Embedded Artificial Intelligence for Tactile Sensing

Electronic tactile sensing becomes an active research field whether for prosthetic applications, robotics, virtual reality or post stroke patients rehabilitation. To achieve such sensing, an array of sensors is used to retrieve human-skin like information, which is called Electronic skin (E-skin). Humans through their skins, are able to collect different types of information e.g. pressure, temperature, texture, etc. which are then passed to the nervous system, and finally to the brain in order to extract high level information from these sensory data. In order to make E-skin capable of such task, data acquired from E-skin should be filtered, processed, and then conveyed to the user (or robot). Processing these sensory information, should occur in real-time, taking in consideration the power limitation in such applications, especially prosthetic applications. The power consumption itself is related to different factors, one factor is the complexity of the algorithm e.g. number of FLOPs, and another is the memory consumption. In this thesis, I will focus on the processing of real tactile information, by 1)exploring different algorithms and methods for tactile data classification, 2)data organization and preprocessing of such tactile data and 3)hardware implementation. More precisely the focus will be on deep learning algorithms for tactile data processing mainly CNNs and RNNs, with energy-efficient embedded implementations. The proposed solution has proved less memory, FLOPs, and latency compared to the state of art (including tensorial SVM), applied to real tactile sensors data. Keywords: E-skin, tactile data processing, deep learning, CNN, RNN, LSTM, GRU, embedded, energy-efficient algorithms, edge computing, artificial intelligence

Gene Silencing Using Chitosan Based siRNA Delivery Systems in Cells and Animals

Le développement de vecteurs de livraison non viraux à des fins thérapeutiques a pris de l’ampleur dans les dernières années. Le chitosane est un polymère cationique naturel ayant la capacité de former des nanoparticules lorsqu’il est mélangé à des molécules polyanioniques comme les petits ARN interférents (pARNi). Les efforts pour identifier les paramètres moléculaires favorisant une bioactivité optimale n’ont pas été concluants en raison de différences expérimentales, d’un manque d’uniformité des protocoles de transfection, de la faible caractérisation du polymère et des différences au niveau des sources de chitosane utilisé. Cette thèse a été entreprise afin de répondre aux objectifs suivants: 1) Tester et valider l’efficacité de transfection de formulations, précédemment identifiées comme optimales pour la livraison d’ADN plasmidique, 2) Examiner l’influence des paramètres intrinsèques (dégrée de deacetylation (DDA), la masse moléculaire (Mn) et le ratio N:P) et extrinsèques (sérum, pH, force ionique et conditions de mélange) sur les caractéristiques physicochimiques des particules, leur internalisation dans les cellules, l’efficacité de silençage, la toxicité métabolique, la génotoxicité et l’hémocomptabilité, en utilisant une chimiothèque de chitosanes hautement caractérisés, et 3) Sélectionner des formulations ayant des caractéristiques optimales relatives à la taille, le potentiel zêta, l’intégrité des nanoparticules et la capacité de ces dernières à induire un silençage spécifique du gène en question tout en étant sécuritaire, et 4) Caractériser la biodistribution des nanoparticules, leurs toxicités et leurs potentiel de silençage génique suite à des injections intraveineuses chez la souris. Une étude initiale a démontré que le chitosane interfère avec l’extraction d’acide nucléique de cellules transfectées in vitro. Une méthode enzymatique simple et peu coûteuse a permis de récupérer l’ARN totale pour des applications moléculaires tel que la PCR en temps réel. De plus, cette étude a permis de réduire le biais (~ 10-15 %) associé aux nanoparticules adsorbées à la surface des cellules lors de mesures du niveau d’internalisation par cytométrie en flux. En outre, la digestion enzymatique du chitosane pourrait être effectuée en présence de guanidium, un agent chaotropique présent dans le tampon de lyse, démontrant ainsi l’efficacité et la simplicité de cette méthode. Avec la résolution de cet obstacle technique, nous avons sélectionné des formulations ayant démontré, auparavant, une efficacité de transfection élevée pour la livraison d’ADN plasmidique. Ces formulations ont été caractérisés pour leur taille, leur forme, leur potentiel surfacique (potentiel zêta), leur capacité de protéger les pARNi contre les nucléases et leur efficacité à transfecter, de façon non toxique, différentes lignées cellulaires. Les nanoparticules ainsi formées étaient sphériques et leur taille variait entre 40 et 100 nm. De plus, les résultats ont démontré que la protection contre les nucléases dépendait de la masse moléculaire et du ratio N:P. Par ailleurs, une haute efficacité (~80%) de silençage génique, en présence de sérum (10%), a pu être atteinte dans plusieurs lignées cellulaires. Pour la première fois, des nanoparticules avaient pu être obtenues à un faible ratio N:P marquant ainsi une différence frappante avec la littérature. Nos résultats ont pu démontrer la cause de ce bais favorisant la sélection de nanoparticules à haut ratio N :P testée dans la littérature. Dans la perspective de comprendre l’influence du degré de désacétylation (DDA) et de la masse moléculaire (Mn) du chitosane ainsi que du ratio N :P sur l’efficacité de transfection in vitro, la toxicité, la génotoxicité, l’hémocompatibilité et la biodistribution in vivo, une chimiothèque de chitosans hautement caractérisés a été produite à de différents DDA (98%, 92%, 80% et 72%) et Mn (5, 10, 40, 80 et 120 kDa) et mélangée avec des pARNi à des ratios N :P de 5 :1 et 30 :1. Les nanoparticules, ainsi formées, ont été caractérisées pour leurs tailles et potentiel surfacique en présence de 10 et 150 mM de sel. L’efficacité d’encapsulation (EE) et de transfection a été mesurée à pH 6.5 et 8 (EE) et à pH 6.5 et 7.4 respectivement. Les formulations les plus performantes ont été sélectionnées pour une caractérisation plus poussée de l’influence de la Mn et du ratio N:P sur l’internalisation des nanoparticules, l’activité métabolique cellulaire, la génotoxicité et l’efficacité de transfection in vitro en présence de sérum. L’hémocompatibilité et la biodistribution in vivo ont également été examinées pour différents Mn, ratios N :P et doses. Nos résultats ont démontré que l’internalisation des nanoparticules et l’efficacité de silençage étaient positivement corrélées à l’augmentation du potentiel surfacique, obtenu en augmentant le DDA et la Mn. Une longueur minimale de ~60-70 monomères (Mn ~10 kDa) était requise pour garantir une stabilité et un silençage en présence ou absence de sérum. L’efficacité de silençage a atteint des niveaux équivalents (~ 80-90%) à ceux du contrôle positif (DharmaFECT®) sans toxicité métabolique ou génotoxicité démontrant ainsi la supériorité de notre système comparativement aux lipides cationiques qui ont diminué l’activité métabolique des cellules. La présence de concentration croissante de sérum a négativement influencé la transfection in vitro. Nos résultats indiquent que l’influence négative du sérum est inversement proportionnelle à une augmentation du DDA, de la Mn et du ratio N :P. L’hémocompatibilité s’est révélée être dépendante de la dose, du DDA et de la masse moléculaire suggérant ainsi l’utilisation d’acide hyaluronique (HA), un polymère anionique et biocompatible, pour diminuer l’interaction avec les composantes du sang et améliorer la stabilité colloïdale. Les études de toxicité in vivo ont démontré que les nanoparticules de chitosane formulées à N:P 5 pourraient être tolérées jusqu’à une dose de 2.5mg/kg siRNA, tandis que celles revêtues de HA améliorent la tolérabilité par un facteur d’au moins 4. Contrairement aux nanoparticules lipidiques, les nanoparticules avec ou sans revêtement n’ont ni entraîné l’expression de cytokines pro-inflammatoires (ex. IL-1β, IL-6, TNF-α, IFN-γ et KC) ni l’augmentation de biomarqueurs sérologiques tels que l’ALT, AST, ALP, l’urée sanguine, et la créatinine. Une diminution des thrombocytes a été uniquement observée avec les formulations lipidiques soulignant ainsi des différences majeures avec le chitosane. L’analyse histopathologique des tissus et le suivit des masses corporelles ont confirmés le profil d’innocuité observé avec le chitosane. L’étude de biodistribution chez la souris démontre une accumulation spécifique de nanoparticules dans les tubules épithéliaux proximaux du rein où 40-50% de silençage a été observé, suggérant ainsi des applications potentielles du système au niveau des maladies rénales.----------ABSTRACT Research to develop safe and efficient non-viral gene delivery vectors for clinical applications has gained momentum in recent years. Chitosan is a natural cationic polymer with a characteristic property of self-assembly with small interfering RNA (siRNA) to form nanoparticles with high in vitro and in vivo transfection efficiencies. Previous efforts to identify molecular parameters favoring optimal bioactivity failed to produce conclusive results because of experimental discrepancies, lack of uniformity in transfection protocols, differences in chitosan sources, and poor characterization. In the light of these lacunae, The project presented in this thesis was carried out with the following objectives 1) Test and validate the transfection efficiency of formulations, previously identified as optimal for plasmid DNA, 2) Investigate the effect of intrinsic (DDA, Mn and N:P ratio) and extrinsic parameters (serum, pH, ionic strength and mixing conditions) on nanoparticle physicochemical characteristics, in vitro cell uptake, knockdown efficiency, metabolic toxicity, genotoxicity and hemocompatibility using a library of precisely characterized chitosans, and 3) Identify formulations with optimal characteristics with respect to size, surface charge, integrity, knockdown, toxicity followed by the characterization of their in vivo biodistribution; toxicity and gene knockdown potential following intravenous administration. An initial study demonstrated that chitosan interferes with column based extractions of total RNA from low cell numbers. The digestion of chitosan using a relatively simple and inexpensive enzymatic method permitted total recovery of high-quality RNA. In addition, surface bound chitosan was shown to bias flow cytometry data, evaluating nanoparticle uptake through fluorescently labeled siRNA. Treatment of cells using the chitosanase method reduced false positive events by around 10-15%. Surprisingly, enzymatic digestion could be performed in guanidium, a chaotropic agent, containing lysis buffer demonstrating the convenience of the method and allowing for the extracted RNA to be used in quantitative PCR experiment. With the technical hurdle solved, specific formulations based on designs parameters for plasmid DNA were characterized for their size, shape, surface charge, nuclease protection and ability to transfect different cell lines and produce non-toxic target specific knockdown. In contrast to plasmid DNA, nanoparticles formed with siRNA were all spherical, and their size ranged from 40-100 nm. For the first time, nanoparticles could be obtained at low N:P ratio in striking difference with the literature. Nuclease protection was found to be molecular weight dependent, and gene silencing in the presence of 10 % serum reached around 80%. This study demonstrated that nanoparticles formulated at low N:P ratio were able to form stable nanoparticles and induce target knockdown. In an attempt to understand the influence of chitosan molecular weight and degree of deacetylation on in vitro transfection efficiency, toxicity, genotoxicity, hemocompatibility and in vivo biodistribution, a library of precisely characterized chitosans was produced at different DDAs (98%, 92%, 80% and 72%) and Mn (5, 10, 40, 80 and 120 kDa). They were then mixed with siRNA at N:P ratios of 5:1 and 30:1, and nanoparticles were characterized for their size and surface charge in the presence of 10 and 150 mM salt. Encapsulation (EE) and transfection efficiencies were characterized at pH 6.5 and 8 for EE and pH 6.5 and 7.4 for in vitro transfection. Formulations were selected for further characterization of the influence of Mn and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection in the presence of increasing concentrations of serum. Hemocompatibility and in vivo biodistribution were also investigated for several Mn, N:P ratio, and dose. Nanoparticle uptake and gene silencing correlated positively with increased surface charge, which in turn was obtained at high DDA and high Mn. A minimum polymer length of ~60-70 monomers, or Mn of ~10kDa, was required for stability and in vitro knockdown in the presence or absence of serum. In vitro knockdown reached levels equivalent to the DharmaFECT® (~ 80-90%) with no metabolic toxicity or genotoxicity, the former in contrast to the lipid-based control which severely impaired metabolic activity. Serum had negative dose-dependent effects on biological performance, which correlated inversely with increased DDA, Mn and N:P. The poor in vitro performance above 50% serum concentration is believed to be multifactorial in cause and could not be elucidated. Despite the negative effect of serum on in vitro transfection efficiency, several reports have demonstrated in vivo efficacy. Hemocompatibility was found to be dose-dependent and increased with both Mn and DDA prompting the use of hyaluronic acid (HA), a biocompatible and negatively charged polymer, to coat nanoparticles for limited blood interaction and improved colloidal stability. Single ascending dose toxicity studies showed that uncoated chitosan-formulated at N:P 5 could be tolerated up to 2.5mg/kg siRNA dose, with nanoparticle coating improving tolerability by at least 4-folds. In contrast to commercially available, and liver-restricted lipid nanoparticles (LNPs), both uncoated and HA-coated did not induce pro-inflammatory cytokines such as. IL-1β, IL-6, TNF-α, IFN-γ, and KC, nor had obvious effects on the liver (ALT, AST, ALP) and kidney (BUN, Creatinine) biomarkers. Thrombocytopenia was only observed with the LNPs formulated with a native siRNA sequence confirming previous reports and highlighting differences with chitosan. Repeated administration and histopathological analysis confirmed the safety profile of chitosan versus LNPs. In vivo biodistribution in mice showed accumulation of nanoparticles in the proximal epithelial tubules of the kidney, where 40-50% functional knockdown was observed and confirmed using multiple techniques, suggesting potential applications in kidney diseases

Élaboration d’un bioessai à haut débit pour la découverte de nouveaux ligands péptidiques chez les végétaux

Suite au projet de séquençage du génome d’Arabidopsis thaliana, plus de 400 récepteurs de types serine/thréonine kinases (Protein Receptor Kinase ou PRK) ont été prédits. Par contre, seulement sept paires de récepteurs/ligands ont été caractérisées jusqu’à présent par des techniques de biochimie et d’analyse, de mutants. Parmi ceux-ci figurent les PRK : BRI1, CLV1, SRK, SR160, Haesa-IDA et PEPR1 qui jouent un rôle important dans le développement, l’auto-incompatibilité sporophytique et les mécanismes de défense. Le but de mon projet de maîtrise était de développer un bioessai à haut débit qui permettra la découverte de ligands peptidiques. Le bioessai utilisera des PRK chimériques composés du domaine extracellulaire (l’ectodomaine) de la PRK à l’étude fusionnée au domaine intracellulaire d’une PRK qui agira comme rapporteur. Deux stratégies sont présentement développées dans notre laboratoire : la première consiste à fusionner la PRK à l’étude avec le domaine intracellulaire (l’endodomaine) du récepteur tyrosine kinase animal EGFR (Epidermal Growth Factor Receptor). Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, une transphosphorylation de l’endodomaine (le domaine kinase) serait détectable. La seconde stratégie utilise l’endodomaine du récepteur BRI1, un récepteur répondant aux brassinostéroïdes. Suite à l’interaction avec une fraction protéique contenant un ligand correspondant à la PRK étudiée, cette fois-ci nous devrions être en mesure de mesurer l’activation d’un gène rapporteur répondant normalement à une activation par les brassinostéroïdes.The complete sequence of the genome of Arabidopsis thaliana was achieved in year 2000 and has resulted in the prediction of more than 400 receptor serine/threonine kinase or Plant Receptor Kinase (PRK). Despite this tremendous work, only seven pairs of ligand/receptor have been characterized through conventional techniques such as mutant analysis and biochemical characterization. These receptors have been found to play an important role in plant defense (SP160), development (BRI1, CLV1) and sporophytic autoincompatibility (SRK). The aim of the project was to develop a high throughput bioassay in order to find new ligands for known receptors. In order to do so, the bioassay will use chimeric protein technology, by fusing the ectodomain of a receptor to a known endodomaine. The latter will play the role of a reporter. Two strategies were developed in our laboratory and are being tested. The first strategy is to fuse the ectodomain of an unknown PRK to the phylogeneticaly unrelated kinase domain of the animal Epidermal Grown Factor Receptor (EGFR). When tested with a crude protein extract containing the specific ligand of the unknown PRK, a transphosphorylation should occur and be detected. The second strategy will use the endodomain of BRI1 as a reporter, a receptor responding to the brassinosteroid phytohormone, which will relay the message to a second construct used as a reporter gene once the ligand has bound the PRK ectodomain fused to the BRI1 endodomain

Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR

Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines

Ionizable lipid nanoparticles for in utero mRNA delivery.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing

Hand-Object Interaction: From Human Demonstrations to Robot Manipulation

Human-object interaction is of great relevance for robots to operate in human environments. However, state-of-the-art robotic hands are far from replicating humans skills. It is, therefore, essential to study how humans use their hands to develop similar robotic capabilities. This article presents a deep dive into hand-object interaction and human demonstrations, highlighting the main challenges in this research area and suggesting desirable future developments. To this extent, the article presents a general definition of the hand-object interaction problem together with a concise review for each of the main subproblems involved, namely: sensing, perception, and learning. Furthermore, the article discusses the interplay between these subproblems and describes how their interaction in learning from demonstration contributes to the success of robot manipulation. In this way, the article provides a broad overview of the interdisciplinary approaches necessary for a robotic system to learn new manipulation skills by observing human behavior in the real world

Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Mohamad Alameh, Myriam Jean, Diogo DeJesus, Michael D Buschmann, Abderrazzak Merzouki1Institute of Biomedical Engineering, Department of Chemical Engineering, École Polytechnique, Station Centre-ville, Montréal, QC, CanadaAbstract: Chitosan, a well known natural cationic polysaccharide, has been successfully ­implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR.Keywords: chitosan, chitosanase, siRNA, DPP-IV gene silencing, RIN, qPC

Vaccine Technologies and Platforms for Infectious Diseases: Current Progress, Challenges, and Opportunities

Vaccination is a key component of public health policy with demonstrated cost-effective benefits in protecting both human and animal populations. Vaccines can be manufactured under multiple forms including, inactivated (killed), toxoid, live attenuated, Virus-like Particles, synthetic peptide, polysaccharide, polysaccharide conjugate (glycoconjugate), viral vectored (vector-based), nucleic acids (DNA and mRNA) and bacterial vector/synthetic antigen presenting cells. Several processes are used in the manufacturing of vaccines and recent developments in medical/biomedical engineering, biology, immunology, and vaccinology have led to the emergence of innovative nucleic acid vaccines, a novel category added to conventional and subunit vaccines. In this review, we have summarized recent advances in vaccine technologies and platforms focusing on their mechanisms of action, advantages, and possible drawbacks