Thèse réalisée dans le cadre d'une collaboration avec UCB Pharma et la région Wallonne s'inscrivant dans le cadre du projet SAS. Le but de ce projet était de développer des implants sous-cutanés imprimés en trois dimensions pour permettre une libération d’anticorps thérapeutique de manière prolongée au cours du temps. En effet, les thérapies disponibles sont souvent administrées par voie intraveineuse, ce qui peut réduire la compliance des patients dû à l’inconfort et à la fréquence de ces administrations. Les systèmes de délivrance, tels que des implants, peuvent limiter les fréquences d’administration grâce à l’insertion d’un dispositif qui libèrera le principe actif au cours du temps durant une période donnée. Les implants s’inscrivent comme une alternative aux microsphères qui sont également des dispositifs développés et investigués en vue de favoriser l’adhésion et la compliance des patients. L’avènement du 3D dans le milieu pharmaceutique a montré une certaine frénésie liée au développement de la médecine personnalisée et à l’innovation du procédé dans ce secteur. La sélection d’un matériau biocompatible et biorésorbable tel que le PLGA représente une véritable plus-value dans le développement d’implant. Etant donné que ces implants sont biodégradables, le retrait n’est pas à envisager, ce qui limite les désagréments du patient à un seul acte chirurgical lors de l’implantation. Au cours de ce travail, une approche pragmatique a d’abord été abordée sur les procédés d’extrusion à chaud et de l’impression 3D en utilisant un polymère couramment employé dans l’impression grand public, le PLA. L’investigation des paramètres d’impressions (température d’impression, epaisseur de couche et vitesse d’impression) et l’usage de divers plastifiants (la triacétine (TA), le polyethylène glycol 400 (PEG 400), le citrate de triéthyle (TEC) et l’acétyle citrate de triéthyle (ATEC)) pour faciliter les procédés à chaud et dans l’idée de réduire les températures d’extrusion et d’impression du matériau ont été évalués. Ces essais ont démontré l’effet de la température d’impression sur la qualité de l’impression et principalement sur les propriétés du matériau comme la force de traction et la ductilité. De plus, l’ajout de plastifiant à la matrice du PLA a permis de diminuer sa température de transition vitreuse. Par exemple, la température de transition vitreuse du PLA a été diminuée de 53 °C à 34 °C par l’ajout de PEG 400. Cette approche avait pour but d’évaluer la possibilité de diminuer les températures d’impression dans l’optique d’encapsuler à chaud un anticorps sensible à la chaleur pour la suite de ce travail.Ensuite, le développement de filaments imprimables contenant des anticorps a été abordé et mis en place à l’aide d’un modèle d’anticorps polyclonal disponible en grandes quantités et à des coûts relativement faibles. Un anticorps à l’état solide a été favorisé dans le procédé car il est largement accepté que les protéines sous forme solide sont plus stables au cours du temps en comparaison aux solutions d’anticorps. De plus, cet état solide facilite les manipulations précédant l’extrusion comme l’étape de mélange. Pour la réalisation des filaments, différents types de PLGA ont été investigués afin d’atteindre les propriétés nécessaires à l’impression en termes de diamètre mais également de comportement physique. Ces dérivés étaient caractérisés par des masses moléculaires différentes comme pour le PDLG5004 (44 kDa), le RG502 (7-17 kDa) et parmis eux, un copolymère PEG-PLGA (2 kDa-20 kDa). Un PLGA de faible masse moléculaire a été sélectionné pour développer ce filament. En effet, les extrusions étaient réalisables à une température maximum de 90 °C et les impressions à 113 °C minimum. L’un des enjeux cruciaux du développement de filament imprimable contenant un anticorps à haute concentration, au minimum 15% (w/w), était d’en assurer l’homogénéité. Cependant, l’usage de températures aussi élevées lors de l’impression a induit la dégradation de l’anticorps par la formation d’agrégats et principalement de fragments. Ces derniers sont généralement produits lors de procédé à haute température ou par l’usage de conditions drastiques telles que l’acidification du milieu. Cette plateforme a été adaptée à l’encapsulation d’anticorps thérapeutique fournit par UCB Pharma. L’usage d’un anticorps monoclonal possédant une stabilité supérieure à celle du modèle initialement utilisé permettrait d’identifier l’impact du procédé sur l’intégrité de l’anticorps. La formulation de l’anticorps a été réalisée en utilisant différents stabilisants conventionnels (sucrose (Suc), trehalose (Tre), 2-Hydroxypropyl-beta-cyclodextrine (HP-β-CD), inuline (Inu) et sorbitol (Sor)) et reconnus pour la stabilisation des protéines. A côté des excipients ajoutés, différentes quantités d’excipients ont été investigués. Ces manipulations ont montré que la stabilité de l’anticorps était privilégiée à l’aide du sucrose et du tréhalose à un ratio anticorps monoclonal:excipient de 2.0:1. En gardant ce ratio, l’ajout d’un acide aminé (leucine) aux deux disaccharides précédemment cités, a amélioré la stabilité de l’anticorps vis-à-vis des procédés à chaud (extrusion et impression 3D). L’homogénéité au sein des filaments imprimables et des pièces 3D a été confirmée tout au long du procédé. En effet, les charges en anticorps étaient similaires à la charge théorique de 15% (w/w). Aucune fragmentation de l’anticorps n’a été observée à l’issue des procédés à chaud. Cependant, une augmentation des agrégats de 2.6% en solution à 3.6% après impression 3D a été constatée à la fin du processus. Après avoir stabilisé l’anticorps, le but premier étant d’en promouvoir une libération prolongée au cours du temps. Les profils ont révélé une libération en trois phases au cours du temps mais avec un relargage après 24h relativement faible (< 5%) dû à la densité des matrices polymériques. Ensuite, la dégradation du polymère représente l’élément limitant la libération de l’anticorps au cours du temps. En effet, l’érosion du polymère joue un rôle clé dans la libération de l’anticorps encapsulé. La libération au cours du temps a été démontrée sur une période allant jusqu’à 15 semaines. La stabilité de l’anticorps dans le milieu de dissolution a été évaluée et une dégradation de celui-ci au cours du temps a été observée. Cette dégradation est principalement liée à l’érosion du polymère et à l’acidification du milieu au cours du test de dissolution. Après avoir optimisé la formulation de l’anticorps et avoir démontré la libération prolongée de celui-ci, son affinité restait à être étudiée. La capacité de l’anticorps à se lier à sa cible a pu être démontrée après 24h de dissolution mais cette affinité s’est réduite au cours de la durée de la dissolution avec une augmentation de l’agrégation et de la fragmentation de l’anticorps. Une étude de stabilité a également démontré que les implants imprimés en 3D sont stables à une température 5 °C sur une durée de 6 mois. Aucun élément de dégradation n’a été observé au cours du temps et l’affinité de l’anticorps a été préservée au cours de l’étude. Finalement, cette plateforme a également été évaluée pour l’encapsulation d’une troisième molécule biologique, un fragment d’anticorps monoclonal, pour d’une part en estimer la stabilité et l’applicabilité et d’autre part envisager une prochaine étude pré-clinique sur rongeurs. Le fragment d’anticorps a montré une stabilité supérieure à celle de l’anticorps monoclonal avec une faible agrégation après l’extrusion et l’impression. La libération prolongée du fragment a été évaluée sur 8 semaines et une libération du fragment de 79% a été observée avec une formulation contenant du tréhalose et de la leucine. En effet, les fragments d’anticorps ont une demi-vie plasmatique relativement faible, de l’ordre de 28 minutes, ce qui donne tout son sens à des systèmes à libération prolongée. Pour finir, la réalisation d’une étude pré-clinique permettrait de valider le modèle. En conclusion, ce travail a démontré la faisabilité de l’usage de l’impression 3D en vue de développer des systèmes à libération prolongée contenant des anticorps et en utilisant des procédés à hautes températures. Ces implants ont été caractérisés par une stabilité favorable et une libération intéressante qui feront l’objet d’investigation lors d’études pharmacocinétiques. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

The present study focuses on the Vindelälven-Juhttátahkka Biosphere Reserve (BR) in Northern Sweden, which serves as an implementation site of UNESCO’s Man and the Biosphere (MAB) programme. In response to a series of environmental and social problems identified in the specific locale, the area inhabited by both “Swedish” and “Sami” people, is designed to serve henceforth as a learning site for sustainable development. Taking Michel Foucault’s work on discourse and power as a reference point, in this study we analyze the discourse that permeates life in the specific milieu, to understand how power operates in the BR and look for resistance. Following Foucault’s theorization of discourse, the study has employed Foucauldian Discourse Analysis (F.D.A.) on the empirical material, gathered from official documents and interviews with people in the BR. Our research has concluded that by employing its scientific programme, UNESCO’s discourse has managed to a large extent to construct in this place a reality that does not allow a future to be imagined outside of the context of sustainable development. This study has found its participants to be influenced by UNESCO’s discourse and has constrained them from perceiving present or imagining future realities that do not sustain the power of the Swedish state under the global neoliberal rule. Lastly, our research has illustrated that the participants who have been found to respond to their experiences have also been found to resist the discourse permeating the BR.

This study of Shelley's intellectual development as it is reflected in these philosophical poems is offered in the hope that knowledge of Shelley's idealism may inspire faith in the beauty which life can possess and trust in the high ideals which alone can create such beauty.

Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have been proven to be highly sensitive and reliable in many applications. With increasing public recognition of threats from bacteria-induced diseases and their potential outbreak among densely populated communities, an intrinsic, low-cost biosensor device that can perform quick and precise identification of the infection type is in high demand to respond to such challenging situations and control the damage those diseases could possibly cause. This dissertation describes the development of a biosensor platform that utilizes polymer thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity- enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross- linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface of the ISAM-coated fiber, which form the core component of the biosensor. By immersing such biosensor fiber into a sample suspension, the immobilized antibody molecules will bind the specific antigen and capture the target cells or cell fragments onto the surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and changing the refractive index of the cladding. This change occurring at the surface of the fiber results in a decrease of optical power emerging from the LPG section of the fiber. By comparing the transmitted optical power before and after applying the sample suspension, we are able to determine whether or not certain bacterial species have attached to the surface of the fiber, and as a consequence, we are able to determine whether or not the solution contains the targeted bacteria. This platform has the potential for detection of a wide range of bacteria types. In our study, we have primarily investigated the sensitivity and specificity of the biosensor to methicillin- resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies have also been conducted on infected mouse tissues and clinical swab samples from human ears, noses, and skin, and in each case, the system was in full agreement with the results of standard culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA infections in clinical swab samples taken from infected patient wounds. It is proposed that nonspecific binding due to insufficient blocking methods is the key issue. Other bacterial strains, such as Brucella and Francisella tularensis have also been studied using a similar biosensor platform with DNA probes and antibodies, respectively, and the outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3 Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml, while for negative control samples at the same concentration, it only produces a signal reduction of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide range of bacteria in a rapid and relatively inexpensive manner. / Ph. D.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...

Abrin is a potent toxin obtained from the seeds of Abrus precatorius. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a disulfide bond. The toxicity of the protein comes from the A subunit harboring RNA-N-glycosidase activity which cleaves the glycosidic bond between the ribose sugar and the adenine at position 4324 in 28S rRNA. The depurination of a specific adenine residue at position 4324 results in loss of conformation of the 28S rRNA at the α sarcin/ricin loop to which elongation factor-2 (EF-2) binds, during the transloction step of translation, leading to inhibition of protein synthesis. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the toxin to gain entry inside cells on binding to receptors with terminal galactose. After entering cells, a few molecules of abrin reach the endoplasmic reticulum (ER) via the retrograde transport, where the disulfide bond between the A and the B subunits gets cleaved. Then the A chain escapes into the cytosol where it binds to its target, the α-sarcin loop of the 28S ribosomal RNA and inhibits protein synthesis. Apart from inhibition of protein synthesis, exposure of cells to abrin leads to the loss of mitochondrial membrane potential (MMP) resulting in the activation of caspases and finally apoptosis. However, whether apoptosis is dependent on the inhibition of protein synthesis has not been elucidated. The major objectives of this study are therefore to delineate the signaling pathways involved in abrin-induced apoptosis. The thesis is divided into 4 Chapters: Chapter 1. provides a overview of the general properties of RIPs, with a brief history, classification, trafficking and biological activities of the toxins. This chapter also discusses their potential use in bio-warfare and the treatments available for management of toxicity. Chapter 2 and 3 discuss the results obtained on studies aimed at gaining insights into the signaling pathways involved in abrin-induced apoptosis. Chapter 4 focuses on the research carried out to understand the mechanisms of neutralization of abrin by the mAb D6F10. Towards the first objective, chapter 2 elucidates the role of endoplasmic reticulum (ER) stress signaling in abrin-induced apoptosis using the human T-cell line, Jurkat as a model system. It could be concluded that the inhibition of protein synthesis by the catalytic A subunit of abrin could result in accumulation of unfolded proteins in the ER leading to ER stress which triggers the unfolded protein response (UPR) pathway. The ER resident trans-membrane sensors IRE1 (Inositol-requiring enzyme 1), PERK (PKR-like ER kinase) and ATF6 (Activating transcription factor 6) are the important players of UPR in mammalian cells. These sensors inhibit translation and increase the levels of chaperones to restore protein homeostasis. However, if the ER stress is prolonged, apoptotic pathways get activated to remove severely damaged cells in which protein folding defects cannot be resolved. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis by activating initiater caspases such as caspase-2 and -8 which eventually trigger mitochondrial membrane potential loss and activation of downstream effector capases-9 and -3. Phosphorylation of eukaryotic initiation factor 2α and upregulation of CHOP [CAAT/enhancer binding protein (C/EBP) homologous protein], important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. However, abrin-induced apoptosis was found be dependent on p38 MAPK but not JNK. We also observed that abrin induced activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. Few toxins belonging to the family of ribosome inactivating proteins such as Shiga toxin have been observed to induce DNA damage in human endothelial cells and activate p53/ATM-dependent signaling pathway in mammalian cells. To further investigate the role of abrin on activation of DNA damage signaling pathway, we analysed the phosphorylation of H2AX and ATM, which are markers for double strand DNA breaks. We observed phosphorylation of H2AX and ATM upon abrin treatment but not when cells were pretreated with the broad spectrum pan caspase inhibitor. This study suggested that the DNA damage observed was an indirect effect of caspase-activated DNase. We concluded from the studies in chapter 2 that inhibition of protein synthesis by abrin can trigger endoplasmic reticulum stress leading to mitochondria-mediated apoptosis. Further studies were conducted to understand the dependence of ER stress on inhibition of protein synthesis and are presented in chapter 3. For this study, we have used an active site mutant of abrin A chain (R167L) which exhibits lower protein synthesis inhibitory activity than the wild type abrin A chain. Recombinant wild type and mutant abrin A chains were expressed in E.coli and purified. Since, abrin A chain requires the B chain for internalization into cells, both wild type and mutant abrin A chains were conjugated to native ricin B chain to generate a hybrid toxin. Next, we have compared the toxic effects of the two conjugates in cells. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild type ricin B-rABRA conjugate but it could trigger ER stress leading to mitochondrial mediated apoptosis in cells though delayed, suggesting that inhibition of protein synthesis is the major factor contributing to abrin-mediated apoptosis. Abrin is extremely lethal and considered as a potential agent for use in biological warfare. Currently, there are no antidotes or effective therapies available for abrin poisoning. Antibody based antitoxins function by either preventing toxin binding to cell surface receptors or by translocation. Antibodies against the B chain of RIPs function by inhibiting the binding of B chain of the toxin to cells, whereas the exact mechanism by which antibodies against A chain function is still not clear. The only known neutralizing monoclonal antibody against abrin A chain, namely, D6F10, was generated in our laboratory and was shown to rescue cells and mice from abrin intoxication. Earlier experiments with confocal microscopy suggested that mAb D6F10 could internalize in HeLa cells along with abrin, suggesting that the antibody can function intracellularly. Chapter 4 discusses the work carried out to delineate the mechanism of intracellular neutralization of abrin by the mAb D6F10. We observed significant reduction in binding and delay in abrin internalization in the presence of the neutralizing monoclonal antibody (mAb) D6F10. Considering that the majority of the abrin after internalization is removed by lysosomal degradation, we studied the fate of abrin in the presence of mAb D6F10. Confocal images did not show any difference in the distribution of abrin in the lysosomes in the absence or presence of antibody. However, the antibody remained persistently colocalized with abrin in the cells, suggesting that the antibody might inhibit enzymatic activity of abrin at its cellular site of action.

Epidemiological, experimental, and clinical observations provide support for a colorectal cancer chemopreventive role for aspirin. We have evaluated the effects of aspirin on proliferation biomarkers in normal-risk and high-risk human subjects for colorectal cancer. Colorectal biopsies were obtained at baseline and at 24h after 28 daily doses of 81mg of aspirin from 13 high-risk and 15 normal-risk subjects for colorectal cancer. We evaluated aspirin's effects on proliferating cell nuclear antigen (PCNA) immunohistochemistry and epithelial mucin histochemistry using the lectin, Amaranthus caudatus agglutinin (ACA) in crypt sections from rectal biopsies. The baseline whole crypt PCNA LIs differed significantly between normal-risk and high-risk subjects. PCNA LIs are not affected by 28 days of aspirin at 81mg daily. ACA LIs are decreased by 28 days of aspirin at 81mg daily in both normal-risk and high-risk subjects. Aspirin's effects on ACA LIs may have mechanistic and biological implications that deserve further attention. PCNA and ACA LIs are not useful as proliferation biomarkers for aspirin's chemopreventive activity in morphologically normal human colorectal mucosa.