1,720,975 research outputs found
Biophysical mechanisms of membrane perturbation and signal transduction produced by proteins and peptides
No full textMy primary research interest is focused on the field of cellular electrical activity, ranging from the ion channels that generates it, up to the study of intracellular processes regulating it, and new generation of drugs. For this purpose, during my Ph.D. I have learnt and improved different cutting-edge techniques, i.e. the patch-clamp technique, the fluorescence imaging, and the synthesis and use of model membranes. Moreover, to explore particular aspects of these molecular mechanisms and to overcome the issues raised during the investigations, non-conventional strategies were employed, even requiring the development of specific devices not commercially available.
In summary, my Ph.D. thesis is focused on two projects: the biophysical characterization of a particular class of membrane active peptides, and the modulation of visual phototransduction in vertebrate cones.
In the first project, I investigated the mechanism of membrane perturbation of cell-penetrating and antimicrobial peptides using the patch-clamp technique. Cell-penetrating peptides (CPPs) are short peptides that are able to cross the cell membrane via energy-dependent and/or independent mechanisms, with low toxicity and without the use of specific receptors. This ability is preserved even when CPPs are conjugated with a large cargo, thus representing an innovative pharmacological tool for the diffusion of large and hydrophilic drugs into the cells. Despite the mechanism of cellular uptake is still debated in literature, it has been proved that it can occur by either direct translocation or endocytosis. In the latter case, though, the cargo-peptide complex often remains trapped inside the endocytic vesicles and is not able to reach its therapeutic target. A possible solution to this problem could be found in another class of small peptides, similar to CPPs, i.e. the antimicrobial peptides (AMPs). AMPs are 12-50 amino acids long peptides, which represent an essential part in the innate immune system in most organisms. Indeed, they are among the first defensive molecules released during infections and their activity is direct thorough the membrane of bacteria, causing its destruction and consequently the death of the pathogen. Therefore, the ability of AMPs to disrupt biological membranes could be exploited to improve the CPPs escape from the endocytic vesicles in addition to, of course, their application as a novel class of antibiotics. The idea is to conjugate the CPP with a molecule that possess an antimicrobial activity, which can destroy the vesicle membrane, and help the complex to reach its target once it has been internalized in the cell. On this ground, the first project I carried out regards the study of a novel chimeric peptide, CM18-Tat11, composed of the antimicrobial peptide CM18 (a cecropin-mellitin hybrid peptide) linked to the cell-penetrating peptide Tat11 (derived from the basic domain of HIV-1 Tat protein). In particular, I investigated the membrane perturbing activity of this peptide (and of its elements) using the patch-clamp technique and operating under strictly physiological conditions. This study has been carried out by recording the ion current flowing through the channels formed by these peptides (if any), once inserted in the membrane of Chinese hamster ovary (CHO) cells. In these experiments, the peptides were applied to (and removed from) the extracellular CHO membrane in ~50 ms with a
computer-controlled microperfusion system. Therefore, besides assessing ion channel characteristics, the dynamics of pore formation and disaggregation could be precisely evaluated as well. I found that CM18-Tat11 produces a large and irreversible plasma membrane lysis, at concentration where CM18 and Tat11 give instead a nearly reversible membrane permeabilization and no perturbation, respectively. Furthermore, using the same method, I studied the biophysical characteristic of another antimicrobial peptide, called CM12, which sequence was obtained from the optimization of CM18. When applied on CHO, CM12 and CM18 produce voltage-independent membrane permeabilization, and no single-channel events were detected at low peptides concentration. These results indicate that both peptides form pores according to a toroidal model, in which the lipid layer bends continuously through the pore so that the core is formed by both lipid head groups and the peptides. Finally, I have studied the single-channels properties generated by the pore-forming peptide alamethicin (Alm) F50/5 and its [L-Glu(OMe)7,18,19] analog inserted in a natural membrane and in giant unilamellar vesicles (GUVs). The possibility to compare the channel activity in the precisely controlled lipid environment of GUVs, with the one recorded in a natural membrane, will open new possibilities in the biophysical characterization of the pores.
The second project of this thesis is focused on the study of the physiological role of the calcium sensor GCAP3 (guanylate cyclase activated protein 3) in the phototransduction cascade in zebrafish. I pursued this study simulating the over expressions and the knockdown of this protein, through the delivery of zGCAP3, or of its monoclonal antibody, into zebrafish cone cytoplasm, while recording their photorensponses with the patch-clamp technique. The proteins were administered inside the cone via the patch pipette thanks to an intracellular perfusion system developed in this thesis. This system allows the delivery of exogenous molecules inside the cell with a controlled timing, by expelling them with a small teflon tube inserted into the pipette lumen controlled by a microperfusion apparatus. Results indicated that the increase in the concentration in zGCAP3 did not altered significantly the light response, while the perfusion with the antibody anti-zGCAP3 caused the progressive fall of the dark current, together with the progressive slowing down of the flash response kinetics. The surprising lack of an effect of zGCAP3 incorporation, suggests that the endogenous number of zGCAP3 is saturating, therefore any further increase of this sensor is ineffective. However, the effects of the antibody can be explained as an inhibition of the target enzyme of zGCAP3, which is the guanylate cyclase (GC).
Finally, no experiments mentioned above would have been accomplished without the development of a “pressure-polishing” system, which makes it possible to modify the geometry of the patch-clamp pipette. The pipette shank (the final part of the pipette) is, in fact, very long and tapered, thus generating a high resistance to the passage of ions and molecules, and making very difficult to perfuse efficiently the cell with the internal perfusion. The pressure polishing setup I developed enlarged the patch pipette shank, using a calibrated combination of heat and air pressure. These pipettes minimized errors in membrane potential control and allowed the insertion of teflon tubes in the pipette lumen very close to its tip
Characterization of Zebrafish Green Cone Photoresponse Recorded with Pressure-Polished Patch Pipettes, Yielding Efficient Intracellular Dialysis.
No full textThe phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment. Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+
Remote Collaborative Childcare in the Workplace: Sharing Childcare with Colleagues during COVID-19 Emergency
No full textThe COVID-19 emergency and consequent lockdowns left many parents struggling to balance childcare and paid employment, exposing the crucial role of work-life balance policies and practices. As a consequence, co-production initiatives has seen a sharp rise, with the creation of many different grassroots activities, also related to childcare services. Although it is not clear whether these initiatives will continue after the pandemic, they provide an interesting laboratory to investigate digitally mediated co-production of services. This paper presents a case study of a grassroot initiative based on the co-production of childcare services within an organizational context. We report on how this initiative has been adapted during the COVID-19 emergency, discussing how the community of colleagues reacted to the new challenges of the pandemic, and the technological and organizational arrangements that have accompanied such changes. Results from interviews and focus groups revealed the mediating role that digital technology played in the co-production, presenting challenges and opportunities to working parents while dealing with a period of emergency and isolation. These findings suggest implications for organizations to promote remote collaborative childcare practices and support workers wellbeing and work-life balance, considering motivational and social aspects and the role of technology in fostering co-production practices
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
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
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
Pressure-Polished Borosilicate Pipettes are “Universal Sealer” Yielding Low Access Resistance and Efficient Intracellular Perfusion
No full textWhole-cell recording is the most widely used configuration of the patch recording technique, mainly because it allows to manipulate the intracellular environment while recording membrane current. However, the patch pipette tapered shank and the small tip opening give high access resistances and preclude efficient exchange between pipette solution and cell cytosol. Independently by the recording configuration, another problem of this technique is to gain consistently tight seals.
Here it is described a method to enlarge the pipette shank without affecting the tip opening diameter, through the calibrated combination of heat and air pressure, with a custom made inexpensive set-up. These pressure polished pipettes give small access resistances, and allow the accommodation of pulled quartz or plastic perfusion tubes very close to the pipette tip (to deliver exogenous molecules into the cytosol with a controlled timing). Finally, it is also described a method to consistently attain seals with pipettes made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes composed by many different lipid mixtures
- …
