Several factors direct the placement of specific nucleosomes, which in turn have the ability to regulate DNA accessibility. These factors include, but are not limited to, nucleotide sequence preference, nucleotide modifications, the type of histones present within the nucleosome, and the presence of additional transcription factor or chromatin remodelers. A combination of these and other factors are responsible for tightly controlled efficient transcription within the eukaryotic cell. In order to contribute to the understanding of these complicated processes, three separate hypothesis-driven investigations were carried out. First, we looked into nucleosome positioning and phasing within closely related cells lines. Second, we examined domain level nucleosome occupancy on various portions of the chromosome. Finally, we generated a novel method that significantly reduces data loss in in vitro nucleosome reconstitution experiments caused by nucleosome fragment-end bias. All three of our investigations yielded separate results. First, by examining positions and phasing patterns within similar cell types we find common patterns and minor differences within similar cell types. The presence of minor differences in nucleosome positions may cause unique expression patterns. Secondly, we found that decreased domain level nucleosome occupancy at the chromosome arms is not caused by the presence of a class of nucleosomes, termed fragile nucleosomes. Finally, we found that when our nucleosome recovery method is applied conservatively to our dataset, it is possible to recover 80% of the lost nucleosome reconstitution data.

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Immunotherapy offers a promising approach to cancer treatment by harnessing a patient’s own immune system to fight malignant cells. However, the clinical application of immunotherapy has been hindered by the immunosuppressive tumor microenvironment generated by cancer cells as a mechanism to impede immune function and evade immune detection. Clinically used immunotherapies, such as immune checkpoint inhibitors and adoptive cell therapy, aim to overcome the immunosuppressive tumor microenvironment by blocking key regulatory pathways and exogenously activating immune cells. While effective against some cancers, these therapies are still limited by systemic toxicity, poor delivery kinetics, and continuous tumor adaptation that leads to immune escape. Herein, we propose the synthesis of nanoscale branching DNA architectures, known as dendrons, to (1) encode and deliver a DNA sequence, termed G3YSD, capable of activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway; and (2) deliver epigenetic modifiers to reprogram immunosuppressive cues in tumor cells. This solution exploits the modularity, programmability, and ease of control over DNA synthesis to generate architectures that exhibit improved delivery kinetics and favorable presentation of cargo to enhance immunomodulatory effects. Our proposed solution directly targets immunosuppressive mechanisms in tumor cells to sensitize them to immune attack and make them more easily recognized by the immune system. Delivery of G3YSD-encoding dendrons to murine B16 melanoma significantly increased the expression of major histocompatibility complex I (MHC I) and programmed cell death-ligand 1 (PD-L1) surface-bound receptors, which are critical for immune signaling pathways. The chemical conjugation of romidepsin, a histone deacetylase inhibitor, to G3YSD-encoding dendrons resulted in more than a 2-fold increase in MHC I expression compared to unconjugated G3YSD sequences and free romidepsin, indicating that the spatial arrangement and presentation of romidepsin has a synergistic impact on cGAS-STING signaling. In addition, pretreatment of B16 melanoma cells with zebularine, a DNA methyltransferase inhibitor, followed by G3YSD-encoding dendrons significantly increased levels of cytotoxic T lymphocyte-mediated lysis in a physiologically relevant co-culture. Developing novel architectures capable of interacting with tumor cells to remodel and overcome immunosuppressive cues will lead to significant advances in the field of immunotherapeutic design and cancer treatment. / 2026-05-23T00:00:00Z

This dissertation presents applications of machine learning and statistical approaches to infer protein-DNA bindings in the presence of epigenetic modifications. Epigenetic modifications are alterations to the DNA resulting in gene expression regulation where the structure of the DNA remains unaltered. It is a heritable and reversible modification and often involves addition or deletion of certain chemical compounds to the DNA. Histone modification is an epigenetic change that involves alteration of the histone proteins – thus changing the chromatin (DNA wound around histone proteins) structure – or addition of methyl-groups to the Cytosine base adjacent to a Guanine base. Epigenetic factors often interfere in gene expression regulation by promoting or inhibiting protein-DNA bindings. Such proteins are known as transcription factors. Transcription is the first step of gene expression where a particular segment of DNA is copied into the messenger-RNA (mRNA). Transcription factors orchestrate gene activity and are crucial for normal cell function in any organism. For example, deletion/mutation of certain transcription factors such as MEF2 have been associated with neurological disorders such as autism and schizophrenia. In this dissertation, different computational pipelines are described that use mathematical models to explain how the protein-DNA bindings are mediated by histone modifications and DNA-methylation affecting different regions of the brain at different stages of development. Multi-layer Markov models, Inhomogeneous Poisson analyses are used on data from brain to show the impact of epigenetic factors on protein-DNA bindings. Such data driven approaches reinforce the importance of epigenetic factors in governing brain cell differentiation into different neuron types, regulation of memory and promotion of normal brain development at the early stages of life. / Doctor of Philosophy / A cell is the basic unit of any living organism. Cells contain nucleus that contains DNA, self replicating material often called the blueprint of life. For sustenance of life, cells must respond to changes in our environment. Gene expression regulation, a process where specific regions of the DNA (genes) are copied into messenger RNA (mRNA) molecules and then translated into proteins, determines the fate of a cell. It is known that various environmental (such as diet, stress, social interaction) and biological factors often indirectly affect gene expression regulation. In this dissertation, we use machine learning approaches to predict how certain biological factors interfere indirectly with gene expression by changing specific properties of DNA. We expect our findings will help in understanding the interplay of these factors on gene expression.

Transgenerational epigenetic inheritance in mamals is a widely discussed topic in today's biology. Epigenetic modifications are molecules that play a crucial role in regulation of gene transcription. Epigenetic modifications regulate another epigenetic modification's establishment. The extrinsic and the intrinsic cellular or organismal environment is involved within the establishment of epigenetic state. The molecules involved in epigenetic processes are able to regulate gene transcription in reaction to the environment and therefore these molecules partly shape the phenotype. Most importantly, epigenetic processes are affected by cellular or organismal history. A question emerges: Are these molecules able to transfer information through germline to subsequent generations? Does transgenerational epigenetic inheritance in mammals exist? Experimental data show it is so. What consequences this can mean in our understanding of evolution? Powered by TCPDF (www.tcpdf.org)

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Des changements de méthylation de l’ADN peuvent affecter l’expression des gènes et pour certains être transmis au travers des générations. De telles « épimutations » qui concernent des groupes de cytosines à proximité ou dans les gènes sont donc une source potentielle de variation phénotypique héritable en absence de changements de la séquence de l’ADN. Chez les plantes la méthylation de l’ADN est cependant principalement observée au niveau des séquences répétées. Il reste à déterminer dans quelle mesure les changements de méthylation au niveau de ce type de séquences peuvent être héritées et affecter les phénotypes. Afin de répondre à ces questions, plus de 500 épiRIL (epigenetic Recombinant Inbred Lines) quasi-isogéniques a été générée chez Arabidopsis thaliana. Cette population a été obtenue par le croisement d’un parent sauvage et d’un parent mutant pour le gène DDM1 présentant une très forte réduction du taux de méthylation de l’ADN. Après un rétrocroisement de la F1 avec une plante sauvage, les individus sauvages pour le gène DDM1 ont été sélectionnés et propagées sur 6 générations par autofécondation. Nous avons montré par l’analyse du méthylome de plus de 100 épiRIL que l’hypométhylation induite par ddm1 présente selon les séquences affectées différents degrés de transmission au travers des générations. La réversion de l’hypométhylation concerne des régions associées à une abondance élevée en sRNA de 24 nt. Nous avons utilisé l’hypométhylation stablement transmise dans les épiRIL induite par ddm1 afin de détecter des QTL (Quantitative Trait Loci) affectant le temps de floraison et la longueur de la racine primaire, deux caractères pour lesquels les variations observées dans les épiRIL présentent une héritabilité importante. En dernier lieu, nous avons recherché par différentes approches les variations causales de ces QTL. / Loss or gain of DNA methylation can affect gene expression and is sometimes transmitted across generations. Such epigenetic alterations, which concern clusters of cytosines located near or within genes, are thus a source of heritable phenotypic variation in the absence of DNA sequence change. In plants however, DNA methylation targets repeat elements predominantly and it remains unclear to which extent DNA methylation changes over repeat sequences can be inherited and affect phenotypes. To address these issues, a population of near-isogenic, epigenetic Recombinant Inbred Lines (epiRILs) was generated in Arabidopsis thaliana. These were derived from a cross between a wild type and an isogenic ddm1 mutant line, in which DNA methylation is compromised specifically over repeat elements. After backcrossing of the F1 and selection of the progeny homozygous for wild-type DDM1, the epiRILs were propagated through six rounds of selfing. Analysis of the methylomes of more than 100 epiRILs and of the parents, indicates that ddm1-induced hypomethylation exhibit different patterns of inheritance through generations. Reversion of ddm1-induced hypomethylation is observed for regions associated with high level of 24 nt siRNA. Based on these findings, stable ddm1-induced hypomethylated regions were used to map quantitative trait loci (QTL) for flowering time and primary root length, two complex traits for which substantial heritable variation is observed in the epiRIL population. We finally analysed these QTL by different approaches to find their causal variations.

Le neuroblastome à haut risque est caractérisé par un faible taux de survie (~30%) et des récidives fréquentes, malgré les traitements multimodaux existants. Généralement, les études d’évaluation des médicaments utilisent la culture cellulaire en 2D, mais elle ne reflète pas la biologie tumorale du neuroblastome in situ, incluant les caractéristiques associées à l’hypoxie et la densité cellulaire. En comparaison aux patients, la culture cellulaire conventionnelle semble altérer le phénotype cellulaire du neuroblastome, le transcriptome et l’épigénome qui affecteront, à leur tour, les résultats des études pharmacologiques. Ainsi, un nouveau modèle de culture cellulaire en 3D a été développé avec plusieurs lignées cellulaires de neuroblastome afin d’atteindre une culture de sphéroïdes à long terme (un mois) avec un taux de viabilité convenable. L’hypothèse de recherche est que les changements épigénétiques et transcriptionnels seront induits par l’adaptation en 3D et vont s’amplifier dans le temps lors de la culture en 3D. Ces changements ont été mesurés en 3D dans le temps pour identifier le moment où l’épigénome des sphéroïdes ressemble le plus à celui des patient à haut risque. Le changement de l’expression des régulateurs épigénétiques survient 24 jours après la mise en sphéroïde, ce qui se traduit par une différence dans la sensibilité aux médicaments épigénétiques par rapport à la culture 2D. En conclusion, notre étude nous a permis de dériver des nouveaux modèles de neuroblastome qui sont plus représentatifs des patients d’un point de vue épigénétique et pharmacologique. / High-risk neuroblastoma is characterized by a low survival rate (~30%) and frequent recurrences, despite all the multimodal treatments available to date. Typically, drug evaluation studies use 2D cell culture which does not reflect well the tumor biology of neuroblastoma in situ, including features associated with hypoxia and cell density. Thus, compared to patients data, the conventional 2D cell culture seems to alter the neuroblastoma cell phenotype, transcriptome and epigenome which in turn will affect the results of pharmacological studies. A new 3D cell culture model was developed with several neuroblastoma cell lines to achieve long term spheroid culture (up to one month) with a suitable viability rate. We hypothesized that transcriptional and epigenetic changes will be induced by 3D adaptation and will amplify over time in the cells cultured in 3D. All these changes were measured by Western Blot in 2D, in short term 3D and in long term 3D to identify the timepoint when the epigenome of the spheroids most closely resembles that of the patient. We found that the occurrence of changes in epigenetic regulator expression occurs after 24 days of spheroid culture, resulting in a difference in a drug sensitivity compared to 2D culture. In summary, we developed new neuroblastoma models that are more representative of patient’s epigenome and pharmacological sensitivity.

Developing behaviours for interaction with objects close to the body is a primary goal for any organism to survive in the world. Being able to develop such behaviours will be an essential feature in autonomous humanoid robots in order to improve their integration into human environments. Adaptable spatial abilities will make robots safer and improve their social skills, human-robot and robot-robot collaboration abilities. This work investigated how a humanoid robot can explore and create action-based representations of its peripersonal space, the region immediately surrounding the body where reaching is possible without location displacement. It presents three empirical studies based on peripersonal space findings from psychology, neuroscience and robotics. The experiments used a visual perception system based on active-vision and biologically inspired neural networks. The first study investigated the contribution of binocular vision in a reaching task. Results indicated the signal from vergence is a useful embodied depth estimation cue in the peripersonal space in humanoid robots. The second study explored the influence of morphology and postural experience on confidence levels in reaching assessment. Results showed that a decrease of confidence when assessing targets located farther from the body, possibly in accordance to errors in depth estimation from vergence for longer distances. Additionally, it was found that a proprioceptive arm-length signal extends the robot’s peripersonal space. The last experiment modelled development of the reaching skill by implementing motor synergies that progressively unlock degrees of freedom in the arm. The model was advantageous when compared to one that included no developmental stages. The contribution to knowledge of this work is extending the research on biologically-inspired methods for building robots, presenting new ways to further investigate the robotic properties involved in the dynamical adaptation to body and sensing characteristics, vision-based action, morphology and confidence levels in reaching assessment.