<p>Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. It was hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could treat bone-metastatic prostate cancer and also exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, <i>in vivo</i> experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), or a therapeutic gene (IL-27). Ultrasound was used to aid gene delivery. Various gene delivery methods were tested and optimized through <i>in vivo</i> studies, with microbubbles in combination with ultrasound (sonoporation) emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing and cytokine arrays were used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone health by improving osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach should enable a significant reduction in chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and simultaneously improving bone quality.</p>

This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials. / Master of Science / Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.

Die vorliegende Arbeit behandelt das biologische Phänomen der Dormanz mit Hilfe mathematischer Modellierung. Dormanz beschreibt dabei einen reversiblen Zustand von Individuen, in dem die metabolische Aktivität reduziert wird und die Resistenz gegen Natureinflüsse erhöht ist. Der erste Teil der Arbeit widmet sich den ökologischen Eigenschaften. Hier wird zunächst ein Moranmodell vorgestellt, welches verschiedene Modellierungsarten von Dormanz aus der Populationsgenetik vereint und unter verschiedenen Skalierungen den schwachen seed-bank Koaleszenten und den starken seed-bank Koaleszenten als anzestralen Prozess innehat. Dadurch werden die Parameter der Koaleszenten vergleichbar. Als Anwendung betrachten wir die sogenannte species abundance distribution, welche mithilfe von Koaleszenten beschrieben werden kann. Der zweite Teil beschäftigt sich mit den Auswirkungen von Dormanz auf evolutionäre Eigenschaften und beginnt mit einer Einführung in die Theorie von adaptive dynamics. Dort werden auch verschiedene Möglichkeiten der Modellierung von Dormanz in individuenbasierten Modellen besprochen. Danach befassen wir uns mit der Erweiterung eines Modells für sympatrische Speziation um den Aspekt der Dormanz. Die canonical equation of adaptive dynamics wird - motiviert durch ein Modell mit Dormanz - für schnellere Mutationsraten aus dem sogenannten power-law Mutationsregime für einen Grenzfall hergeleitet. Die Arbeit schließt mit dem dritten Teil, in welchem ein individuenbasiertes Modell für die Entwicklung von Krebs unter dem Einfluss von Chemotherapie und unter Berücksichtigung von Dormanz vorgestellt wird. In Simulationsstudien wird untersucht, inwiefern Dormanz zu Misserfolg einer Therapie beiträgt. Ein weiteres Ziel ist die Analyse von Kombinationsbehandlung mit einem Medikament welches mit dormanten Zellen interagieren kann insbesondere unter Betrachtung verschiedener Therapieansätze zur Behandlung von dormanten Krebszellen. / The present thesis uses mathematical modelling to investigate the consequences of dormancy. Dormancy describes a reversible and protected state of reduced metabolic activity which enhances an individual's resilience to hazardous conditions. In this sense, dormancy acts as a protection mechanism against habitats with unfavourable environments. The thesis considers the impact of dormancy on ecological, evolutionary and in its broadest sense pathogenic properties of microbial populations. The first part is concerned with studying the impact of dormancy on ecology. For this, a Moran model is presented which unifies different models of dormancy from population genetics and exhibits the weak seed-bank coalescent and the strong seed-bank coalescent as the scaling limit of the ancestral process. As an application we consider the species abundance distribution which can be described using coalescent theory. In the second part we consider the influence of dormancy on evolutionary properties. The modelling framework for this is the theory of adaptive dynamics. We then show that competition-induced dormancy may favour sympatric speciation. A key aspect in the derivation of this result is the canonical equation of adaptive dynamics. We extend this equation - motivated by a model including dormancy - to power-law mutations in a limiting case. We conclude the thesis with the third part where we provide an individual-based model for the treatment of cancer with chemotherapy under consideration of dormant cancer cells. Using simulation studies, we investigate how dormancy may contribute to treatment failure. Another goal of this chapter is to analyse combination treatment with a drug which directly targets dormant cancer cells and to formulate general observations regarding various strategies to counter cancer cell dormancy.

DNA is wrapped around proteins called histones, whose modification regulates numerous cellular processes. Therefore it is not surprising that mutations in the genes that modify the histones are frequently associated with human cancer. For example, mutations in SETD2, encoding the sole enzyme that catalyses histone H3 lysine 36 trimethylation (H3K36me3), occur frequently in multiple cancer types. This identifies H3K36me3 loss as an important event in cancer development, and also as a potential therapeutic target. This thesis investigates the following questions: (1) how does the loss of H3K36me3 contribute to cancer development; and (2) what therapy can be used to kill cancers that have already lost H3K36me3. To answer the first question, this thesis shows that H3K36me3 facilitates the accurate repair of DNA double-stranded breaks (DSBs) by homologous recombination (HR). H3K36me3 promotes HR by recruiting CtIP to the site of DSBs to carry out resection, allowing the binding of HR proteins (such as RPA and RAD51) to the damage sites. Thus it is proposed that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions suppresses genetic mutations which could promote tumourigenesis. To answer the second question, this thesis reveals a clinically relevant synthetic lethal interaction between H3K36me3 loss and WEE1 inhibition. WEE1 inhibition selectively kills H3K36me3-deficient cells by inhibiting DNA replication, and subsequent fork stalling results in MUS81 endonuclease-dependent DNA damage and cell death. The mechanism is found to be synergistic depletion of RRM2 (ribonucleotide reductase small subunit), the enzyme that generates deoxyribonucleotides (dNTPs). This work reveals two pathways that regulate RRM2: one involves transcriptional activation of RRM2 by H3K36me3, and the other involves RRM2 degradation regulated by Cyclin-Dependent Kinase, CDK1 (which is controlled by WEE1, CHK1 and ATR). Based on this mechanism, the synthetic lethal interaction is expanded, from between two genes, to between two pathways. Supported by in vivo experiments, the study suggests that patients with cancers that have lost H3K36me3 could benefit from treatment with the inhibitors of WEE1, CHK1 or ATR.

Das Zytokin Interleukin-7 (IL-7) ist für die Entstehung und das Überleben reifer T Zellen von zentraler Bedeutung. Die Gabe von IL-7 führt sowohl in der Maus als auch im Menschen zu erhöhten T Zellzahlen und einem veränderten T Zellphänotyp. Folglich könnte sich die therapeutische Gabe von IL-7 bei Patienten mit geschwächtem Immunsystem positiv auswirken. Diese Hypothese wird derzeit in mehreren klinischen Studien untersucht. Bisher wurde allerdings nur die Wirkung von IL-7 auf T-Zellen studiert. Zu dessen Wirkung auf andere Immun- oder Stromazellen sowie deren IL-7-abhängigen Beitrag zur Regulation der T-Zellhomöostase ist nur wenig bekannt. Daher war es Ziel der Arbeit, den Einfluss einer therapeutischen Gabe von IL-7 auf adoptiv-transferierte T-Zellen in IL-7-Rezeptor (IL-7R)-kompetenten und defizienten lymphopenischen Mäusen zu studieren. Die Untersuchungen bestätigen, dass die Gabe von IL-7 T-Zellantworten unterstützt, zeigen jedoch auch, daß viele dieser Effekte von IL-7R-exprimierenden Wirtszellen abhängig sind. Dies weist darauf hin, dass IL-7R-vermittelte Signale in Wirtszellen indirekt T-Zellantworten beeinflussen. Zudem zeigte sich, dass effiziente anti-Tumor-T Zellantworten von IL 7R-vermittelten Signalen in Wirtszellen abhängen. Vor allem nicht-hämatopoetische Wirtszellen fungieren hier als Regulatoren der IL-7-Therapie-vermittelten T Zelldifferenzierung. Unsere Ergebnisse bestätigen außerdem, dass Stromazellen in verschiedenen Organen il-7 exprimieren und zeigen darüber hinaus, dass diese Zellen durch die Gabe von IL-7 beeinflusst werden. Wir folgern daraus, dass die Effekte der IL-7-Therapie auf T Zellhomöostase teilweise indirekt über il-7-exprimierende Stromazellen vermittelt werden. Um diese Zellen genauer identifizieren und untersuchen zu können, haben wir ein neues transgenes Mausmodell charakterisiert, was es erleichtern wird, die beteiligten molekularen Signalwege zu analysieren und den Erfolg der adoptiven T Zelltherapie zu verbessern. / Interleukin-7 (IL-7) is an essential cytokine required for the development and maintenance of mature T cell. Its availability is limited under normal conditions, but rises during lymphopenia, leading to increased T cell proliferation. The administration of recombinant IL-7 to normal or lymphopenic mice and humans results in increased T cell numbers and altered T cell phenotype. Hence, IL-7 administration could mediate therapeutic benefits in immunocompromised patients and is currently tested in several clinical trials. However, besides its well-studied effects on T cells little is known about the effect of IL-7 on other immune and non-immune cells and their influence on T cell homeostasis. Therefore, we evaluated the effect of IL-7 therapy on adoptively transferred T cells in IL-7 receptor (IL-7R)-competent and IL-7R-deficient lymphopenic mice. We confirm the benefits of IL-7 therapy on T cell responses but additionally show that many of these effects are dependent on IL-7R expression by host cells, indicating that IL-7R signaling in host cells modulates T cell responses. We show that efficient T cell responses against cancer are dependent on host IL-7R signaling. Based on studies in bone-marrow chimeric mice, we identify non-hematopoietic host cells as main regulators of IL-7 therapy-modulated T cell differentiation. We conclude from these data that IL-7 therapy affects non-hematopoietic stromal cells that modulate the success of adoptive T cell therapy. Our results confirm that stromal cells in various organs express il-7 and show that these cells are targeted by IL-7 therapy in vivo. Hence, we propose that il-7-expressing cells regulate IL-7 therapy-modulated T cell homeostasis. To identify and study these il-7 expressing stromal cells in more detail, we characterized a new transgenic mouse model that will facilitate determining the molecular pathways to improve the success of adoptive T cell therapy.

No Brasil, o Sistema de Saúde é Universalista, com o tratamento integral gratuito ao paciente com câncer, cujo direito é assegurado por Lei e regulamentado pelo Ministério da Saúde / Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA), por meio de Decreto Presidencial e Portarias. A habilitação e credenciamento de Hospitais em Unidades ou Centros de Alta Complexidade em Oncologia segue critérios rígidos, que determina como sendo obrigatória a presença do Cirurgião-Dentista na equipe multiprofissional e multidisciplinar na oncoterapia. O tratamento odontológico é compulsório e imprescindível na terapia antineoplásica, para prevenir e tratar as complicações orais: hemorragia, infecção, mucosite oral, xerostomia, cárie de radiação, trismo, alterações periodontais, osteonecrose avascular, osteorradionecrose; que podem levar à interrupção da oncoterapia, acarretando um aumento considerável nos custos da terapia implementada, a piora da qualidade de vida, e em alguns casos, podendo levar o paciente a óbito. O protocolo de cuidados orais na oncoterapia é normatizado e padronizado pelo INCA. Desta forma, foi realizado estudo do tratamento odontológico em pacientes com diagnóstico de câncer atendidos no Serviço de Odontologia Oncológica do UNACON do Hospital Geral de Palmas/Tocantins, no período de abril de 2011 a dezembro de 2016. Os dados foram obtidos de forma individualizada e estruturados segundo Variáveis Demográficas (VD), Procedimento Odontológico (PO), Procedimento Odontológico por Dente (POD) e Outras Variáveis de Interesse (OVI). O impacto financeiro de PO e POD foi avaliado pela comparação entre a tabela SIGTAP do Sistema Único de Saúde (SUS) e a tabela VRPO/SOESP (Valores Referenciais para Procedimentos Odontológicos/Sindicato dos Odontologistas do Estado de São Paulo). A maior incidência de pacientes foi observada na 5ª década de vida, com tendência à proporção homem/mulher de 1:1. Foram realizados 910 procedimentos preventivos/profilaxia/atividade educativa; 1826 raspagens supra e subgengival e aplicação tópica de flúor por hemiarcada; 932 restaurações de uma e duas faces com resina fotopolimerizável; 909 exodontias; 2746 sessões de laserterapia de baixa potência. O impacto financeiro mostrou uma defasagem significativa da Tabela SIGTAP/SUS. O estudo realizado revela a importância de uma base de dados estruturada para o registro do tratamento odontológico realizado em pacientes com diagnóstico de câncer atendidos no Sistema Único de Saúde; para que com isso seja possível fomentar, nos Sistemas de Saúde no Mundo, a elaboração e padronização de protocolo de cuidados orais na terapia antineoplásica, e o planejamento dos recursos humanos e financeiros destinados ao tratamento odontológico nos pacientes oncológicos. / In Brazil, the Health System is Universalist, with free comprehensive treatment for the cancer patient, whose right is guaranteed by Law and regulated by the Ministry of Health / National Cancer Institute José Alencar Gomes da Silva (INCA), through a Presidential Decree and Ordinances. The accreditation of Hospitals in Units or Centers of High Complexity in Oncology follows rigid criteria, which determines as mandatory the presence of the Dentist in the multiprofessional and multidisciplinary team in oncotherapy. Dental treatment is compulsory and essential in antineoplastic therapy to prevent and treat oral complications: hemorrhage, infection, oral mucositis, xerostomia, radiation caries, trismus, periodontal changes, avascular osteonecrosis, osteorradionecrosis; which can lead to interruption of oncotherapy, leading to a considerable increase in the costs of the therapy implemented, worsening of quality of life, and in some cases, leading to death. The protocol of oral care in oncotherapy is standardized by INCA. In this way, a study of the dental treatment was carried out in patients diagnosed with cancer attended at the UNACON Oncological Dentistry Service of the General Hospital of Palmas / Tocantins, from april 2011 to december 2016. Data were obtained individually and structured according to Demographics Variables (VD), Dental Procedure (PO), Dental Procedure by Tooth (POD) and Other Variables of Interest (OVI). The financial impact of PO and POD was evaluated by comparing the SIGTAP table of the Unified Health System (SUS) and the VRPO / SOESP table (Reference Values for Dental Procedures / Union of Dental Practitioners of the State of São Paulo). The highest incidence of patients was observed in the 5th decade of life, with a tendency to male to female ratios of 1: 1. Nine hundred and ten (910) preventive procedures / prophylaxis / educational activity were carried out; 1826 supra and subgingival scaling and topical application of fluoride by hemiarcate; 932 single and double sided restorations with photopolymerizable resin; 909 exodontia; 2746 sessions of low power laser therapy. The financial impact showed a significant lag in the SIGTAP / SUS Table. The study reveals the importance of a structured database for the registry of dental treatment performed in patients diagnosed with cancer treated in the Unified Health System; so that it is possible to promote, in the World Health Systems, the elaboration and standardization of oral care protocol in antineoplastic therapy, and the planning of human and financial resources for dental treatment in cancer patients.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Herkömmliche Zytostatika greifen vornehmlich in den Zellzyklus ein und somit werden Zellen mit hoher Proliferationsrate geschädigt. Allerdings fallen hierunter nicht ausschließlich Krebszellen, sondern auch gesunde, schnell proliferierende Gewebearten. Auf Grund dessen verursacht eine klassische Chemotherapie schwerwiegende Nebenwirkungen. Neuere Therapieansätze greifen daher geno- sowie phänotypischer Unterschiede zwischen malignen und gesunden Zellpopulationen auf und können selektiv den zytotoxischen Wirkstoff in die Tumorpopulation einbringen. Dazu werden sogenannte Prodrug-Konzepte verfolgt, bei denen ein möglichst „untoxisches” Prodrug gezielt im entarteten Gewebe enzymatisch zum zytotoxischen Wirkstoff (Drug) aktiviert wird. In diesem Rahmen werden Substrate für die Antibody-Directed Enzyme Prodrug Therapy (ADEPT) hergestellt. Bei diesem Konzept wird eine hohe Tumorspezifität durch Konjugate aus Enzymen und Antikörpern erlangt, indem das Immunglobulin selektiv an tumorassoziierte Antigene bindet und durch das konjugierte Enzym die Drugfreisetzung ermöglicht wird. Die natürlichen zytotoxischen Antibiotika (+)-CC 1065 und (+)-Duocarmycin SA dienen hierbei als Leitstrukturen für die Synthese entsprechender Prodrugs. Im Rahmen der vorliegenden Arbeit wurden insgesamt zwei neue Duocarmycin-analoge Prodrugs sowie neun neue seco-Drugs synthetisiert, wobei vier Vertreter eine terminale Alkinfunktion aufweisen. Für die Darstellung der Prodrugs wurden auf die Galaktose als Glykosideinheit zurückgegriffen. Zudem wurde ein neuartiges dimeres seco Drug hergestellt, das aus zwei pharmakophoren Einheiten sowie einem verbrückenden Linker mit Alkineinheit besteht. Die jeweiligen Substanzen wurden auf ihre In-vitro-Zytotoxizitäten sowie die Eignung für eine Anwendung im ADEPT-Ansatz evaluiert. Neun der neuen Duocarmycin-Analoga wurden in Form von seco- und Prodrugs wurden im Rahmen des aktivitätsbasierten Protein-Profilings untersucht. Hierbei konnte die Aldehyddehydrogenase 1 als wichtiges Angriffsziel der Duocarmycin-Familie verifiziert werden.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

La compréhension des mécanismes moléculaires de différentes pathologies a permis la caractérisation de gènes et de protéines impliqués dans la pathogénèse et l'identification de cibles thérapeutiques intracellulaires. La nature hydrophobique de la membrane cellulaire empêche le passage des médicaments dans les cellules. Les Cell-Penetrating Peptides (CPP) ou domaines de transduction protéiques (PTD) sont des peptides qui permettent l'internalisation de macromolécules hydrophiles in cellulo et in vivo. Un nouveau peptide issu du facteur de transcription ZEBRA du virus Epstein-Barr, et qui possède des propriétés de transduction a été caractérisé récemment dans notre laboratoire. Des études par mutagénèse de délétion de la protéine ZEBRA ont permis d'identifier la région d'acides aminés (nommé ainsi MD) impliquée dans la pénétration cellulaire. Ce peptide traverse les membranes des cellules de mammifères par un mécanisme de translocation directe, même lorsqu'il est fusionné à des molécules telles que la protéine reportrice eGFP. Le mécanisme de pénétration directe représente un grand avantage pour les applications thérapeutiques: les molécules cargos peuvent être internalisées directement dans le cytoplasme cellulaire sans dégradation et sous une forme biologiquement active. L'objectif de cette thèse est d'étudier les propriétés de pénétration cellulaire du peptide MD et d'évaluer ses applications thérapeutiques comme système de vectorisation des protéines. Ce travail est structuré en trois parties. La première partie porte sur l'étude de l'optimisation de la séquence peptidique MD par réduction de taille et l'évaluation du rôle de sa composition en acides aminés dans le processus de translocation à travers la membrane cellulaire. Cette étude a conduit à l'identification d'une séquence plus courte MD (MD11) possédant une efficacité et un mécanisme de translocation inchangés. La deuxième partie décrit une approche thérapeutique basée sur MD11 visant à la complémentation protéique d'un dysfonctionnement identifiée dans la plupart des cancers. Les cellules tumorales présentent des altérations dans la machinerie de traduction résultant dans une prolifération cellulaire incontrôlée. Parmi les différents facteurs intervenant dans la régulation de ce processus, le facteur eucaryote d'initiation 3 (eIF3) contribue à l'oncogenèse et au maintien de l'état cancéreux. Ce complexe est composé de 13 sous-unités, désignées eIF3 a-m. L'expression de certaines sous-unités est altérée dans plusieurs cancers, et en particulier la sous-unité f (eIF3f) est significativement diminuée dans le mélanome, les cancers du pancréas, de la vulve, du sein, de l'intestin et de l'ovaire. L'expression ectopique par transfection transitoire du gène eIF3f inhibe la synthèse protéique et induit l'apoptose dans le mélanome et dans les cellules cancéreuses pancréatiques. A partir de ces observations, nous avons développé une approche thérapeutique innovante pour le traitement des cancers dans lesquels la protéine manquante eIF3f est produite sous forme recombinante fusionnée à la séquence de MD11, et ensuite internalisée dans les cellules cibles tumorales. Ces résultats démontrent que le système de transfert de eIF3f basé sur MD11 représente une stratégie efficace pour supprimer la prolifération des cellules tumorales. La dernière partie de cette thèse explore la propriété de pénétration de MD11 dans les cellules de levure, et en particulier dans le champignon pathogène Candida albicans. Les résultats obtenus démontrent la polyvalence de MD11, qui fonctionne comme vecteur de protéines à activité biologique aussi bien dans la levure que dans les cellules de mammifères. Le potentiel de MD11 comme système de transport et de relargage des protéines a donc été établis, toutefois certaines améliorations en ce qui concerne la formulation des protéines de fusion et des études in vivo doivent être réalisées afin de valider son efficacité thérapeutique. / In recent years, the understanding of disease molecular mechanisms has led to the identification of genes and proteins that are altered in disease state and many therapeutic targets have been found located within cells. The protective and hydrophobic nature of plasma membrane prevents therapeutic drugs from entering cells. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as a group of non-invasive delivery vectors for various hydrophilic macromolecules, and several in vitro and in vivo applications as pharmaceutical carriers have been reported. A novel cell-penetrating peptide deriving from the Epstein-Barr virus ZEBRA transcription factor has been recently characterized in our laboratory. A reductionist study of full-length ZEBRA protein has allowed to identify the amino acid region (named as Minimal Domain, MD) implicated in cellular uptake. This peptide is able to cross the mammalian cell membranes via a direct translocation mechanism even when fused to cargo molecules such as eGFP reporter protein. The direct penetration mechanism represents a great advantage for therapeutic applications as the cargo molecules can be directly delivered into cells cytoplasm in a biological active form. The aim of this thesis is to explore the cell-penetrating properties of the MD peptide and evaluate its applications as therapeutic protein delivery system. This work is structured in three parts.The first part describes the study on the optimization of MD peptide sequence by size-reduction and the evaluation of its amino acid composition role in the translocation process across the cell membrane. This study has led to the identification of a shorter MD sequence (MD11) with unvaried mechanism of translocation. The second section describes a MD11-based therapeutic approach aiming at repair a dysfunction of the protein synthesis identified in most cancers. The regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth and subtle defects in the translational machinery can alter the cellular physiology and lead to cell malignancy. Among the different factors intervening in the regulation of this process, the eukaryotic initiation factor 3 (eIF3) contributes to oncogenesis and maintenance of the cancer state. This complex is composed of 13 subunits (designated eIF3 a-m). The expression of eIF3 subunits is altered in several cancers, and in particular the f subunit (eIF3f) is significantly down-regulated in pancreas, vulva, breast, melanoma, ovary and small intestine tumors. The eIF3f ectopic expression by transient gene transfection inhibits cellular protein synthesis and induces apoptosis in melanoma and pancreatic cancer cells. Starting from these observations, we developed an innovative therapeutic approach for cancer treatment in which the missing eIF3f protein is produced in vitro in fusion to MD11, and delivered to cells. These results have demonstrated that the MD11- based eIF3f transfer system may represent a powerful strategy to suppress the tumor-cell proliferation. The last part of this thesis explores the cell-penetrating property of MD11 in yeast cells, and in particular in the pathogenic fungus Candida albicans. The presented results demonstrate the versatility of MD11, functioning as vectors in both yeast and mammalian cells and as carrier for proteins with biological activity.The MD11 potential as protein delivery system is evident; however some improvements regarding the fusion protein formulation and in vivo studies should be realized to validate the effectiveness of its therapeutic application.