro-System devices that can potentially solve common medical problems. Part B is focused on ‘Biological’ Micro-System devices/tools for facilitating/enabling biological research. Specifically, Part A describes two implantable, electronics-free intraocular pressure (IOP) microsensors for the medical management of glaucoma: 1) Near Infrared Fluorescence-based Optomechanical (NiFO) technology - Consists of an implantable, pressure sensor that ‘optically encodes’ pressure in the near infrared (NIR) regime. A non-implantable, portable and compact optical head is used to excite the sensor and collect the emitted NIR light. The thesis discusses optimized device architecture and microfabrication approaches for best performance commercialization. 2) Displacement based Contrast Imaging (DCI) technology - A proof of concept, fluid pressure sensing scheme is shown to operate over a pressure range of 0–100 mbar (∼2 mbar resolution between 0–20 mbar,∼10 mbar resolution between 20–100 mbar), with a maximum error of <7% throughout its dynamic range. The thesis introduces the DCI technology and discusses its application as an IOP sensor. Moreover, Part A also describes a Touch-activated Sanitizer Dispensing (TSD) system for combating community acquired infections. The TSD can be mounted on any surface that is exposed to high human traffic and consists of an array of human-powered, miniaturized valves that deliver a small amount of disinfectant when touch actuated. The device disinfects the person’s hand that is touching it while being self-sterilized at the same time. The thesis describes the design and implementation of a proof of concept TSD that can disinfect an area equivalent to the size of a thumb. A significant (~ 10 fold) reduction in microbiological load is demonstrated on the fingertip and device surface within the first 24 hours. The size and footprint of the TSD can be scaled up as needed to improve hand hygiene compliance. In Part B, we developed a microfluidic chip for immobilizing Drosophila melanogaster larva by creating a cold micro-environment around the larva. After characterizing on chip temperature distribution and larval body movement, results indicate that the method is appropriate for repetitive and reversible, short-term (several minutes) immobilization. The method offers the added advantage of using the same chip to accommodate and immobilize larvae across all developmental stages (1st instar-late 3rd instar). Besides the demonstrated applications of the chip in high resolution observation of sub cellular events such as mitochondrial trafficking in neurons and neuro-synaptic growth, we envision the use of this method in a wide variety of biological imaging studies employing the Drosophila larval system, including cellular development and other studies. Finally, Part B also describes a 3D printed millifluidic device for CO2 immobilization of Caenorhabditis elegans populations. We developed a novel 3D printed device for immobilizing populations of Caenorhabditis elegans by creating a localized CO2 environment while the animals are maintained on the surface of agar. The results indicate that the method is easy to implement, is appropriate for short-term (20 minutes) immobilization and allows recovery within a few minutes. We envision its use in a wide variety of biological studies in Caenorhabditis elegan, including cellular development and neuronal regeneration studies.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144050/1/amritarc_1.pd

Etude spatiotemporelle des biofilms électroactifs multi-espèces à l'échelle microscopique par une approche microfluidique et optique

Les biofilms électroactifs (EABs) multi-espèces sont capables d'échanger des électrons avec la surface d'une électrode. Les EABs sont principalement utilisés dans les systèmes bioélectrochimiques (BES), où la production d'électricité est difficile à maintenir à long terme. En général, l'électroactivité des EABs atteint un maximum (Jmax) qui diminue progressivement après quelques dizaines de jours de fonctionnement du BES. Dans ce contexte, l'utilisation typique de macroélectrodes pour tester les hypothèses liées à la perte d'électroactivité des EABs rend difficile la fixation et le contrôle de conditions homogènes, couplées à des techniques d'analyses destructives ponctuelles, où l'évolution spatio-temporelle des EABs est clairement perdue. La première partie de cette thèse a été consacrée au travail avec des microélectrodes (∅=50µm) en acier inoxydable (SS), afin d'assurer des conditions expérimentales plus homogènes à la surface de l'électrode. Dans un premier temps, la formation des EABs provenant des marais salants sur les microélectrodes a été standardisée, où l'électroactivité distinctive observée dans les macroélectrodes a été reproduite avec succès. Par la suite, quatre étapes temporelles principales de la biocolonisation et de l'électroactivité ont été détaillées. Une viabilité élevée, des taux de croissance maximaux du biofilm et une quantité importante de protéines de substances polymériques extracellulaires (EPS) ont favorisé l'augmentation de l'électroactivité jusqu'à Jmax. Ensuite, le déclin progressif de l'électroactivité est devenu irréversible, alors que la vitesse de croissance du biofilm à diminué avec l'accumulation de cellules mortes et l'augmentation de la quantité de polysaccharides d’EPS. En outre, la population microbienne des EABs a évolué de Marinobacterium spp. à Desulfuromonas spp. Enfin, d'autres études portant sur le rôle de l'EPS dans l'électroactivité des EABs ont montré une quantité constamment élevée de protéines d'EPS et une faible proportion de polysaccharides d'EPS lorsque l'électroactivité a été augmentée. La deuxième partie de cette thèse s'est focalisée sur le développement d'une microBES transparente (V=0.3 mL) avec une microélectrode intégrée en SS, pour l'observation in situ et en temps réel des interfaces microélectrode/EAB et EAB/cellules planctoniques. La dynamique de la formation du biofilm a été corrélée à l'électroactivité du EAB, où la découverte d'une couche dense active de bactéries planctoniques à proximité de l'interface microélectrode/EAB ouvre de nouvelles voies de recherche sur la formation du biofilm et les mécanismes de transfert d'électrons

1-D broadside-radiating leaky-wave antenna based on a numerically synthesized impedance surface

A newly-developed deterministic numerical technique for the automated design of metasurface antennas is applied here for the first time to the design of a 1-D printed Leaky-Wave Antenna (LWA) for broadside radiation. The surface impedance synthesis process does not require any a priori knowledge on the impedance pattern, and starts from a mask constraint on the desired far-field and practical bounds on the unit cell impedance values. The designed reactance surface for broadside radiation exhibits a non conventional patterning; this highlights the merit of using an automated design process for a design well known to be challenging for analytical methods. The antenna is physically implemented with an array of metal strips with varying gap widths and simulation results show very good agreement with the predicted performance