{"id":{"repo_id":"sherbrooke","oai_identifier":"oai:usherbrooke.scholaris.ca:11143/21828"},"canonical_url":"https://search.dev.ndltd.org/etd/sherbrooke/oai:usherbrooke.scholaris.ca:11143/21828","repository":{"repo_id":"sherbrooke","name":"Université de Sherbrooke","base_url":"https://usherbrooke.scholaris.ca/server/oai/request"},"display":{"title":"Explorer l'impact de l'élimination de la charge nette cationique des signaux de localisation nucléaire (NLS) sur le ciblage par l'importine-α","abstract":"The nucleus is the control center of the eukaryotic cells as it contains most of the genetic material (DNA) necessary for host survival. As the DNA and DNA-interacting proteins in the nucleus are highly sensitive to damage, the nucleus is a target for therapeutic intervention for many diseases such as cancer. However, the nucleus is protected by a double membrane that only allows low molecular weight molecules to freely diffuse through nuclear pores, making it challenging to target. Nuclear transport is a vital component of normal functioning in eukaryotic cells. Specialized transport proteins called importins recognize nuclear localization signals (NLSs) on proteins in the cytoplasm requiring nuclear entry. This biological phenomenon has spurred extensive research into NLS-tagged therapeutics to 8actively9 target the nucleus to maximize therapeutic efficacy. Despite many advancements in NLS-therapeutic design, the absence of consistent successes within clinical settings prompts critical questions about the factors contributing to this challenge. Addressing this question drives the rational of this thesis. Typically, NLSs are highly cationic nature due to the importance that lysine and arginine amino acids are required to form crucial contacts with the side chains of importin-ɑ in the major or minor NLS-binding pockets. From a pharmaceutical perspective, it is known that therapeutic agents with increased net-positive molecular charges exhibit poor pharmacokinetics due to significant non-specific ionic interactions with mammalian cell membranes that are rich in anionic phospholipids. As a result, when these agents are administered intravenously, they are rapidly eliminated from the blood stream and insufficiently accumulate at the tumor site to evoke effective therapeutic efficacy. Labeling NLSs to therapeutics requires high NLS-to-antibody ratios required to achieve efficient nuclear import. In the current work, we have designed and developed novel NLSs bearing zero net-charge. This thesis examines the importin-ɑ ability of these net zero NLSs using experimental methods concentrating on biophysical and biochemical techniques. Specifically, crystal structures were obtained with the net zero NLSs complexed to importin-ɑ. Circular dichroism and microscale thermophoresis were employed to evaluate NLS-importin-ɑ complex stabilities and binding affinities. Our results showed that zero net-charge NLSs could be developed with binding affinities and interaction modes comparable to those of natural NLSs. The anionic amino acids in the net zero NLSs formed novel contacts with importin-ɑ, while maintaining the same binding modes as the natural NLS counterparts. Taken together, this research opens the door for the development of more effective NLS-based therapies with potentially improved pharmacokinetics and increased tumor-site accumulation.","abstract_html":"The nucleus is the control center of the eukaryotic cells as it contains most of the genetic material (DNA) necessary for host survival. As the DNA and DNA-interacting proteins in the nucleus are highly sensitive to damage, the nucleus is a target for therapeutic intervention for many diseases such as cancer. However, the nucleus is protected by a double membrane that only allows low molecular weight molecules to freely diffuse through nuclear pores, making it challenging to target. Nuclear transport is a vital component of normal functioning in eukaryotic cells. Specialized transport proteins called importins recognize nuclear localization signals (NLSs) on proteins in the cytoplasm requiring nuclear entry. This biological phenomenon has spurred extensive research into NLS-tagged therapeutics to 8actively9 target the nucleus to maximize therapeutic efficacy. Despite many advancements in NLS-therapeutic design, the absence of consistent successes within clinical settings prompts critical questions about the factors contributing to this challenge. Addressing this question drives the rational of this thesis. Typically, NLSs are highly cationic nature due to the importance that lysine and arginine amino acids are required to form crucial contacts with the side chains of importin-ɑ in the major or minor NLS-binding pockets. From a pharmaceutical perspective, it is known that therapeutic agents with increased net-positive molecular charges exhibit poor pharmacokinetics due to significant non-specific ionic interactions with mammalian cell membranes that are rich in anionic phospholipids. As a result, when these agents are administered intravenously, they are rapidly eliminated from the blood stream and insufficiently accumulate at the tumor site to evoke effective therapeutic efficacy. Labeling NLSs to therapeutics requires high NLS-to-antibody ratios required to achieve efficient nuclear import. In the current work, we have designed and developed novel NLSs bearing zero net-charge. This thesis examines the importin-ɑ ability of these net zero NLSs using experimental methods concentrating on biophysical and biochemical techniques. Specifically, crystal structures were obtained with the net zero NLSs complexed to importin-ɑ. Circular dichroism and microscale thermophoresis were employed to evaluate NLS-importin-ɑ complex stabilities and binding affinities. Our results showed that zero net-charge NLSs could be developed with binding affinities and interaction modes comparable to those of natural NLSs. The anionic amino acids in the net zero NLSs formed novel contacts with importin-ɑ, while maintaining the same binding modes as the natural NLS counterparts. Taken together, this research opens the door for the development of more effective NLS-based therapies with potentially improved pharmacokinetics and increased tumor-site accumulation.","abstract_has_math":false,"creators":["Abdoli, Amirabbas"],"institution":"Université de Sherbrooke","degree_name":"M. Sc.","degree_level":"Maîtrise","degree_discipline":"Sciences des radiations et imagerie biomédicale","degree_department":null,"school":null,"contributors":[],"advisors":["Leyton, Victor Jeffrey","Lavigne, Pierre"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024","date_published":"2024","updated_at":"2026-07-27T21:07:57Z","subjects":["NLS","Importin-ɑ","Zero net-charge","MST","Circular dichroism","TAF8"],"languages":["fr","en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/11143/21828","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Leyton, Victor Jeffrey","Lavigne, Pierre"]},{"key":"dc:creator","label":"Author","values":["Abdoli, Amirabbas"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-08-15T14:07:18Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-08-15T14:07:18Z"]},{"key":"dc:date.issued","label":"Date","values":["2024"]},{"key":"dc:publisher","label":"Institution","values":["Université de Sherbrooke"]},{"key":"dc:type","label":"Dc Type","values":["Mémoire de maîtrise"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Sciences des radiations et imagerie biomédicale"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Maîtrise"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M. 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As the DNA and DNA-interacting proteins in the nucleus are highly sensitive to damage, the nucleus is a target for therapeutic intervention for many diseases such as cancer. However, the nucleus is protected by a double membrane that only allows low molecular weight molecules to freely diffuse through nuclear pores, making it challenging to target. Nuclear transport is a vital component of normal functioning in eukaryotic cells. Specialized transport proteins called importins recognize nuclear localization signals (NLSs) on proteins in the cytoplasm requiring nuclear entry. This biological phenomenon has spurred extensive research into NLS-tagged therapeutics to 8actively9 target the nucleus to maximize therapeutic efficacy. Despite many advancements in NLS-therapeutic design, the absence of consistent successes within clinical settings prompts critical questions about the factors contributing to this challenge. Addressing this question drives the rational of this thesis. Typically, NLSs are highly cationic nature due to the importance that lysine and arginine amino acids are required to form crucial contacts with the side chains of importin-ɑ in the major or minor NLS-binding pockets. From a pharmaceutical perspective, it is known that therapeutic agents with increased net-positive molecular charges exhibit poor pharmacokinetics due to significant non-specific ionic interactions with mammalian cell membranes that are rich in anionic phospholipids. As a result, when these agents are administered intravenously, they are rapidly eliminated from the blood stream and insufficiently accumulate at the tumor site to evoke effective therapeutic efficacy. Labeling NLSs to therapeutics requires high NLS-to-antibody ratios required to achieve efficient nuclear import. In the current work, we have designed and developed novel NLSs bearing zero net-charge. This thesis examines the importin-ɑ ability of these net zero NLSs using experimental methods concentrating on biophysical and biochemical techniques. Specifically, crystal structures were obtained with the net zero NLSs complexed to importin-ɑ. Circular dichroism and microscale thermophoresis were employed to evaluate NLS-importin-ɑ complex stabilities and binding affinities. Our results showed that zero net-charge NLSs could be developed with binding affinities and interaction modes comparable to those of natural NLSs. The anionic amino acids in the net zero NLSs formed novel contacts with importin-ɑ, while maintaining the same binding modes as the natural NLS counterparts. Taken together, this research opens the door for the development of more effective NLS-based therapies with potentially improved pharmacokinetics and increased tumor-site accumulation.","Le transport nucléaire est un processus vital pour le fonctionnement des cellules eucaryotes, jouant un rôle central dans la biogenèse cellulaire et la régulation de l’expression génique. Les importines permettent le passage des cargos contenant des signau x de localisation nucléaire (NLS) à travers le complexe de pores nucléaires, ce qui facilite le transport actif des protéines du cytoplasme vers le noyau. L’importine-α est majeure dans ce processus, sa structure comprend 10 répétitions ARM pour former une construction cylindrique contorsionnée et une poche de liaison NLS principale et mineure. Les importines -α se calculent en trois clades : α1, α2 et α3, avec des niveaux de séquence d’acides aminés hautement conservés, et des protéines nucléaires avec un NLS monopartite ou bipartite interagissent avec les résidus acides de celles -ci. Malgré leur potentiel thérapeutique, les NLS traditionnels sont limités par leur nature hautement cationique, entraînant une élimination rapide du sang et une pharmacocinétique médiocre. Cette limitation est due à des interactions ioniques non spécifiques entre les molécules cationiques et les membranes cellulaires, augmentant ainsi leur clairance. Le marquage des NLS avec des anticorps monoclonaux nécessite un rapport élevé de NLS/anticorps, produisant souvent des espèces cationiques \"surchargées\" peu idéales pour une utilisation thérapeutique. Pour surmonter ces défis, nous avons développé de nouveaux NLS sans charge nette, visant à améliorer la pharmacocinétique en réduisant les interactions non spécifiques. Dans nos travaux, nous avons conçu et caractérisé ces nouveaux NLS en utilisant des approches informatiques et biophysiques pour cibler efficacement l'importine -α. Les interactions entre les NLS et l'importine-α ont été étudiées par cristallographie, dichroïsme circulaire et thermophorèse à l'échelle microscopique. Les résultats ont montré qu'il est possible de développer des NLS à charge nette nulle avec des affinités de liaison et des modes d'interaction comparables à ceux des NLS naturels. Cette avancée ouvre la voie à des thérapies basées sur les NLS plus efficaces, avec une pharmacocinétique améliorée et un taux de dégradation réduit, offrant ainsi un potentiel prometteur pour les applications thérapeutiques."]},{"key":"dc:title","label":"Title","values":["Explorer l'impact de l'élimination de la charge nette cationique des signaux de localisation nucléaire (NLS) sur le ciblage par l'importine-α"]}]}],"canonical_facts":{"dc:contributor.advisor":["Leyton, Victor Jeffrey","Lavigne, Pierre"],"dc:creator":["Abdoli, Amirabbas"],"dc:date.accessioned":["2024-08-15T14:07:18Z"],"dc:date.available":["2024-08-15T14:07:18Z"],"dc:date.issued":["2024"],"dc:description.abstract":["The nucleus is the control center of the eukaryotic cells as it contains most of the genetic material (DNA) necessary for host survival. As the DNA and DNA-interacting proteins in the nucleus are highly sensitive to damage, the nucleus is a target for therapeutic intervention for many diseases such as cancer. However, the nucleus is protected by a double membrane that only allows low molecular weight molecules to freely diffuse through nuclear pores, making it challenging to target. Nuclear transport is a vital component of normal functioning in eukaryotic cells. Specialized transport proteins called importins recognize nuclear localization signals (NLSs) on proteins in the cytoplasm requiring nuclear entry. This biological phenomenon has spurred extensive research into NLS-tagged therapeutics to 8actively9 target the nucleus to maximize therapeutic efficacy. Despite many advancements in NLS-therapeutic design, the absence of consistent successes within clinical settings prompts critical questions about the factors contributing to this challenge. Addressing this question drives the rational of this thesis. Typically, NLSs are highly cationic nature due to the importance that lysine and arginine amino acids are required to form crucial contacts with the side chains of importin-ɑ in the major or minor NLS-binding pockets. From a pharmaceutical perspective, it is known that therapeutic agents with increased net-positive molecular charges exhibit poor pharmacokinetics due to significant non-specific ionic interactions with mammalian cell membranes that are rich in anionic phospholipids. As a result, when these agents are administered intravenously, they are rapidly eliminated from the blood stream and insufficiently accumulate at the tumor site to evoke effective therapeutic efficacy. Labeling NLSs to therapeutics requires high NLS-to-antibody ratios required to achieve efficient nuclear import. In the current work, we have designed and developed novel NLSs bearing zero net-charge. This thesis examines the importin-ɑ ability of these net zero NLSs using experimental methods concentrating on biophysical and biochemical techniques. Specifically, crystal structures were obtained with the net zero NLSs complexed to importin-ɑ. Circular dichroism and microscale thermophoresis were employed to evaluate NLS-importin-ɑ complex stabilities and binding affinities. Our results showed that zero net-charge NLSs could be developed with binding affinities and interaction modes comparable to those of natural NLSs. The anionic amino acids in the net zero NLSs formed novel contacts with importin-ɑ, while maintaining the same binding modes as the natural NLS counterparts. Taken together, this research opens the door for the development of more effective NLS-based therapies with potentially improved pharmacokinetics and increased tumor-site accumulation.","Le transport nucléaire est un processus vital pour le fonctionnement des cellules eucaryotes, jouant un rôle central dans la biogenèse cellulaire et la régulation de l’expression génique. Les importines permettent le passage des cargos contenant des signau x de localisation nucléaire (NLS) à travers le complexe de pores nucléaires, ce qui facilite le transport actif des protéines du cytoplasme vers le noyau. L’importine-α est majeure dans ce processus, sa structure comprend 10 répétitions ARM pour former une construction cylindrique contorsionnée et une poche de liaison NLS principale et mineure. Les importines -α se calculent en trois clades : α1, α2 et α3, avec des niveaux de séquence d’acides aminés hautement conservés, et des protéines nucléaires avec un NLS monopartite ou bipartite interagissent avec les résidus acides de celles -ci. Malgré leur potentiel thérapeutique, les NLS traditionnels sont limités par leur nature hautement cationique, entraînant une élimination rapide du sang et une pharmacocinétique médiocre. Cette limitation est due à des interactions ioniques non spécifiques entre les molécules cationiques et les membranes cellulaires, augmentant ainsi leur clairance. Le marquage des NLS avec des anticorps monoclonaux nécessite un rapport élevé de NLS/anticorps, produisant souvent des espèces cationiques \"surchargées\" peu idéales pour une utilisation thérapeutique. Pour surmonter ces défis, nous avons développé de nouveaux NLS sans charge nette, visant à améliorer la pharmacocinétique en réduisant les interactions non spécifiques. Dans nos travaux, nous avons conçu et caractérisé ces nouveaux NLS en utilisant des approches informatiques et biophysiques pour cibler efficacement l'importine -α. Les interactions entre les NLS et l'importine-α ont été étudiées par cristallographie, dichroïsme circulaire et thermophorèse à l'échelle microscopique. Les résultats ont montré qu'il est possible de développer des NLS à charge nette nulle avec des affinités de liaison et des modes d'interaction comparables à ceux des NLS naturels. Cette avancée ouvre la voie à des thérapies basées sur les NLS plus efficaces, avec une pharmacocinétique améliorée et un taux de dégradation réduit, offrant ainsi un potentiel prometteur pour les applications thérapeutiques."],"dc:identifier.uri":["http://hdl.handle.net/11143/21828"],"dc:language.iso":["fr","en"],"dc:publisher":["Université de Sherbrooke"],"dc:subject":["NLS","Importin-ɑ","Zero net-charge","MST","Circular dichroism","TAF8"],"dc:title":["Explorer l'impact de l'élimination de la charge nette cationique des signaux de localisation nucléaire (NLS) sur le ciblage par l'importine-α"],"dc:type":["Mémoire de maîtrise"],"thesis:degree_discipline":["Sciences des radiations et imagerie biomédicale"],"thesis:degree_level":["Maîtrise"],"thesis:degree_name":["M. Sc."],"thesis:institution_name":["Faculté de médecine et des sciences de la santé"]},"updated_at":"2026-07-27T21:07:57Z"}