Differential resistance to cell entry by porcine endogenous retrovirus subgroup A in rodent species

Background: The risk of zoonotic infection by porcine endogenous retroviruses (PERV) has been highlighted in the context of pig-to-human xenotransplantation. The use of receptors for cell entry often determines the host range of retroviruses. A human-tropic PERV subgroup, PERV-A, can enter human cells through either of two homologous multitransmembrane proteins, huPAR-1 and huPAR-2. Here, we characterised human PARs and their homologues in the PERV-A resistant rodent species, mouse and rat ( muPAR and ratPAR, respectively). Results: Upon exogenous expression in PERV-A resistant cells, human and rat PARs, but not muPAR, conferred PERV-A sensitivity. Exogenously expressed ratPAR binds PERV-A Env and allows PERV-A infection with equivalent efficiency to that of huPAR-1. Endogenous ratPAR expression in rat cell lines appeared to be too low for PERV-A infection. In contrast, the presence of Pro at position 109 in muPAR was identified to be the determinant for PERV-A resistance. Pro109. was shown to be located in the second extracellular loop (ECL2) and affected PERV-A Env binding to PAR molecules. Conclusion: The basis of resistance to PERV-A infection in two rodent species is different. Identification of a single a. a. mutation in muPAR, which is responsible for mouse cell resistance to PERV-A highlighted the importance of ECL-2 for the viral receptor function

Forward Modeling and validation of a new formulation to compute self-potential signals associated with ground water flow

The classical formulation of the coupled hydroelectrical flow in porous media is based on a linear formulation of two coupled constitutive equations for the electrical current density and the seepage velocity of the water phase and obeying Onsager's reciprocity. This formulation shows that the streaming current density is controlled by the gradient of the fluid pressure of the water phase and a streaming current coupling coefficient that depends on the so-called zeta potential. Recently a new formulation has been introduced in which the streaming current density is directly connected to the seepage velocity of the water phase and to the excess of electrical charge per unit pore volume in the porous material. The advantages of this formulation are numerous. First this new formulation is more intuitive not only in terms of establishing a constitutive equation for the generalized Ohm's law but also in specifying boundary conditions for the influence of the flow field upon the streaming potential. With the new formulation, the streaming potential coupling coefficient shows a decrease of its magnitude with permeability in agreement with published results. The new formulation has been extended in the inertial laminar flow regime and to unsaturated conditions with applications to the vadose zone. This formulation is suitable to model self-potential signals in the field. We investigate infiltration of water from an agricultural ditch, vertical infiltration of water into a sinkhole, and preferential horizontal flow of ground water in a paleochannel. For the three cases reported in the present study, a good match is obtained between finite element simulations performed and field observations. Thus, this formulation could be useful for the inverse mapping of the geometry of groundwater flow from self-potential field measurements

Cell entry and exit of porcine endogenous retrovirus A: receptors and release inhibitor

Following the discovery that porcine endogenous retrovirus (PERV) can infect human cells, the potential risk of a zoonotic infection by PERV has been a major obstacle in the xenotransplantation field. The aim of this thesis is to gain a better understanding of PERV biology, so as to help assess and reduce the risk of PERV zoonosis. PERV subgroup A can enter human cells through two human PERV-A receptors (huPAR-1 and -2). To determine critical regions in the receptor for PERV-A infection, chimeric receptors between huPAR-2 and the non functional murine PAR (muPAR) have been analysed. A single amino acid difference (amino acid 109) was found responsible for the inability of muPAR to mediate PERV-A binding and infection. These results were then applied to the evaluation of PERV infection of non-human primates (NHP). NHP could represent an ideal animal model for assessing the risk of zoonosis following long-term exposure to porcine material. However, PERV does not infect NHP cells with the same efficiency as it does human cells. The data presented in this thesis suggests that in some NHP species the poor infectivity is due to mutation of the same critical amino acid (a.a.109) described for muPAR. However, African green monkey cells express two functional receptors and other mechanisms are likely to be responsible for the low susceptibility to PERV-A infection. Secondly, I evaluated the effect of a release inhibitor as a possible strategy to reduce PERV dissemination from pig cells. Human tetherin can inhibit retrovirus production from cells. I showed that overexpression of human and newly cloned porcine tetherin in pig cells can reduce the release of PERV. My data suggests that tetherin-expressing transgenic pigs could represent a safer donor in xenotransplantation

“Let the algorithm decide”: is human dignity at stake?

The goal of this article is to argue that the debate regarding algorithmic decision-making and its impact on fundamental rights is not well-addressed and should be reframed in order to allow for adequate regulatory policies regarding recent technological developments in automation. A review of the literature on algorithms and an analysis of Articles 6, IX and 20 of the Brazilian Federal Law n° 13.709/2018 (LGPD) lead to the conclusion that claims that algorithmic decisions are unlawful because of profiling or because they replace human analysis are imprecise and do not identify the real issues at hand. Profiles are nothing more than generalizations, largely accepted in legal systems, and there are many kinds of decisions based on generalizations which algorithms can adequately make with no human intervention. In this context, this article restates the debate about automated decisions and fundamental rights focusing on two main obstacles: (i) the potential for discrimination by algorithmic systems and (ii) accountability of their decision-making processes. Lastly, the arguments put forward are applied to the current case of the covid-19 pandemic to illustrate the challenges ahead

Pharmacological studies of novel antitumoral compounds

Over the years biomedical research was focused on the development of new anticancer agents able to selectively target cancer cells at low concentration efficacy. Based on the idea that oncogenes and tumour suppressor genes are a critical force in the malignant transformation of cells, research efforts have focused on developing drugs that directly target these genes. In the first study we focused on B-cell acute lymphoblastic leukaemia (B-ALL), that is one of the most common paediatric malignant disorders characterized by an accumulation of B-cell blasts reminiscent of normal stages of differentiation and by infiltration of various extramedullary sites. Excessive cell proliferation induced by aberrant entry into the cell cycle is considered an hallmark of cancer. Recent findings have revealed that CDK4/6 and its regulatory subunit cyclin D1 are potentially oncogenes and are overexpressed in a diverse set of human cancers, including B-cell acute lymphoblastic leukemia (B-ALL). Moreover, CDK6 is essential for MLL-rearranged leukemias. These findings suggest that CDK4/6 may be effective targets for therapeutic intervention. To test this possibility, we have inhibited CDK4 and CDK6 in four cell lines of B-ALL, characterized by different genetic rearrangements, using Ribociclib, an orally bioavailable, small molecule inhibitor of both CDK4 and CDK6. At low concentration, Ribociclib potently inhibit CDK4/6 without induction of apoptosis; therefore, standard treatment for newly diagnosed childhood B-ALL patients includes glucocorticoids (GC) treatment, but the molecular basis of GC sensitivity and resistance remains largely unknown; for this reason we have tested if Ribociclib in combination with Dexamethasone, a glucocorticoid that is currently used as antitumour agent in the treatment of leukemia, may lead to synergistic killing of leukemia cells. B-ALL patients that respond poorly to glucocorticoid therapy when diagnosed are usually predicted to undergo relapse. Therefore, understanding the biological mechanisms underlying this poor responsiveness is crucial for the development of more effective therapies

WHO collaborative study to assess the suitability of the 1st International Standard and the 1st International Reference Panel for antibodies to Ebola virus

A WHO international collaborative study was undertaken to evaluate preparations of Ebola virus disease (EVD) convalescent plasmas for their suitability to serve as the WHO 1st International Standard (IS) and the WHO 1st International Reference Panel (IRP) for Ebola virus antibodies for use in the standardization and control of assays. The study involved participants testing the convalescent plasma sample preparations and additional monoclonal antibody samples in a blinded manner alongside the WHO International Reference Reagent (NIBSC code 15/220) using anti-EBOV assays established in their laboratories. The candidate 1st IS for Ebola virus antibodies (study sample code 92, NIBSC 15/262) consists of ampoules containing the freeze-dried equivalent of 0.5 mL pooled convalescent plasma obtained from six Sierra Leone patients recovered from EVD. The candidate 1st IRP of anti-Ebola virus convalescent plasmas (NIBSC 16/344) consists of freeze-dried preparations of single donations of convalescent plasma obtained from four patients and one healthy blood donor. Each panel member is an ampoule containing the equivalent of 0.25mL plasma. All convalescent plasmas are confirmed PCR-negative for Ebola virus and underwent, along with the negative plasma, solvent detergent (SD) treatment prior to their development into candidate WHO biological reference materials. In this collaborative study, 17 laboratories from 4 countries used a range of live Ebola virus neutralization assays, pseudotyped virus neutralisation assays and enzyme immunoassays to test the collaborative study samples. Surface plasmon resonance and Western blot assessments were also undertaken. The study found that the candidate International Standard has the highest absolute titre among the convalescent plasma samples, although the geometric mean titres of all the convalescent plasmas fall within ~5-fold of each other. The potencies of three of the convalescent samples fall near the detection limit of some assays. This study also demonstrated that the agreement between laboratories for potencies relative to the candidate International Standard represents an improvement compared to the agreement in absolute titres; however, there is poor agreement between relative potencies for some assays. The results obtained from accelerated thermal degradation studies at 1year indicate that the candidate IS is stable and suitable for long-term use. The results of the collaborative study indicate the suitability of the candidates to serve as WHO reference materials and it is proposed that 15/262 is established as the WHO 1st IS for EBOV antibodies with an assigned potency of 1.5 IU/mL when reconstituted as directed in the instructions for use. It is also proposed that 16/344 is established as the WHO 1st IRP of anti-EBOV convalescent plasmas with panel member code 95 (NIBSC 15/280) assigned a unitage of 1.1 IU/mL when reconstituted as directed in the instructions for use. The other panel members have not been assigned a unitage. The implementation and use by laboratories of the proposed WHO reference materials for EBOV antibodies will facilitate the characterization of the factors that contribute to assay variability and standardization of results across assays and laboratorie

Early Potent Protection against Heterologous SIVsmE660 Challenge Following Live Attenuated SIV Vaccination in Mauritian Cynomolgus Macaques

Background: Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation. Methodology/Principal Findings: Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I /II haplotype or TRIM5α polymorphism and study outcome was identified. Conclusion/Significance: This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system

Sviluppo di un set-up di misura innovativo per lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS

Il lavoro contenuto in questa tesi riguarda lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS, in particolare in switch dielectric-less. Viene in oltre presentato un set-up di misura innovativo per l’attuazione dei dispositivi suddetti, indipendentemente dalla tensione elettrostaticaopenEmbargo per motivi di priorità nella ricerca (previo accordo con terze parti

Etude du transcriptome kératinocytaire au cours du programme de différenciation de l'épiderme

Au sein de l'épiderme, la fonction de barrière est assurée par la partie la plus superficielle, la couche cornée, constituée de cornéocytes, des cellules mortes très cohésives entourées d'une matrice lipidique. Un processus complexe de mort cellulaire programmée, la cornification, aboutit à la transformation des dernières cellules vivantes, les kératinocytes granuleux, en cornéocytes dépourvus d'activité transcriptionnelle et traductionnelle. Les kératinocytes granuleux constituent donc à la fois le stade ultime de la différenciation épidermique, mais aussi celui où sont produits les différents acteurs de la cornification et de la desquamation, donc de la fonction barrière. Très peu d'études moléculaires systématiques ont été consacrées à l'épiderme humain, et seulement quatre facteurs de transcription spécifiques requis pour la fonction barrière ont été identifiés. Le travail présenté ici combine plusieurs techniques à grande échelle afin d'identifier de nouveaux gènes impliqués dans les étapes tardives de ce programme. Un procédé de purification de cellules à partir d'épiderme humain normal a été développé afin d'obtenir des fractions enrichies en kératinocytes de différents stades de différenciation. Ce procédé, couplé à la technique des ORESTEs, a permis dans un premier temps de produire 22 000 séquences et d'identifier 3387 gènes exprimés dans la couche granuleuse, parmi lesquels de nouveaux marqueurs de la différenciation impliqués dans diverses fonctions (protéine structurale, métabolisme et transport des lipides, protéase et inhibiteurs, etc.) Les profils d'expression génique des kératinocytes granuleux ont également été comparés avec ceux des kératinocytes basaux au moyen de puces à ADN pangénomiques, mettant en évidence 200 candidats. Afin d'identifier des facteurs de transcription supplémentaires, une recherche bioinformatique de sites de fixation conservés évolutivement a été réalisée sur les promoteurs de 52 marqueurs de la différenciation. Les résultats issus de ces trois techniques ont été combinés et analysés afin de réduire à 300 gènes le nombre de candidats à valider. Pour 155 d'entre eux, leur profil d'expression au sein de l'épiderme a pu être analysé par PCR quantitative, permettant d'identifier 49 nouveaux marqueurs de la différenciation. De plus, sur 94 gènes quantifiés codant pour des facteurs de transcription, 37 apparaissent positivement ou négativement régulés, suggérant un rôle dans le contrôle des étapes tardives du programme de différenciation. Ces résultats ouvrent de nombreuses voies et constituent donc une avancée importante pour la compréhension des mécanismes complexes mis en jeu dans ce réseau de régulation génique, tant dans le cadre physiologique que pour certaines pathologies caractérisées par une altération de la différenciation, comme dans le cas du psoriasis ou des ichthyoses.The barrier function of the epidermis is provided by its outermost part, the cornified layer, formed by corneocytes, strongly interconnected dead cells surrounded by a lipid-rich extracellular environment. A complex programmed cell death, named cornification, turns the last living cells, granular keratinocytes, into dead corneocytes devoid of transcriptional and translational activity. Since all the constituents of corneocytes as well as the enzymes regulating their release leading to the desquamation are produced by granular keratinocytes, they do not only represent the last step of epidermal differentiation, but also its climax. Very few large-scale studies have been performed on human epidermis, and until now only four transcription factors required for the barrier function were described. The present work combines several large-scale techniques in order to identify new genes, especially transcription factor (TF)-coding genes, potentially involved in the last step of epidermal differentiation. Based on a purification process using iterative trypsinizations of chilled epidermis fragments, we recovered cell fractions enriched in keratinocytes from various differentiation states. This process, used together with the ORESTEs technique, allowed the production of more than 22000 sequences corresponding to 3387 genes expressed in the granular layer, including new differentiation markers involved in various functions (structural protein, lipid metabolism and transport, protease and inhibitors, etc). Gene expression profile of granular keratinocytes was also compared with basal keratinocytes' one using pangenomic microarrays, highlighting 200 candidates. To identify some additional TF-coding genes, a study of conserved TF binding site was performed by bioinformatics on the promoters of 52 differentiation markers. The results produced by these three techniques were combined and analyzed to select 300 candidates to be validated by real-time PCR. Among them, 155 could be reliably quantified, including 49 new differentiation-associated genes. Moreover, 37 of 94 TF-coding genes appeared to be up- or downregulated in granular keratinocytes, suggesting a function in the regulation of the differentiation. These advances made in the field of normal keratinocyte differentiation should improve the knowledge of the complex regulatory networks driving the formation of the epidermal barrier, and may also contribute to the deciphering of complex diseases such as atopic dermatitis, psoriasis and ichthyoses, which are characterized by deregulated differentiation programs