<div> <div> <div> <p>The exploratory profiling and quantitative bioassays of lipids, small metabolites, and peptides have always been challenging tasks. The most popular instrument platform deployed to solve these problems is chromatography coupled with mass spectrometry. However, it requires large amounts of instrument time, intensive labor, and frequent maintenance, and usually produces results with bias. Thus, the pace of exploratory research is one of poor efficacy and low throughput. The work in this dissertation provides two practical tactics to address these problems. The first solution is multiple reaction monitoring profiling (MRM-profiling), a new concept intended to shift the exploratory research from current identification-centered metabolomics and lipidomics to functional group screening by taking advantage of precursor ion scan and product ion scan. It is also demonstrated that MRM-profiling is capable of quantifying the relative amount of lipids within the same subclass. Besides, an application of the whole workflow to investigate the strain-level differences of bacteria is described. The results have zeroed in on several potential lipid biomarkers and corresponding MRM transitions. The second strategy is aimed to increase the throughput of targeted bioassays by conducting induced nanoelectrospray ionization (nESI) in batch mode. A novel prototype instrument named "Dip-and-Go" system is presented. Characterization of its ability to carry out reaction screening and bioassays exhibits the versatility of the system. The distinct electrophoretic cleaning mechanism contributes to the removal of salt during ionization, which assures the accuracy of measurement.</p></div></div></div>

Produktionen av rekombinanta biofarmaceutiska läkemedel, exempelvis monoklonala antikroppar, är en oumbärlig men ansträngande process. Under 2019 var sju av de tio mest sålda läkemedlen globalt baserade på rekombinant producerade monoklonala antikroppar. En betydande flaskhals i utvecklingen av stabila cellinjer är screening och selektion av högproducerande singelcellkloner. Tekniker som utspädningskloning (limiting dilution) och fluorescensaktiverad cellsortering (FACS) når idag medelmåttiga resultat som bäst, men saknar förmåga att både isolera produktiva singelcellkloner och hålla cellviabilitet på en acceptabel nivå. Samtidigt kan CellCelectorn™, ett helt automatiserat instrument utformat för att detektera, selektera och isolera singelcellkloner, screena celler för att kartlägga deras produktivitet. CellCelectorn™ är utrustad både med brightfield- och fluorescerande kameror och kan ranka samtliga cellkloner i en provplatta efter produktivitet för att sedan överföra de mest lovande klonerna till en destinationsplatta för vidare analyser och singelklonexpansion. Med avstamp i detta instrument var syftet med projektet att utveckla ett automatiserat arbetsflöde för screening och selektion av högproducerande singelcellkloner med hög genomströmning vid generering av stabila cellinjer. Inledande tester med redan utvecklade och produktiva cellinjer genomfördes för att undersöka CellCelectorns™ prestanda och för att upprätta och optimera sekundära parametrar relevanta för instrumentets kapacitet. Då dessa inledande tester var framgångsrika utfördes en polyklonal selektion i syfte att utveckla tre stabila cellinjer producerandes olika antikroppar. Dessa cellinjer skulle sedan användas för att undersöka CellCelectorns™ förmåga att isolera och överföra de mest lovande singelklonerna till en destinationsplatta. Dessa test kunde tyvärr inte genomföras, då kontaminering i de polyklonala poolerna hade uppstått, men de övergripande resultaten av detta projekt indikerar att CellCelectorn™ är kapabel till att screena för och ranka singelkloner efter deras produktivitet samt att selektera högproducerande klonkandidater under en enda arbetsvecka. Framtida tester är dock nödvändiga för att säkerställa CellCelectorns™ förmåga att isolera singelcellkloner med hög genomsnittlig cellviabilitet för att kunna genomföra singelklonexpansion. / The production of recombinant biopharmaceuticals, such as monoclonal antibodies (mAbs), from stable mammalian cell lines is an indispensable yet strenuous process. In 2019, seven of the top ten most sold drugs globally were based on monoclonal antibodies produced recombinantly. A prominent bottleneck in stable cell line development is the screening and selection of high target protein producing single clone candidates. Today, techniques such as limiting dilution and Fluorescence Activated Cell Sorting (FACS) receive moderate success at best at isolating single clones while keeping cell viability high. Simultaneously, the CellCelector™ is a fully automated instrument designed for the detection, selection, and isolation of single cell clones. Containing both a brightfield and fluorescence-detecting camera, the CellCelector™ can screen cells to measure their productivity and rank them accordingly, meaning high target protein producing clones can be selected and transferred to a destination plate for single clone expansion. Thus, this project aimed at developing an automated high-throughput workflow using the CellCelector™ to streamline the screening and selection steps of stable mammalian cell line generation. Several tests with stable proof-of-concept cells were performed to evaluate the performance of the CellCelector™ and to establish favorable secondary parameter settings. As initial proof-of-concept tests showed promise, a polyclonal selection to obtain three stable Human Embryonic Kidney (HEK) cultures expressing different antibodies was carried out in order to test the CellCelector™ proficiency in clone selection and picking. Unfortunately, no clone picking or single clone expansion could be executed due to bacterial contamination in the cell cultures. Nevertheless, the overall results of this project indicate high potential of the CellCelector™ to detect and identify promising stable clone candidates for single clone expansion over the course of a single work week. Future tests are however required to solidify CellCelector™ ability to isolate monoclonal clones while preserving cell viability for single clone expansion.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.

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Gegenwärtig ist das Interesse und der Bedarf von Proteinbindern insbesondere in der Biotechnik und Pharmaforschung sehr groß. Kombinatorische, Partikel-basierte (One-Bead-One-Compound) Peptidbibliotheken sind eine Technik, um selektiv bindende Proteine zu identifizieren. Allerdings beinhaltet das Screening dieser Peptidbibliotheken aufwendige Schritte, wie die Separation, Sequenzierung und Charakterisierung von identifizierten Bindern. In dieser Arbeit wurde ein Chip-System entwickelt, auf dem alle Schritte eines Screenings durchgeführt werden können. Dafür wurde ein Glasobjektträger mit einem magnetisch leitenden, doppelseitigen Klebeband versehen. Die Partikel der Bibliothek wurden durch ein Sieb aufgetragen. Dies führte zu einer geordneten Immobilisierung der Partikel auf dem Chip. Über 30.000 Partikel konnten so auf einem Chip immobilisiert werden. Für die Identifizierung von selektiven Protein-bindenden Peptiden wird die immobilisierte Peptidbibliothek mit einem Fluorophor-markierten Protein inkubiert, bindende Partikel mittels Fluoreszenzscan identifiziert und die Peptidsequenz direkt auf dem Chip mittels Matrix-Assisted-Laser-Desorption/Ionization-(MALDI)-Flugzeit-(TOF)-Massenspektroskopie (MS) bestimmt. Die Durchführung einer Abbruchsequenz-Methode erlaubt die eindeutige Bestimmung der Peptidsequenzen mit einer nahezu 100 % Genauigkeit. Die entwickelte Technologie wurde in einem FLAG-Peptid-Modell validiert. Bei dem Screening wurden neue anti-FLAG-Antikörper-bindende Peptide identifiziert. Anschließend wurden in einem Screening von ca. 30.000 Partikeln IgG-bindende Peptide mit mittleren mikromolaren Dissoziationskonstanten identifiziert. Für die Identifizierung stärkerer Binder wurde eine magnetische Anreicherung entwickelt, die dem Chip-Screening vorgeschaltet werden kann. Hiermit wurden aus ca. 1 Million gescreenter Partikel, Peptide mit Dissoziationskonstanten im niedrigen mikromolaren Bereich identifiziert. / The screening of one-bead-one-compound (OBOC) libraries is a well-established technique for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The combinatoric peptide screening traditionally involves tedious steps such as affinity selection, bead picking, sequencing and characterization. In this thesis, a high-throughput “all-on-one chip” system is presented to avoid slow and technically complex bead picking steps. Beads of a combinatorial peptide library are immobilized on a conventional glass slide equipped with an electrically conductive tape. The beads are applied by using a precision sieve, which allows the spatially ordered immobilization of more than 30,000 beads on one slide. For the target screening, the immobilized library is subsequently incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS), high-affinity binders are directly and unambiguously sequenced directly from the bead. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to 100 %. This new technique was validated by employing a FLAG-based model system. In a first step, new peptide binders for the M2 anti-FLAG monoclonal antibody were identified. Finally, this system was utilized to screen for IgG-binding peptides. The screening of about 30.000 peptides on one chip led to the identification of peptide binders in the mid micromolar range. A magnetic enrichment technique was developed to increase the number of screened beads. By combining the magnetic enrichment strategy with the chip system, 1 million beads were screened and IgG-binders in the low micromolar range were identified.

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