Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

Biological studies on in vitro cell cultures are of fundamental importance for investigating cell response to external stimuli, such drugs for specific treatments, or for studying communication between cells. In the electrophysiology field, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. Thus, their improvement is a central topic nowadays in neuroscience and cardiology [1]. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate[2], allowing for monitoring action potentials from many cells simultaneously. In fact, nanoscale biomaterials were able to overcome the limitations of previous technologies, paving the way to the development of platforms for interfacing the electrogenic cells at unprecedented spatiotemporal scales. These devices, together with microfluidics, are starting to be used for drug screening and pharmaceutical drug development since they represent a powerful tool for monitoring cell response when cultures are stimulated by target compounds. Many pharmaceutical agents, however, including various large molecules (enzymes, proteins, antibodies) and even drug-loaded pharmaceutical nanocarriers, need to be delivered intracellularly to exercise their therapeutic action inside the cytoplasm[3]. Nanoscale electrodes offer individual cell access and non-destructive poration of the cellular membrane enabling high capability in the delivery of biomolecules. Among all the techniques, electroporation have proven encouraging potential as alternative to the carrier mediated methods for molecular delivery into cultured cells[4]. In this regard, different groups [5][6][7] exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. These efforts provided powerful tools for advancing applications in therapeutics, diagnostics, and drug discovery, in order to reach an efficient and localized delivery on a chip. Despite these new tactics, there is still a critical need for the development of a functional approach that combines recording capabilities of nanostructured biosensors with intracellular delivery. The device should provide for tight contact between cells and electrode so as to enable highly localized delivery and optimal recording of action potentials in order to attain a high degree of prediction for the disease modeling and drug discovery. This \u201con-chip\u201d approach will help to gain deeper insight in several bio-related studies and analyses, providing a comprehensive knowledge of the entire cellular dynamics when selectively stimulated by the desired bio-molecules. In the first part of this dissertation, a solution will be proposed in order to fill this gap and respond to this need in the biology field. In the first chapter, I will describe briefly the principles of action potentials and how neurons and cardiomyocyte are composed, together with the development of electrophysiology and the advent of multielectrode arrays. In the second chapter, more details about fabrication and cell-electrode system modelling will be explained. In the same chapter, I will explore the development of multielectrode arrays up to the present days, along with the advent of nanotechnologies and the related techniques for improving the previous platforms. The different cell poration techniques will be described in order to reach the best recording capabilities without damaging cells. Electroporation, optoporation and spontaneous poration will be presented and the chosen technique for our application (electroporation) will be reviewed more in detail. In the third chapter, different methodologies for intracellular delivery will be explained, focusing also on the electroporation technique. A small paragraph about the integration of these techniques on chip will be inserted to illustrate the state of the art of these devices. The fourth chapter will explicate in details the Microfluidic multielectrode array idea, the approach used in order to fabricate this novel platform from scratch, the experiments carried out to verify its capabilities and the associated results. In the last paragraph, I will discuss how the proposed platform could became suitable for the day to day uses in research activity by employing nanoporous materials. In fact, big efforts are carried out in order to find appropriate metamaterials as substitutes of the 3D counterparts so as to decrease the cost of device manufacturing that makes them unfitting with research activity. As a novel electrode material, nanoporous metals possess unique properties, such as a low fabrication cost, high plasmonic enhancement and large surface-volume ratio[8]. Nanoporous gold behaves like a metamaterial whose effective dielectric response can be tuned accordingly to the wanted use. These properties make the material suitable for multiple biosensing application, from a high-performance and reliable SERS (surface enhanced raman scattering) substrate [9] to an electrode in CMOS MEAs capable of intracellular recordings[10]. All these properties were explored in the last years, but it could be interesting to further study if the characteristics of this material could make it a good photoelectrical modulating material for eliciting electrogenic cells firing activity. In this way, this technology could be in principle easily implemented on commercial CMOS devices, consenting stimulation and recording at single cell level with high-resolution sensors, opening the way to new methodologies for studying electrogenic cells and tissues. Electrical stimulation of excitable cells is the basis for many implantable devices in cardiac treatment and in neurological studies for treating debilitating neurological syndromes. In order to make the technique less invasive, optical stimulation was widely investigated [11]. The non-genetic photostimulation is starting to make its way in the field since it allows to avoid changing the biological framework by using transient thermal or electrochemical outputs from synthetic materials attached to the target cells[12]. If stimulated with impinging light these materials could inject free charges into the solution resulting in an ionic current at the interface able to eliciting of neurons[13] or cardiomyocyte action potentials. Plasmonic porous materials have all the suitable properties to be considered as an appealing tools for charge injection and consequently for stimulation of electrically active cells [14]. Thus, the second part of this dissertation will exploit the capabilities of these plasmonic metamaterials, placing particular emphasis on the possibility of photoelectrochemical modulation. In particular, in the fifth and last chapter I will describe all the properties and application of the porous material and the mechanism of photoemission. In the experimental paragraphs, the free charge photoemission properties of porous gold will be explored together with plasmonic non-genetic photostimulation of the cardiac cells on commercial CMOS MEAs

Hydrological cycle during droughts: large-scale analyses for process understanding and modelling

Droughts strongly affect the environment and human activities with long-term and far-reaching impacts that will increase in the next decades under global changes. Thus, we need an in-depth understanding of drought processes and their robust modelling to cope with drought risk. For hydrologists, recurring challenges include predicting the impacts of precipitation (P) deficits in the form of soil moisture, streamflow (Q), or groundwater deficits. Water stored in catchments and evapotranspiration (ET) regulate drought evolution, that is the propagation of P deficits through the hydrological cycle and the subsequent recovery. Yet, analyses explicitly considering the joint contribution of storage and ET to drought evolution across different hydroclimatic regimes are rare. Furthermore, many hydrological models poorly simulate Q during droughts, but previous studies have rarely assessed model performances during droughts in multi-variable and spatially-distributed evaluations. This PhD thesis aimed to answer two main research questions: (i) do storage changes and ET affect drought evolution across climates and landscapes?; (ii) does a distributed hydrological model properly represent Q, ET, and storage during droughts? I performed a large-sample data-based analysis of Q, ET, and changes in the subsurface storage (in soil and groundwater) over the period 2010-2019 for 102 Italian catchments to answer the first question. To address the second question, I evaluated Q, ET, and storage simulations from the process-based distributed hydrological model Continuum over the Po river basin (northern Italy) during recent droughts, including the severe 2022 event. From the large-sample data-based analysis, I found that annual subsurface storage changes represented on average 11% of annual P across the study catchments, and mostly buffered Q deficits during drought years and their recovery. ET, instead, both buffered and aggravated Q deficits, and it had a decoupled response to P. These results revealed the prominent role of subsurface storage in driving the evolution of annual droughts. From model evaluation, I showed worse model performances in simulating Q for severe than for moderate droughts (mean KGE across the 38 study sub-catchments = 0.55±0.25 during moderate droughts and 0.18±0.69 in 2022) and I linked them to a degraded simulation of ET, rather than storage, especially in the human-affected croplands (mean r = -0.03 and nRMSE = 1.8 across the croplands in 2022). By calibrating the model during a moderate drought, I showed similar model performances during the severe event (mean KGE = 0.18±0.63), which further point to specific human-water processes during this event. Therefore, I delineated possible ways forward for model improvement during severe droughts, such as an enhanced consideration of human interference, especially in ET. The findings of the thesis provided a consistent picture of the different role ET and storage have in drought evolution and in our modelling capabilities, coherently with recent literature, also on multi-year droughts. Moreover, these results emphasized the need for a holistic approach across the hydrological cycle for process understanding and model evaluation during droughts, with the ultimate goal of improving drought modelling for water resources management, disaster risk reduction, and climate change impact assessments

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

In electrophysiology, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate. In parallel, studies exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. Delivery and recording have independently led to great advances in neurobiology, however, their integration on a single chip would give complete insights into pathologies development and fundamental advancements in drug screening methods. In this work, we demonstrate how a microfluidic-MEA technology may be used to record both spontaneous and chemically induced activity in vitro. We propose a device that can deliver molecules to only a few chosen cells and detecting the response in cellular activity at multiple sites simultaneously. In addition, will be discussed how the adoption of nanoporous metamaterial in place of nanostructures might lower costs and speed up production. Furthermore, this same material, will be identified for the first time in this work as photoelectrical modulating material for eliciting electrogenic cells firing activity. Specifically, by converting NIR laser pulses into stimulatory currents, plasmonic metamaterials may be employed to induce action potentials. This method enables remote access to optical pacing with precise spatiotemporal control, allowing to be used as a valid alternative of the traditional genetic-based optical stimulation techniques. Therefore, in addition to pharmaceutical applications, these final characteristics may pave the way for a new generation of minimally invasive, cellular type-independent all-optical plasmonic pacemakers and muscle actuators

A tomographic approach to non-Markovian master equations

We propose a procedure based on symplectic tomography for reconstructing the unknown parameters of a convolutionless non-Markovian Gaussian noisy evolution. Whenever the time-dependent master equation coefficients are given as a function of some unknown time-independent parameters, we show that these parameters can be reconstructed by means of a finite number of tomograms. Two different approaches towards reconstruction, integral and differential, are presented and applied to a benchmark model made of a harmonic oscillator coupled to a bosonic bath. For this model the number of tomograms needed to retrieve the unknown parameters is explicitly computed.Comment: 15 pages, 2 figure

Reconstruction of Markovian Master Equation parameters through symplectic tomography

In open quantum systems, phenomenological master equations with unknown parameters are often introduced. Here we propose a time-independent procedure based on quantum tomography to reconstruct the potentially unknown parameters of a wide class of Markovian master equations. According to our scheme, the system under investigation is initially prepared in a Gaussian state. At an arbitrary time t, in order to retrieve the unknown coefficients one needs to measure only a finite number (ten at maximum) of points along three time-independent tomograms. Due to the limited amount of measurements required, we expect our proposal to be especially suitable for experimental implementations.Comment: 7 pages, 3 figure

Perfectly secure steganography: hiding information in the quantum noise of a photograph

We show that the quantum nature of light can be used to hide a secret message within a photograph. Using this physical principle we achieve information-theoretic secure steganography, which had remained elusive until now. The protocol is such that the digital picture in which the secret message is embedded is perfectly undistinguishable from an ordinary photograph. This implies that, on a fundamental level, it is impossible to discriminate a private communication from an exchange of photographs.Comment: 5 pages, 3 figures + appendix : 5 pages, 6 figure

An ontology-based model for SME network contracts

Even if collaboration is considered an effective solution to improve business strategies, SMEs often lack common principles and common forms of contractual coordination. Several policies implemented by E.U. have addressed the setup of a comprehensive SME policy framework. However, European institutions seem to have focused more on organizational devices to conduct business activities rather than on contractual forms of coordination. In April 2009, Italy adopted a law in network contract to promote the development of interfirm cooperation strategies to foster enterprises’ innovation and growth. Even if this law represents a novelty in Europe and may offer new challenges and hints, it still presents some lacks in its formulation. The current research aims at presenting the Italian law for network contract, by highlighting both its potentialities and its defects. A formal model to support the design of a SME network was proposed, by providing both an ontology-based model to help the definition of the contract in a structured way, and a basic workflow to identify the important phases of the network design, i.e., the feasibility study and the negotiation. In this way, the network rules and criteria for controlling the network members’ contributions are defined. Mathematical tools derived from performance optimization were exploited

flexible reconfiguration of avs rs operations for improved integration with manufacturing processes

Abstract The improvements in connectivity and data availability enable to fully integrate all the components of a production system. Manufacturing processes are frequently reconfigured over time, due to changes in lot sizes, process parameters and product customization. Despite this, warehousing operations are often disregarded: usually, automation systems for warehouses are set-up during the installation and their management is hardly ever reviewed. As a consequence, the manufacturing process is adapted to the capabilities of the warehousing system, rather than the other way round. To overcome this issue, this paper aims to propose a method capable to support an easy reconfiguration of warehousing operations based on the current state of the manufacturing process. The method is applied to an Autonomous Vehicle Storage and Retrieval Systems (AVS/RS), one of the most recent and promising automation technologies for warehouses. The proposed approach is based on both discrete-event simulation and analytical techniques and is applied to a real case of an Italian company

Sudden cardiac death in young athletes: Literature review of molecular basis

Intense athletic training and competition can rarely result in sudden cardiac death (SCD). Despite the introduction of pre-participation cardiovascular screening, especially among young competitive athletes, sport-related SCD remains a debated issue among medical personnel, sports communities and laypersons alike, and generates significant media attention. The most frequent cause of SCD is a hidden inherited cardiomyopathy, the athletes may not even be aware of. Predictive medicine, by searching the presence of pathogenic alterations in cardiac genes, may be an integrative tool, besides the conventional ones used in cardiology (mainly electro and echocardiogram), to reach a definitive diagnosis in athletes showing signs/symptoms, even borderline, of inherited cardiomyopathy/ channelopathy, and in athletes presenting family history of SCD and/or of hereditary cardiac disease. In this review, we revised the molecular basis of the major cardiac diseases associated to sudden cardiac death and the clinical molecular biology approach that can be used to perform risk assessment at DNA level of sudden cardiac death, contributing to the early implementation of adequate therapy. Alterations can occur in ion channel genes, in genes encoding desmosomal and junctional proteins, sarcomeric and Z-disc proteins, proteins for the cytoskeleton and the nuclear envelope. The advent of next generation sequencing (NGS) technology has provided the means to search for mutations in all these genes, at the same time. Therefore, this molecular approach should be the preferred methodology for the aforementioned purpose