1,720,964 research outputs found
Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology
No full textThe development of novel approaches to stimulate neuronal circuits is crucial to
understand the physiology of neuronal networks, and to provide new strategies
to treat neurological disorders.
Nowadays, chemical, electrical or optical approaches are the main exploited
strategies to interrogate and dissect neuronal circuit functions. However,
although all these methods have contributed to achieve important insights into
neuroscience research field, they all present relevant limitations for their use in
in-vivo studies or clinical applications. For example, while chemical stimulation
does not require invasive surgical procedures, it is difficult to control the
pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation
provides high temporal bandwidth, but it has low spatial resolution and it
requires implantation of electrodes; optical stimulation provides subcellular
resolution but the low depth penetration in dense tissue still requires the invasive
insertion of stimulating probes.
Due to all these drawbacks, there is still a strong need to develop new
stimulation strategies to remotely activate neuronal circuits as deep as possible.
The development of remote stimulation techniques would allow the combination
of functional and behavioral studies, and the design of novel and minimally
invasive prosthetic approaches.
Alternative approaches to circumvent surgical implantation of probes include
transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among
v
these methods, the use of magnetic and ultrasound (US) fields represents the
most promising vector to remotely convey information to the brain tissue. Both
magnetic and low-intensity US fields provide an efficient mean for delicate and
reversible alteration of cells and tissues through the generation of local
mechanical perturbations.
In this regard, advances in the mechanobiology research field have led to the
discovery, design and engineering of cellular transduction pathways to perform
stimulation of cellular activity. Furthermore, the use of US pressure fields is
attracting considerable interest due to its potential for the development of
miniaturized, portable and implantation-free US stimulation devices.
The purpose of my PhD research activity was the establishment of a novel
neuronal stimulation paradigm adding a cellular selectivity to the US stimulation
technology through the selective mechano-sensitization of neuronal cells, in
analogy to the well-established optogenetic approach. In order to achieve the
above mentioned goal, we propose the cellular overexpression of
mechanosensitive (MS) ion channels, which could then be gated upon the
application of an US generated pressure field. Therefore, we selected the bacterial
large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion
channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL
with its extensive characterization represents a malleable nano-valve that could
be further engineered with respect to channel sensitivity, conductance and gating
mechanism, in order to obtain the desired biophysical properties to achieve
reliable and efficient remote mechanical stimulation of neuronal activity.
In the first part of the work, we report the development of an engineered MscL
construct, called eMscL, to induce the heterologous expression of the bacterial
protein in rodent primary neuronal cultures. Furthermore, we report the
structural and functional characterization of neuronal cells expressing the eMscL
channel, at both single-cell and network levels, in order to show that the
functional expression of the engineered MscL channel induces an effective
vi
neuronal sensitization to mechanical stimulation, which does not affect the
physiological development of the neuronal itself.
In the second part of the work, we report the design and development of a water
tank-free ultrasound delivery system integrated to a custom inverted
fluorescence microscope, which allows the simultaneous US stimulation and
monitoring of neuronal network activity at single resolution.
Overall, this work represents the first development of a genetically mechanosensitized
neuronal in-vitro model. Moreover, the developed US delivery system
provides the platform to perform high-throughput and reliable investigation,
testing and calibration of the stimulation protocols.
In this respect, we propose, and envisage in the near future, the exploitation of
the engineered MscL ion channel as a mature tool for novel neuro-technological applications
Retinal and brain organoids: bridging the gap between in vivo physiology and in vitro micro-physiology for the study of Alzheimer’s diseases
Get PDFRecent progress in tissue engineering has led to increasingly complex approaches to investigate human neurodegenerative diseases in vitro, such as Alzheimer's disease, aiming to provide more functional and physiological models for the study of their pathogenesis, and possibly the identification of novel diagnostic biomarkers and therapeutic targets. Induced pluripotent stem cell-derived cortical and retinal organoids represent a novel class of in vitro three-dimensional models capable to recapitulate with a high similarity the structure and the complexity of the native brain and retinal tissues, thus providing a framework for better mimicking in a dish the patient's disease features. This review aims to discuss progress made over the years in the field of in vitro three-dimensional cell culture systems, and the benefits and disadvantages related to a possible application of organoids for the study of neurodegeneration associated with Alzheimer's disease, providing a promising breakthrough toward a personalized medicine approach and the reduction in the use of humanized animal models
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors
No full textWe describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Modulation of Neural Network Activity through Single Cell Ablation: An in Vitro Model of Minimally Invasive Neurosurgery
No full textThe technological advancement of optical approaches, and the growth of their applications in neuroscience, has allowed investigations of the physio-pathology of neural networks at a single cell level. Therefore, better understanding the role of single neurons in the onset and progression of neurodegenerative conditions has resulted in a strong demand for surgical tools operating with single cell resolution. Optical systems already provide subcellular resolution to monitor and manipulate living tissues, and thus allow understanding the potentiality of surgery actuated at single cell level. In the present work, we report an in vitro experimental model of minimally invasive surgery applied on neuronal cultures expressing a genetically encoded calcium sensor. The experimental protocol entails the continuous monitoring of the network activity before and after the ablation of a single neuron, to provide a robust evaluation of the induced changes in the network activity. We report that in subpopulations of about 1000 neurons, even the ablation of a single unit produces a reduction of the overall network activity. The reported protocol represents a simple and cost effective model to study the efficacy of single-cell surgery, and it could represent a test-bed to study surgical procedures circumventing the abrupt and complete tissue removal in pathological conditions
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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