Proteins provide evidence that a given gene is expressed, and machine learning algorithms can be applied to various proteomics problems in order to gain information about the underlying biology. This dissertation applies machine learning algorithms to proteomics data in order to predict whether or not a given peptide is observable by mass spectrometry, whether a given peptide can serve as a cell penetrating peptide, and then utilizes the peptides observed through mass spectrometry to aid in the structural annotation of the chicken genome. Peptides observed by mass spectrometry are used to identify proteins, and being able to accurately predict which peptides will be seen can allow researchers to analyze to what extent a given protein is observable. Cell penetrating peptides can possibly be utilized to allow targeted small molecule delivery across cellular membranes and possibly serve a role as drug delivery peptides. Peptides and proteins identified through mass spectrometry can help refine computational gene models and improve structural genome annotations.

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles). The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I). Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV. The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency. Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs. Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The inflammatory arthritis Ankylosing Spondylitis (AS) is linked to the human leucocyte antigen HLA-B27. HLA-B27 is thought to drive AS because it misfolds during assembly in the endoplasmic reticulum (ER), inducing ER cell stress. Modulating HLA-B27 folding in the ER is therefore a therapeutic target pathway. The recent discovery of polymorphisms in the ER-resident peptidase ERAP1 that can impact on HLA-B27 and AS, makes ERAP1 one such target. Exosomes are small, typically 50-200 nm sized particles, formed in the endosomal recycling pathway, which can be released into the extracellular environment. Exosomes have a wide range of biological activities depending on the cell type of origin, and on the delivered cargo, which can include bio-active proteins, lipids, mRNA and miRNA. There is interest in the use of exosomes as drug delivery agents. Here, exosomes were studied as a delivery agent to modulate ERAP1, as a potential therapeutic tool for the treatment of AS. Exosomes, isolated from cell lines including CEM and Jurkat (T cell lineage), Jesthom (B cell lineage), U937 (monocyte lineage) and the epithelial HeLa cell line, were characterized by nanoparticle tracking analysis, flow cytometry and immunoblotting using markers including CD9, CD63, CD81 and TSG101. Differential expression of these markers in the immune cell lines indicated the complexity of defining exosomes. EVs were then tested using cell penetrating peptides, electroporation, lipid transfection and sonication for their ability to load FITC-siRNA or FITC-antibody as cargo. Significantly, post-loading RNase A or trypsin incubation demonstrated that many techniques do not lead to efficient cargo loading of exosomes. Sonication proved the most effective technique, with up to 30% efficiency. Loading of exosomes with ERAP1-targetted siRNA did not however lead to notable ERAP1 inhibition. The data indicates that external loading of exosomes with cargo remains a significant challenge in developing exosomes as therapeutic tools.

Les « cell penetrating peptides » (CPPs) sont des vecteurs peptidiques capables de délivrer diverses molécules (acides nucléiques, protéines, peptides, petites molécules, etc.) à l'intérieur des cellules de mammifères. Notre laboratoire a élaboré le vecteur amphipatique secondaire CADY capable de transporter, indépendamment de toute voie d'endocytose, des molécules thérapeutiques telles que les petits ARN interférents (siRNA). En effet, leur potentiel thérapeutique réside dans leur capacité à inhiber de manière spécifique l'expression de protéines dérégulées dans un cadre pathologique.Le sujet de ma thèse s'est centré sur la caractérisation et l'optimisation des complexes CADY/siRNA. Au cours de mes travaux, nous avons pu mettre en évidence que CADY adoptait une structure en hélice alpha en présence du siRNA ce qui conduit à la formation des nanoparticules. Notre étude a eu pour but d'optimiser la séquence de CADY et de contrôler sa formulation pour permettre le transfert de notre système de vectorisation d'une application in cellulo à une application in vivo. En premier lieu, nous avons mené une étude de relations structure-activité avec six peptides analogues de CADY, en réalisant des mutations sur les résidus tryptophanes (PSF1, PSF2, PSF3 et PSW) et sur la zone initiatrice de l'hélice alpha (PG9, PG16). L'analyse approfondie de ces analogues a permis de confirmer que la limitation du caractère amphipathique et du polymorphisme structural des vecteurs conduisaient à une réduction de l'efficacité d'internalisation. Parmi les 6 analogues, seules les nanoparticules à base de PG9 et PG16 présentent des résultats in cellulo comparables à ceux obtenus avec les nanoparticules CADY/siRNA. A ce jour, la séquence primaire de CADY étant la plus adaptée pour la transfection de siRNA, nous avons établi une procédure de formulation standardisée permettant un autoassemblage CADY/siRNA reproductible et homogène, dont la taille moyenne est de 106 ± 31 nm et l'indice de polydispersité de 0,357 ± 0,053. De plus, nous avons mis en place une procédure d'extrusion/lyophilisation afin de stocker les nanoparticules sous forme de poudre. Celle-ci peut être resuspendue en milieu aqueux sans modifications des propriétés colloïdales des nanoparticules ni de leur capacité de transfection.Dans le but d'améliorer la spécificité tissulaire et la biodisponibilité des nanoparticules CADY/siRNA pour une application in vivo, nous avons greffé des motifs de ciblage (YIGSR-S) et de furtivité (PEG) sur la séquence de CADY. L'ajout de ces deux entités, de natures très différentes, ne modifie que faiblement les caractéristiques physico-chimiques (ex. taille moyenne des complexes) et biologiques (transfection cellulaire) des nanoparticules. Ces résultats sont très encourageants pour le développement de nanoparticules dites de 3ème génération, sur lesquelles on peut greffer plusieurs sortes de molécules d'intérêts (ciblage, polymère, agent de contraste etc.).L'ensemble des résultats obtenus au cours de ma thèse marque un réel progrès dans l'optimisation de la formulation du vecteur CADY, et nous incitent à exploiter davantage son potentiel pour le transfert de siRNA in vivo. / Cell penetrating peptides (CPPs) are short peptides that can enter many cell types and transduce into cells a wide range of molecular therapeutics (nucleic acid, proteins, peptides, small molecules, etc.). Our laboratory has developed the secondary amphipathic peptide CADY able to promote the transport of small interfering RNA (siRNA) independently of all endocytotic pathways. Indeed, siRNA therapeutic interest lies on its ability to inhibit specifically deregulated proteins in the context of pathology. The subject of my thesis focused on the characterization and optimization of CADY/siRNA complexes. During my work, we have been able to show that CADY adopts a helical structure while interacting with the siRNA leading to the formation of nanoparticles. The goal of my study was to optimize CADY sequence and control its formulation to consider the transferring from an in cellulo to an in vivo application of our vectorization system. First, we conducted a structure-activity study with six analogues by mutating CADY on tryptophane residues (PSF1, 2, 3 and PSW) and in the area initializing helical structure (PG9, PG16). A thorough analysis of these analogues has confirmed that the limitation of the amphipathic character and structural polymorphism is directly related to the reduction of internalization efficiency of our CPPs. Among the six analogues, only PG16/siRNA and PG9/siRNA nanoparticles show in cellulo results equal to those obtained with CADY/siRNA. Based on the fact, that CADY is the most suitable vector for the transfection of therapeutic molecules such as siRNA, we have established a standard formulation procedure to obtain reproducible and homogeneous CADY/siRNA complexes with an average size of 106 ± 31 nm and a polydispersity index of 0.357 ± 0.053. In addition, we have implemented an extrusion/lyophilization step to allow nanoparticle storage as powder, which can be re-suspended in an aqueous solution without losing their colloidal and transfection properties.In order to improve tissue specificity and bioavailability of CADY/siRNA nanoparticles for an in vivo application, we have grafted ether a targeting sequence (YIGSR-S) or a stealth motif (PEG) to the CADY sequence. These two entities of very different nature provoke only few changes in the physicochemical (e.g. average size) and biological (cell transfection) characteristics of the nanoparticles formed with a siRNA. These results are very encouraging for the development of the so-called 3rd nanoparticle generation which includes several kinds of molecules (targeting, polymer, contrast agent etc.).These outcomes mark the real progress in CADY formulation optimization, and encourage us to further exploit its potential for the in vivo transfer of siRNA.

<p>Pathogenic bacteria present a critical threat to modern medicine. Therapeutic strategies to target and eliminate resilient bacteria are not advancing at the same rate as the emergence of bacterial resistance. An associated urgent concern regarding antibiotic resistance is the existence and proliferation of intracellular bacteria, which find refuge from bactericidal mechanisms by hiding within mammalian cells. Therefore, many once-successful antibiotics become ineffective through the development of resistance, or through failure to reach intracellular locations in therapeutic concentration. To overcome these challenges, the covalent combination of a conventional antibiotic with an antibiotic, cell-penetrating peptide was explored to develop dual-action antibiotic conjugates. </p> <p>Herein, we utilized a strategy in conjugating the antibiotics by a cleavable linkage to cationic amphiphilic polyproline helices (CAPHs) to improve vancomycin and linezolid antibiotics. This approach enables the conjugate to penetrate cells and deliver two potent monomeric antimicrobial drugs. The vancomycin-CAPH conjugate, <b>VanP14S</b>, showed enhanced mammalian cell uptake compared to vancomycin, a poor mammalian cell-penetrating agent; and <b>VanP14S</b> was capable of cleaving and releasing two antibiotics under mimicked physiological conditions. Enhanced antibacterial activity was observed against a spectrum of Gram-positive and Gram-negative pathogens, including drug-resistant strains. Further investigation revealed that this conjugate’s bactericidal activity was not entirely the result of significant membrane perturbation such as a lytic mode of action. Mammalian cell toxicity and red blood cell lysis were insignificant at relevant bactericidal concentrations below 20 µM. The current results suggest an enhanced binding to the peptidoglycan of bacteria, the target of vancomycin, although more work is needed to justify this claim. Preliminary results on <b>VanP14GAPS</b>, a conjugate with a more rigid CAPH, convey similar activity to <b>VanP14S; </b>however,<b> </b>moderate increases in red blood cell lysis and cytotoxicity were observed. </p> <p>Regarding the <b>LnzP14</b> conjugate, preliminary data reveal that the conjugate has Gram-negative activity against <i>Escherichia coli</i>, whereas linezolid is ineffective in killing Gram-negative bacteria. This conjugate showed significant enhancement in cellular uptake compared to the CAPH, and the release of linezolid and CAPH in physiological conditions was confirmed. Overall, arming a conventional antibiotic with an antimicrobial, cell-penetrating peptide appears to be a powerful strategy in providing novel antibiotic conjugates with the propensity to overcome the limitations in treating challenging pathogens.</p>

Over the years, biological drugs have evolved and have made breakthroughs in diseases associated with extracellular proteins. However, intracellular proteins that cause disease progression are still largely inaccessible. Examples of diseases that are caused by an intracellular aggregation of proteins are neurodegenerative diseases such as Parkinson's disease, Huntington's disease (HD), and Alzheimer's disease (AD). The purpose of the work is to find a strategy to reach the neurons intracellularly. The goal is to be able to design a biological drug that enters the neuron by investigating different uptake mechanisms. A systematic review of 43 published studies was reviewed, and the results could be obtained. All result presents data from different receptors, cell-penetrating peptides, and adeno-associated viruses (AAV) that were examined. It showed that there are advantages and disadvantages with all the uptake mechanisms. There are risks of side effects for each uptake mechanism, and further studies are required to consider the risk. AAV2 and the neuron-specific receptors lack specific information about their mechanism, but there is a high potential to develop these strategies. Both AVV and the neuron-specific receptors provide specific uptake into tissues.

No description available.

Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle's internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications - LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine...

<p dir="ltr">In light of new interest in biomaterials for drug formulation and delivery, coiled-coil assemblies have emerged as promising candidates. Characterized by facile synthesis, low toxicity, and biocompatibility, coiled-coil assemblies hold a significant potential for drug encapsulation and cellular delivery. Herein, I will discuss the development of a novel nanoscale metal-mediated coiled-coil assembly with tunable assembly properties, presenting a compelling platform for drug encapsulation and targeted cell delivery, thereby addressing critical challenges in modern pharmaceutical science.</p><p dir="ltr">Moreover, the rise of antimicrobial resistance has contributed to a global health crisis. Many current antibiotics are announced as unviable, and the addition of bacteria that invade the cell further challenges and limits these current drugs. The Chmielewski group has previously shown that cationic amphiphilic polyproline helices (CAPHs) act as dual agents displaying both cell-penetration and antibiotic activity. To further increase these dual properties, new unnatural amino acids and new CAPH peptides were synthesized with new configurations of hydrophobic and hydrophilic moieties. I will discuss the outcomes of these modifications, including cell penetration, subcellular localization, and antibacterial efficacy.</p>

Les peptides pénétrateurs de cellule (CPP) sont des oligopeptides cationiques faisant parti des vecteurs les plus étudiés dans le cadre du développement du transport ciblé de médicament à l’intérieur de l’organisme. Les applications principales sont par exemple le traitement des cancers ou la thérapie génique. Néanmoins, certaines caractéristiques des CPPs rendent leur utilisation médicale compliquée, tels que leur manque de spécificité à l’égard des cellules cibles ou la perte de leurs propriétés pénétrantes lorsqu’un cargo moléculaire leur est greffé. L’une des solutions envisagées pour résoudre ces problèmes est le greffage sur des polypeptides di-blocs auto-assemblés basés sur de l’élastine (ELPBC), des systèmes développés par l’équipe d’Ashutosh Chilkoti à l’Université de Duke (USA). Des travaux précédents ont montré que ces macromolécules, que l’on appelle CPP-ELPBC, retrouvaient les propriétés pénétrantes du CPP même en présence d’un cargo et permettaient également d’induire une spécificité à l’encontre des cellules cancéreuses. En revanche, le mécanisme de pénétration de ces systèmes restait inconnu.Dans cette thèse, je me suis concentré sur l’étude du mécanisme de pénétration des CPP et des CPP-ELPBC au travers de membranes lipidiques modèles, et en particulier sur l’adsorption de ces molécules à la surface de vésicules unilamellaires géantes (GUV). Le développement d’une nouvelle méthode de quantification de la fluorescence en microscopie confocale m’a permis de réaliser des mesures simples de comptage de peptides à la surface des vésicules, ce qui m’a permis par la suite de procéder à des mesures thermodynamiques de l’adsorption des peptides. / Cell-penetrating peptides (CPP) are cationic oligopeptides currently investigated as potential vectors for targeted drug delivery design, for applications in cancer treatment and/or gene therapy. Nevertheless, some drawbacks make the CPP complex for medical applications, such as their lack of specificity toward target cells or the loss of their penetrating properties once they have been grafted with a molecular cargo. One of the solutions studied to overcome these issues is the binding of the CPP unit on a self-assembling elastin-like diblock polypeptide (ELPBC), a macromolecular system designed by the team of Ashutosh Chilkoti from Duke University (USA). While it has already been proven that these molecules, named CPP-ELPBC, recover the penetrating properties of the CPP despite the presence of a cargo and also induce a selectivity toward tumorous cells, the exact mechanism of translocation is still under debate.In this PhD thesis, I focused on the investigation of the translocation mechanism of the CPP and CPP-ELPBC using model lipid membranes, and specifically the adsorption of these molecules at the surface of giant unilamellar vesicles (GUV). The development of a new quantification method of fluorescence in confocal microscopy allowed me to directly count the peptides adsorbed on the surface of the GUVs, which I used to perform thermodynamic measurements on the peptide adsorption.