NCube/papyrus huggingface dataset:
Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.

Keyphrases: Anticorps monoclonal, IgG, N-glycosylation, Sialylation, CHO, Transfection, ST6GalI, ADCC, Monoclonal antibody, Transient expression, IVIG
Études d'ingénierie du ribozyme VS de Neurospora

Ribozymes are catalytic RNAs frequently exploited for the development of biochemistry tools and therapeutic agents. They are particularly interesting in gene inactivation strategies for the degradation of mRNA and viral RNA genome. Currently, the most common ribozymes used in the development of therapeutic agents are the hammerhead and hairpin ribozymes, which can specifically recognize and cleave target single-stranded RNAs through the formation of stable secondary structures. In vivo, single-stranded RNAs can be difficult to target because most RNA adopt complex secondary and tertiary structures. It could be worthwhile to target folded RNA motifs, and an interesting target is the stem-loop structure because stem-loops are important for the overall folding of RNA molecules and they can perform important biological functions. The Neurospora VS ribozyme recognizes its stem-loop folded substrate via a specific kissing-loop interaction, but it has never been exploited for the recognition of target RNA very different from its natural substrate. The goal of the work presented in this thesis is to determine whether the VS ribozyme possesses the necessary adaptability for engineering ribozymes that target RNAs different from its natural substrate. For this thesis, the VS ribozyme was adapted for the recognition of different substrates and kinetic studies were performed to evaluate the effect of these modifications on the cleavage activity. In a first study, the VS ribozyme was modified to recognize and cleave substrates with different stem Ib lengths. The VS ribozyme was successfully adapted to theses substrates of different lengths with a cleavage activity similar to the unmodified ribozyme and substrate. In a second study, the I/V kissing-loop interaction was substituted by rational design, in order to establish if the VS ribozyme variants could recognize and cleave a substrate with a different loop than its natural substrate. The I/V kissing-loop interaction was substituted for the HIV-1 TAR/TAR* and the Deinococcus radiodurans RNA large rRNA subunit L22/L88 kissing-loop interactions. The cleavage reaction was observed for the ribozymes with these new interactions, but with reduced activity. Finally, in vitro selection (SELEX) was used to enable more efficient cleavage of a mutant substrate with a new loop. SELEX experiments enabled v the selection of ribozyme variants that cleave a substrate with a terminal loop mutated to that of the HIV-1 TAR RNA with a very efficient cleavage activity. All of the studies presented in this thesis show that the VS ribozyme can be modified in various ways for the specific recognition of different substrates, while maintaining efficient cleavage activity. These results demonstrate the great potential of VS ribozyme engineering and are very promising for further engineering studies of VS-derived ribozymes for the cleavage of stem-loop folded target RNA completely different from its natural VS ribozyme substrate.

Keyphrases: SHAPE, SELEX, Neurospora VS ribozyme, RNA engineering, kissing-loop interaction, kinetic studies
Rôle du facteur de transcription PITX1 dans les pathogenèses de l'ostéoporose et des maladies parodontales

Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and density leading to an increased risk of fracture. Several studies have linked osteoporosis to the low bone density of the mandibles, the periodontal attachment loss, increasing of the alveolar crest heigh and tooth loss.   This study aims to understand the mechanisms underlying bone loss. During mouse development, PITX1 plays a key role in the identity of the hindlimb and the proper development of the jaws and teeth. Interestingly, PITX1-null mice displayed severe skeletal phenotype. Whereas, ageing PITX1+/- progressively developed OA-like lesions in cartilage associated with a drastic increase in cortical and trabecular bone formation. Conversely, expression analysis of osteoblasts derived from monozygotic twins discordant for osteoporosis reported an 8.6-fold up-regulation of Pitx1 expression in osteoblasts from osteoporotic twins when compared with healthy ones.   Collectively, these data prompted us to investigate the role of PITX1 in normal and pathological bone metabolism. In this context, transgenic Col1α1-Pitx1 mice over expressing Pitx1 specifically in bone tissue under the type-I collagen promoter (2.3kb fragment) were generated and phenotypically characterized.  These data suggested that Pitx1 overexpression induces an osteoporosis like phenotype accompanied with oral bone loss, edentulousness, dental and periodontal problems.   These results suggested that Pitx1 overexpression induces alteration of bone homeostasis via the inactivating of the the Wnt/β-catenin canonical pathway. This hypothesis was supported by the fact that treatment with lithium chloride, a Wnt canonical activator, rescued the phenotype.  Finally, this study establishes a crucial role of PITX1 in the regulation of bone mass and a possible involvement in the developpement of osteoporosis and periodontal disease via the inactivation of the Wnt/β-catenin canonical pathway.

Keyphrases: PITX1, PITX1, Osteoblasts, Bone mass, Osteoporosis, Dental problems, periodontal diseases, Tooth loss, Wnt/β-catenin pathway
The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

induce the production of nitric oxide (NO), which is critical for vascular permeability and angiogenesis. β-catenin is an essential component of the adherens junction as well as Wnt/β-catenin signaling pathway and it binds T-cell factor (TCF)/lymphoid enhancer factor, regulating expression of numerous genes including those involved in angiogenesis. S-nitrosylation (SNO) is a mechanism used by NO to regulate protein activity by adding a nitroso group to cysteine residues. eNOS derived-NO is capable to induce SNO of β-catenin at cell-cell junction and modulate endothelial permeability. Additionally, NO has been implicated in the transcriptional control of gene expression. Therefore, the goals of our studies were to investigate the regulatory roles of NO on transcription in endothelial cells, in particular to the modulation of the transcriptional activity of β-catenin. The objective of the first study is to investigate whether the SNO of β-catenin affect its transcriptional activity. We found that NO inhibits β-catenin transcriptional activity and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, VEGF, which can activate eNOS to produce NO in endothelial cells, repressed Wnt3a-induced expression of Wnt target gene AXIN2 in an eNOS-dependent manner. Moreover, we identified that Cys466 on β-catenin is a critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we showed that Cys466 is responsible for the disruption iv of β-catenin/TCF4 transcriptional complex, and NO-dependant inhibition of Wnt3a-simulated endothelial cell proliferation. Given the known effects of NO on transcription, whole transcriptome sequencing was performed in order to understand the transcriptional regulation of NO in endothelial cells. By analyzing gene differential expression in cells transfected with control and eNOS siRNA, we show that eNOS knockdown upregulates the expression of genes involved in cell polarization, such as PARD3A, PARD3B, PKCZ, CRB1 and TJ3. The up-regulation of these genes was confirmed by qRT-PCR analysis, suggesting that NO may regulate cell polarization. The analysis also showed that genes regulated by eNOS knockdown were involved in endothelial cell integrity and immune response. In addition, KEGG signaling pathway analysis showed that genes regulated by eNOS were enriched in many signal pathways including Ras signaling, which are important for endothelial cell migration. Moreover, clustering of differentially expressed genes in VEGF-treated cells and VEGF-treated eNOS-depleted cells revealed that NO may affect expression of genes in angiogenesis in response to VEGF, including those genes involved in chemotaxis. Our studies show that NO affects transcription in endothelial cells and regulates expression of genes involved in angiogenesis and endothelial cell function.

Keyphrases: Nitric Oxide, Endothelial Cell, Wnt/β-catenin Signaling, Transcriptional Activity, Bioinformatic Analysis
Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between its α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9, ortholog protein in Caenorhabditis elegans. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cancer cells. When these residues are mutated and proteins expressed in cancer cells, cells harbor mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation, events associated with chromosome instability and aneuploidy. To further analyze the effects of Bcl-xL Ser49 and Ser62 in normal cells, the present studies were performed in normal human diploid cells, and in vivo in Caenorhabditis elegans.   First, we studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL(wild type), (S49A), (S49D), (S62A), (S62D) and the dual (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL Ser49 and Ser62 phosphorylation/ dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells.   Second, we undertook experiments in Caenorhabditis elegans to understand the importance of Bcl-xL Ser49 and Ser62 in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual-site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching, variations in the length of their mitotic regions and transition zones, chromosomal abnormalities at their diplotene stages, and increased germline apoptosis. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. The in vivo observations confirmed the importance of Ser49 and Ser62 within the loop domain of Bcl-xL in maintaining chromosome stability.   These studies could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy.

Keyphrases: Bcl-xL, Bcl-xL, Mitosis, Chromosome instability, Aneuploidy, Senescence, Apoptosis
What ‘Security’, whose ‘Rights’ and which ‘Law’? : the Israeli High Court of Justice and the Israeli settlements in the occupied West Bank

This thesis examines how the Israeli High Court of Justice (HCJ) has interpreted and applied principles of the international law of occupation and of international human rights law, in adjudicating petitions filed by Palestinians. The thesis focuses on HCJ judgments that were rendered since the outbreak of the Second Intifada (2000) in relation to petitions challenging the legality of measures implemented by Israeli authorities for the professed need of enhancing the security of Israeli settlements and settlers in the occupied West Bank. The first question that this research seeks to answer is the extent to which the Court has provided Palestinian petitioners with a venue for an effective domestic remedy for alleged violations of their internationally protected rights. The research adopts the position of the HCJ that the law of occupation is guided by an internal logic of balancing between the occupant’s security requirements and the fundamental rights of the occupied population. It also espouses that this logic is reflected in the three normative principles underlying this body of law: stating that occupation is ‘temporary’; that it is a form of ‘trust,’ and that it does not bestow sovereignty by the occupant over the occupied territory. Hence, the second question that the research attempts to answer is whether the Court’s adjudication has promoted or undermined those principles.  Israel’s occupation of the West Bank has been a long one. Coupled with the heightened security threats after 2000 and the active government supported settlement policy, this provides a unique case study for testing the following hypothesis: the domestic courts of democracies, where those in the capacity of the highest judicial body of an occupying power, can successfully uphold fundamental rights and freedoms, and promote the international rule of law.  The first chapter analyses the HCJ judgments that were rendered in relation to petitions challenging the construction of the Wall inside the West Bank and its associated Seam Zone, as well as special security zones around settlements, to assess their implications for the first normative principle. The second chapter examines judgments that were rendered in relation to movement restriction imposed on Palestinians, allegedly for the safety of Israeli settlements and/or settlers, and their implications for the second normative principle. The third chapter looks at decisions rendered in relation to the legality of the Wall’s route in and around annexed East Jerusalem. The research is based on empirical data from Israeli lawyers who regularly petition the HCJ on behalf of Palestinians.

Keyphrases: Effective domestic remedy, International human rights law, International law of occupation, International rule of law, Israeli High Court of Justice, Israeli settlements, Palestinian civilian population, Military security measures, Wall, West Bank
Finding the idea of home

The notion of home is a powerful generator of ideas and imagination in our culture and society. Few can have as significant an impact on the idea of home as an architect. This research thesis explores the concept of home as studied by architects and non-architects alike, in order to further understand its impact on placemaking and the construction of North American domestic environments.   A look at the connotations of space and place, from temporal, behavioural, and social perspectives, further establishes the epistemology of home as an impactful element of contemporary design theory and practice. The hypothetical breaking down of space into its component pieces drives the supposition that architectural devices can be manipulated to shift a physical house into the psychological realm of a home.   Unpacking the maneuverability of the various parts of a building and its surroundings, six themes are applied to three fields of data. The six themes  ̶  In-Between, Border, Pathway, Node, Details, and Representation ̶ exemplify key architectural moments, potentially occurring within the domestic project, that alter corporal and mental behaviour. Within the constrained disciplines of social and affordable housing in Canada, a tripartite examination of photographs of existing low-cost homes, critical writing on this typology, and student-generated research-creation projects, elicits an operational notion of home, and reinforces the value of community and boundary.    The intent of this project is to advance knowledge in architecture by exploring the topic of home within teaching, research, and design. This functional approach to thinking about design places the user at the heart of the domestic environment and emphasizes the belief that home gives meaning and purpose, while reinforcing the ethical responsibility of the architect to make this an everyday reality.

Keyphrases: Architecture, Home, Housing, Social housing, Affordable housing, Device, Architecture
La caractérisation du personnage Jésus dans la narration des Actes des Apôtres

Though Luke’s theology of resurrection is widely studied in the Book of Acts, Jesus is never seen as its main character, unlike the apostles, the Holy Spirit or God. However, the first verse of Acts suggests that the Jesus character will continue to do and teach what he began in the Gospel, even if soon after the opening, he leaves the scene. To resolve this paradox, a thorough analysis of Jesus characterization in Acts is needed. The first chapter of this dissertation introduces the subject, what has already been said, and two narrative scholars’ methodologies selected for this study. In chapter two, the difficult identification of the Lord of Acts is discussed. Then, the epistemological framework of Elizabeth Struthers Malbon is considered. It shows how characterization works, by ranking all episodes involving Jesus in five groups : (1) enacted Christology : what Jesus does; (2) deflected Christology : what Jesus says in response to other characters; (3) projected Christology : what other characters and the narrator say to and about Jesus; (4) mirroring Christology : what other characters do that mirrors what Jesus says and (5) reflected Christology : what other characters do that mirrors what Jesus does. Chapter three discusses the method of John Darr which helps us to understand the why by observing four cognitive activities of the reader: (1) anticipation and retrospection; (2) consistency-building; (3) identification and (4) defamiliarization. In the conclusion in chapter four, the results of the two systems are compared to resolve the case of the paradoxical opening of Acts. The main conclusion of the different observations of this thesis shows that Jesus is not so absent from the story. Thus the narrative rhetoric of characterization leads the reader to understand that Jesus is mainly present through the characters of the story in acts and words.

Keyphrases: Bible, New Testament, Gospel of Luke, Acts, Jesus, Christology, Narrative Criticism, Characterization
Institutions de microfinance et prêteurs informels de l’agriculture familiale en Côte d’ivoire : de la cohabitation à la fusion

The aim of our study was to propose a sustainable financing mechanism. It emerges that food subsistence agriculture is mainly financed by the informal lenders (suppliers of inputs and purchaser of agricultural product) due to their proximity. Anywhere, the results reveal that the collateral, guaranteed and the disbursement periods discourage credit and low down the repayment rate. These results allowed us to propose a new mechanism of financing which amalgamates the informal and formal lenders. This mechanism is based on the MFI by creating a committee of credit made up of all the actors. Thus, the suppliers provide inputs to the farmers. And the repayment is ensured by the purchasers of products after the sale of the harvest. This system increases activities of all actors and legalize those of the informal lenders.

Keyphrases: philosophy, ethics, economics, Informal lender, Co-production, philosophy, ethics, economics, Coproduction
Inequality, Poverty, Two Invariance Conditions, and a Product Rule

Two axioms in the measurement of inequality and poverty which are widely perceived to be innocuous and unexceptionable - although they have both been challenged in the literature - are the Scale Invariance Axiom and the Replication Invariance Axiom. These axioms have endorsed an essentially relative approach (with respect to income-size and population-size respectively) to the measurement of inequality and poverty. The present paper is an expository essay which aims to clarify the logical and ethical limitations of either a purely relative or a purely absolute approach to distributional measurement. In the process, it also reviews two proposals - due to Manfred Krtscha and Eduardo Arriaga respectively - for ‘intermediate’ measures of inequality and poverty, which moderate the ‘extreme’ values underlying relative and absolute measures by combining these opposing values in a simple product formula.

Keyphrases: philosophy, ethics, economics, Scale Invariance, Translation Invariance, Unit Consistency, Replication Invariance, Replication Scaling, Population Replication Principle, philosophy, ethics, economics, Invariance