Prof. Denis Lafontaine

Dr Nicole Borghi

Prof. Johannes Dr Geiselmann

Dr Olivier Destaing

Anouk Emadali

Sara Badawi

on nucleolar structure and function Etudier l'impact

Keywords: mammary epithelial cells, compressive forces, nucleus, nucleolus, nucleolar proteins

I want to thank 'La Ligue Contre le Cancer' for the three-year scholarship and the Institute of Advanced Biosciences for their warm welcoming.

Second, I want to express my gratitude to the entire team led by Christophe GUILLUY and Laurence lAFANECHERE. Here I say, thank you for the warm welcome, for all of our meetings and discussions, and for the scientific exchange. It was an honor to meet you all and learn from your experiences. My sincere gratitude goes out to Dr. Monika DOLEGA. I appreciate the chance to work on this project, the insightful conversations we had, and of

for his advice that was more than work, but also more to me as a PhD student, and here I say, 'Merci pour le conseil, vos mots resteront gravés dans ma tête!'. I would like also to thank my friends in the IAB: Amani Shreim, Khaldoun Gharzeddine, and Nour El Hussein, Line Chaar, thanks for their support and motivation. Truly grateful to all of my friends I've met abroad for their encouragement, which has kept me going. Special thanks to Nour Kourani, Mahdi Shaheen and Hassan El Ali and here I want to say: You have called me Dr from the first year of my PhD study and believed in me. All I can say is thank you! And hopefully, I shall aspire to what I have been waiting all these years. Last but not least, I could not have done this work without the help of my family. I'd like to take this opportunity to express how grateful I am to have them in my life. 'Thank you for always believing in me and supporting me,' I'd like to say. Thank you for always being by my side and never giving up on me, whether I was at the top of the mountain or the bottom. Despite the distances in between, you are far apart but close at heart. lamina is more than nuclear membrane meshwork, and chromatin remodels under stress. II) Regulation of cellular homeostasis under an applied mechanical force: is the nucleolus associated? III) Direction of epithelial folding defines the impact of mechanical forces on the epithelial state. IV) Recent studies investigating the interplay between mechanical cues and nucleolar structure and function.

Contents

3.Thesis objectives 4.Materials and Methods

-I) Cellular model and force application. I.1-Cellular model: 1.2-Methodology for the force application using an in vitro system.60 1.2.1-Flex-cell plate an in vitro system to apply uniaxial compressive forces 1.2.2-4D cell Dynamic confiner an in vitro system to apply confined cells between cell surface II)-Electron microscopy

List of Figures and Tables Figures:

Figure 1: Nucleolar organization of eukaryotic cells.. Figure 2: Localization of nucleolar proteins markers in the three nucleolar sub-compartments in human Hela cell.. Figure 3: Scheme of human ribosomal gene and their transcripts. Figure 4: Scheme showing factors required for transcription initiation by RNA polI in mammals.. Figure 5: Short-term regulation of rDNA transcription under different environmental cues.). Figure 6: Eukaryotic C/D box snoRNA and snoRNPs organization.. Figure 7: A model of FC/DFC organization and the 47S pre-rRNA sorting in the human nucleolus. Figure 8: Schematic illustration of nucleolar assembly by end of telophase.. Figure 9: Nucleolar reorganization upon stress.. Figure 10: rDNA transcription transcription and cell cycle are mutually regulated.. Figure 11: The mammary duct has a bilayered organization of luminal (epithelial) and myoepithelial cells.. Figure 12: Main molecular players acting at Integrin (green) and Cadherin (red) adhesion sites. Figure 13: Molecules contributing in the nuclear-cytoskeletal coupling.. Figure 14: Schematic illustration of Mechanotransduction processes. Figure 15: Development of mammary gland and the mechanical forces exerted on cells during different developmental stages. Figure 16: Mechanical forces during cancer initiation and progression.. Figure 17: In vitro systems used to apply mechanical compression, with significant findings.. Figure 18: Scheme showing the in vitro device used to apply uniaxial compressive and tensile forces.. Figure 19 Scheme showing steps followed to use the 4D in vitro dynamic device.. Figure 20 Creating a semi-quiescent monolayer of MCF10A epithelial cells.. Figure 21 In-plane mechanical compression induces reorganization of β -catenin at the cell-cell junctions Figure 22 Compressive forces impact nuclear morphology and induce lamin A/C remodeling.. Figure 23 Mechanical compression regulates the nucleolar tripartite organization particularly the fibrillar center. Figure 24 Compressive forces induces nucleolar remodeling. Figure 25 Mechanical forces trigger an increase in the abundance of proteins known to regulate nucleolar phase separation Figure 26 Mechanically stretched epithelial cells do not show a significant change in nucleolar structure and morphology. Figure 27 Compressive forces do not activate the p53 nucleolar stress pathways Figure 28 Mechanical compression regulates UBF abundance at 2 hours and localization after 4 hours.. Figure 29 Compressive forces for 4 hours attenuates rDNA transcription .. Figure 30 Compressive induces nucleolin release from the nucleolus.. Figure 31 Nucleolin is released in response to another type of applied compressive forces .

Figure 32 Nucleolin and UBF localization is not impacted by applied tensile stretch for 4 hours..

Tables:

Table 1: Antibodies used for immunofluorescence labeling 63.

Table 2: Antibodies used for protein analysis by western blotting

List of Abbreviations

List of Abbreviations

Preface

Over the past few decades, there has been broader recognition that mechanical signals arising from neighboring cells, the extracellular matrix, and fluid flow contribute to critical aspects of organ formation, tissue regeneration, and disease initiation and progression [START_REF] Paszek | The Tension Mounts: Mechanics Meets Morphogenesis and Malignancy[END_REF]. Among these biophysical signals, changes in tension, including tensile and compressive forces, regulate cellular function and influence the organization of surrounding tissues [START_REF] Chen | Tensile and Compressive Force Regulation on Cell Mechanosensing[END_REF]. In general, biophysical signals are sensed by surrounding cells via a complex network of adhesive receptors, mechanosensitive membrane channels, and cytoskeletal proteins [START_REF] Hoffman | Dynamic Molecular Processes Mediate Cellular Mechanotransduction[END_REF]). In addition, several studies have shown the mechanisms by which forces are transmitted to the nucleus and their ability to remodel the lamin that supports the nuclear membrane and to organize chromatin within the nucleoplasm [START_REF] Kalukula | Mechanics and Functional Consequences of Nuclear Deformations[END_REF]). As a result, these forces can drive cells to migrate, spread, invade, and induce many other cellular behaviors.

Although we know that an externally applied force can reshape the nuclear structure, little is known about the effect of the force on the most prominent organelle within the nucleus: the nucleolus, which in fact, forms a close association with mechanosensitive nuclear components (Buchwalter and Hetzer 2017b;[START_REF] Gupta | Lamin B2 Modulates Nucleolar Morphology, Dynamics, and Function[END_REF][START_REF] Martin | Lamin B1 Maintains the Functional Plasticity of Nucleoli[END_REF]). The nucleolus is a tripartite organelle that lacks a membrane. Each sub-compartment contains a unique component of proteins and rRNA that performs its function, which is the synthesis of ribosomal subunits [START_REF] Mélèse | The Nucleolus: An Organelle Formed by the Act of Building a Ribosome[END_REF]. Notably, besides the primary role of the nucleolus in ribosomal subunits formation, many other functions have been identified, including maintaining cellular homeostasis under various environmental stresses (Grummt 2013). The interplay between biophysical signals and the nucleolus is just emerging. It has recently been demonstrated that limited cell adhesion and extracellular matrix stiffness determine nucleolar structure and function [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF][START_REF] Jaecker | Nucleoli in Epithelial Cell Collectives Respond to Tumorigenic, Spatial, and Mechanical Cues[END_REF]. Only recently, two qualitative studies, showed that limited cell adhesion and extracellular matrix stiffness determine nucleolar structure and function [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF][START_REF] Jaecker | Nucleoli in Epithelial Cell Collectives Respond to Tumorigenic, Spatial, and Mechanical Cues[END_REF]). Yet, a profound characterization of nucleoli in terms of their structural organization and function under mechanical stress has never been addressed.

In recent work obtained by the team, we showed that compressive forces applied in the plane of monolayer of epithelial cells, regulates the transcriptional level of genes that encodes for ribosomal proteins and small non-coding RNA. Interestingly, based on these results, we asked the following question: What would be the common process that ribosomal proteins and snoRNA participates in? In fact, it is the ribosome biogenesis, a process that occurs in the nucleolus. Based on these results, in my thesis, I became interested in studying the effect of biophysical cues on nucleolar structure and function, particularly compressive forces. At the core of this topic, many questions were asked, (1) Can mechanical forces trigger standard pathways of nucleolar stress response. (2) Can external applied compressive forces impact the tripartite organization of the nucleolus, and if I could observe such changes, which subcompartment would be highly impacted (3) Besides nucleolar structural changes, could compressive forces affect nucleolar function and regulate nucleolar proteins localization. Moreover, if I can observe structural and functional changes, can we add

Introduction

Part 1 The Nucleolus: its Structure and Function Over a century passed between the first description of the nucleolus by Wagner in 1835 and Valentina in 1836-1839 and releasing of a definitive experiment showing that the nucleolus is a factory for ribosome subunits formation in the 1960s. After that, many researchers focused on investigating the nucleolar structure, defining its subcompartments, and determining what it does and how it does it. The acquisition of more information about the nucleolus has added more complexity.

Eukaryote's ribosomes are about 40% larger than those of prokaryotes. Hundreds of RNA modifications are found in eukaryotic RNA, which are relatively absent in prokaryotes. Indeed, a eukaryote's rDNA contains multiple copies of genes for rRNA formation. Therefore, for the efficient rDNA transcription, processing, and assembly of ribosomal subunits, the rDNA is carefully packaged within the compact structure of the nucleolus. The nucleolus is considered the most prominent structure within the nucleus of almost all eukaryotic cells.

In the first part of my manuscript, I will describe the structural organization of the nucleolus. Where the nucleolus appears as a tripartite structure, including three sub-compartments: the fibrillar center (FC), bounded by a dense fibrillar center (DFC), then many FC/DFCs are included in the granular center (GC), which represents the largest compartment. The main role of the nucleolus is to produce the ribosomes needed for protein synthesis. Here in the first part, I will briefly mention the major steps of ribosome biogenesis and relate them to each nucleolar compartment. Moreover, I will briefly describe few nucleolar proteins that contribute to different steps during the formation of ribosome subunits. The nucleolus assembly and disassembly are driven by liquid-liquid phase separation (LLPS). In addition, each of the nucleolar subcompartments has a unique complement of proteins and RNAs that execute its function and determine the sub-compartment's relative surface tension, thereby determining which droplets contact one another and which droplets will be embedded or partially contained with the other. Because of that, I will describe nucleolus assembly and disassembly as an example of LLPS. In addition, I will mention how LLPS and ribosome biogenesis impact nucleolar number and size. At the end of Part I, I will give examples of how cells under certain environmental challenges regulate ribosome biogenesis, which leads to the adaptation of the cell to environmental changes.

Structural organization of the nucleolus as a consequence of the dynamics of ribosome biogenesis I) Building blocks of nucleolar compartmentation

During the nineteenth century, numerous cytologists described the variability of nucleolar morphology only by using light microscopy (Montgomery Jr. 1898) (figure1; A). Since that time, the nucleolus has been the subject of several intense investigations, and our interest in the nucleolus has evolved due to several technical progress. With the discovery reported by Max Birnstiel and Ferrucio Ritossa in 1964, where they identified the presence of DNA inside the nucleolus based on nucleic acid hybridization, nucleolus gained much more attention (Ritossa and Spiegelman 1965). Soon after, it was discovered that individual chromosomes tend to occupy certain territories within the nucleus. Among these chromosome territories is the nucleolus. Later, the electron microscopy images of Oscar Miller and Barba Beatty revealed the transcripts of the genes residing in the nucleolus that appear as "Christmas trees" (figure1; C) (Miller and Beatty 1969). Later, the number of chromosomes contributing to nucleolus formation was determined to be five in mammals, where the following chromosomes 13,14,15,21, and 22 contain the rDNA genes on their acrocentric arms (Henderson, Warburton, and Atwood 1972). The ribosomal genes are designated by the nucleolarorganizing regions (NORs).

In addition to the nucleolar gene transcription described by transmission electron microscopy, the basic structure of the nucleolus was revealed using the same technique. The experiment showed that the nucleolus is composed of mainly three building blocks in mammals. These are the fibrillar center (FC), the dense fibrillar center (DFC), and the granular center (GC) (figure1; B). The fibrillar center appears as low-contrast areas around 5 nm in diameter [START_REF] Goessens | Nucleolar Structure[END_REF]. The latter is surrounded by highly contrasted regions corresponding to the DFC (D. [START_REF] Hernandez-Verdun | Structural Organization of the Nucleolus in Mammalian Cells[END_REF]. The FC/DFC is included in the GC, the largest compartment (Hadjiolov 1985;[START_REF] Hernandez-Verdun | Structural Organization of the Nucleolus in Mammalian Cells[END_REF]. By 1960 and1970, the notion that the nucleolus is the site of ribosome biogenesis was well-established in modern molecular and cellular biology. Scientists in the nucleolus field tried to address the details of ribosome subunit formation by linking the stages of this process with the classically defined sub-compartments revealed by electron microscopy. Adapted from [START_REF] Boisvert | The Multifunctional Nucleolus[END_REF]). B) Standard Electron microscopy preparation of Hela cells. The images show the three nucleolar compartments due to their different contrast: the fibrillar center (FC; asterisks), the dense fibrillar center (white arrows), and the granular center (GC). Scale bar: (a) = 0.5 µm and (b) = 0.1 µm. Adapted from (Danièle Hernandez-Verdun et al. 2010). C) rDNA repeats visualized by the Miller spread technique. Transmission electron micrograph of transcription of ribosomal RNA genes from the extrachromosomal nucleoli of a newt oocyte. Active transcription is seen by the tree-like structures "Christmas trees", in which each branch represents a nascent transcript. Adapted from (Miller and Beatty 1969).

The fibrillar center is characterized by specific topology and is well represented by its components. The fibrillar center house the enzymatic apparatus for rDNA transcription, including RNApol I, DNA topoisomerase I, transcription upstream binding factor (UBF), and several other proteins [START_REF] Goessens | Nucleolar Structure[END_REF]. The nucleoli of different cell types contain a variable fibrillar center of different number and sizes [START_REF] Hozák | Three-Dimensional Reconstructions of Nucleolus-Organizing Regions in PHA-Stimulated Human Lymphocytes[END_REF]. The level and efficiency of rDNA transcription determine the number and appearance of fibrillar centers [START_REF] Jordan | The Quantitative Relationship of the Fibrillar Centres and Other Nucleolar Components to Changes in Growth Conditions, Serum Deprivation and Low Doses of Actinomycin D in Cultured Diploid Human Fibroblasts (Strain MRC-5)[END_REF]. For example, in resting lymphocytes (G0), the nucleolar organizing regions (NORs) are in close contact, thus forming a common fibrillar center. Following the activation of cell proliferation and hence rDNA transcription using phytohemagglutinin (PHA), the number of fibrillar centers increases, accompanied by the development of dense fibrillar compartments. It has been anticipated that this increase is due to separating NORs from each other, thereby creating an individual fibrillar centers [START_REF] Hozák | Three-Dimensional Reconstructions of Nucleolus-Organizing Regions in PHA-Stimulated Human Lymphocytes[END_REF]. Interestingly, the authors showed that these multiple formed FCs are localized into one single fibrillar complex surrounded by the dense fibrillar center.

The dense fibrillar center surrounds the fibrillar center, where nascent rRNA is detected as well as protein and processing machinery related to the early event of rRNA processing (Cmarko et al. 2000). The main proteins in the DFC are fibrillarin, NOPP140, and snoRNPs. These are critical players in rRNA processing, hence the mature rRNA formation (Ochs, Lischwe, Spohn, et al. 1985).

Granular components correspond to the largest part detected by electron microscopy (15-20nm). This compartment corresponds to the pre-ribosomal subunit's assembly (the small and the large subunits). The protein markers of the GC are abundant such as nucleophosmin (B23) [START_REF] Biggiogera | Simultaneous Immunoelectron Microscopic Visualization of Protein B23 and C23 Distribution in the HeLa Cell Nucleolus[END_REF]), Bop1 [START_REF] Pestov | Evidence of P53-Dependent Cross-Talk between Ribosome Biogenesis and the Cell Cycle: Effects of Nucleolar Protein Bop1 on G(1)/S Transition[END_REF], and Nop52 [START_REF] Savino | The Nucleolar Antigen Nop52, the Human Homologue of the Yeast Ribosomal RNA Processing RRP1, Is Recruited at Late Stages of Nucleologenesis[END_REF].

Therefore, the nucleolus is a tripartite structure where each compartment is characterized by specific proteins (figure 2) that are needed for ribosome subunits formation. The relative protein quantity of different compartments reflects the dynamic complexity of each step.

.

II) Role of the nucleolus in ribosome biogenesis

As I mentioned above, the main role of the nucleolus is ribosome biogenesis, this process starts in the nucleolus, but additional maturation events occur in the nucleoplasm and cytoplasm. In eukaryotes, ribosome biogenesis is a highly coordinated mechanism that requires the activity of three RNA polymerases (PolI, PolII, PolIII), 75 small nucleolar RNAs (snoRNAs), and more than 250 ribosomal biogenesis factors (RBFs) [START_REF] Brombin | New Tricks for an Old Dog: Ribosome Biogenesis Contributes to Stem Cell Homeostasis[END_REF]. This process is divided into six significant critical steps: (i) synthesis of the components (rRNAs, ribosomal proteins RPs, snoRNAs, assembly factors AFs), (ii) processing of pre-rRNA through cleavage, (iii) covalent modifications of pre-RNAs, RPs and AFs, and (iv) assembly of the mature rRNA with ribosomal proteins, (v) transport and (vi) quality control over the ribosomes in the cytoplasm. This part aims to give an overview of the different steps of the pathway in ribosome biogenesis, how it's affected by cellular environmental changes and the importance of ribosome biogenesis in maintaining cell homeostasis II.1-rDNA organization, transcription, and regulation Ribosomal DNA (rDNA) is organized in repeated sequences with ~400 copies in human cells (figure 3) (Henras et al. 2015). Interestingly, the presence of many strongly transcribed rDNA genes allows the generation of a high number of rRNAs, thereby fulfilling the great demand for ribosome production. However, only a fraction of these repeats are actively transcribed [START_REF] Birch | Structure and Function of Ribosomal RNA Gene Chromatin[END_REF], and the inactive regions have a potential role in maintaining genome integrity [START_REF] Hernandez-Verdun | Ribosomal DNA and the Nucleolus in the Context of Genome Organization[END_REF]. Due to this difference in repeats activity, the rDNA chromatin in mammalian cells can be subdivided into three states according to the transcriptional and chromatin state and epigenetic features: transcriptionally competent, transcriptionally active, and inactive rDNA [START_REF] Hamperl | Chromatin States at Ribosomal DNA Loci[END_REF]. The first two states are euchromatic and may oscillate between facultative and constitutive transcriptional activation, while the third represents heterochromatin stably silenced genes. The silent rRNA genes are characterized by the presence of DNA methylation at the promoter region and are associated with repressive histone marks such as histone 3 lysine 9 dimethylation (H3K9me2), and histone 3 lysine 9 trimethylation (H3K9me3), and deacetylated histones [START_REF] Zhou | The Chromatin Remodeling Complex NoRC Targets HDAC1 to the Ribosomal Gene Promoter and Represses RNA Polymerase I Transcription[END_REF].

Ribosome biogenesis starts with the transcription of rRNA genes (18S, 5.8S, and 28S) at the border of FC/DFC, precisely transcribed by RNA polymerase I (RNA polI). An additional rRNA, the 5S, is transcribed independently in the nucleoplasm from multiple genes by RNA pol III. It is important to note here that the transcriptional machinery of 18S, 5.8S, and 28S is different from 5S (Ciganda and Williams 2011). The mechanism required for 5S transcription is not mentioned in the manuscript. Transcription of rDNA comprises a series of coordinated steps, including the following: transcription initiation, promoter escape, elongation, and termination. rDNA transcription starts with the recruitment and assembly of RNApolI, with several other transcriptional proteins, into the pre-initiation complex at the rRNA gene promoter. In mammalian cells, the pre-initiation complex (PIC) is composed of RNA polymerase I (RNApolI), selectivity factor (SLI), and upstream binding factor (UBF) (figure 4). Since UBF is one of the nucleolar proteins I addressed in my thesis, I will briefly describe this protein, and how certain cellular conditions regulate rDNA transcription through UBF. Indeed, following the binding of UBF to the promoter region, it loops approximately 140 base pairs (bp) of DNA into a single turn through its high mobility binding domain. This remodeling will allow the displacement of nonspecific DNA binding proteins (e.g., histone H1) from the rDNA promoter region, thus rendering rDNA into an open chromatin state, allowing recruitment of RNA polI, therefore, transcriptional activation [START_REF] Kuhn | The Nucleolar Transcription Activator UBF Relieves Ku Antigen-Mediated Repression of Mouse Ribosomal Gene Transcription[END_REF].

Once the pre-initiation complex is formed, the chromatin is in an open state, the nucleotide starts to be incorporated, and transcription is initiated. Promoter escape is a step that describes the RNApolI clearance from the promoter, achieved by several inhibiting interactions, thus allowing elongation of the transcription [START_REF] Panov | A Step Subsequent to Preinitiation Complex Assembly at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA Polymerase I-Dependent Transcription[END_REF]. The rRNA formation has been observed by electron microscopy, which is referred to as the Christmas tree as mentioned above. Indeed, multiple rounds of transcription can occur in parallel due to the re-binding of UBF-SLI to rRNA genes. Part I The Nucleolus: Structure and Function 19 Transcription of rDNA can be efficiently regulated to be responsive to both general metabolic processes and specific environmental challenges. Based on that, rDNA regulations have been categorized into short-and long-term.

Long-term regulation involves control of the active and inactive status of rDNA loci through epigenetics regulations, thereby regulating the number of active genes. These modifications of the chromatin states are associated with specific histone modifications, including methylation and acetylation, that can be mediated by a chromatin complex termed NoRC. The NoRC (nucleolar remodeling complex) is a member of SNF2h-containing chromatin remodeling complexes. TIP5, the large subunit of NoRC, targets histone-modifying enzymes to rDNA to generate a repressive heterochromatic structure [START_REF] Zhou | The Chromatin Remodeling Complex NoRC Targets HDAC1 to the Ribosomal Gene Promoter and Represses RNA Polymerase I Transcription[END_REF].

On the other hand, short-term regulations are triggered in response to environmental changes leading to regulations of the transcription rate of rRNA genes (figure 5,A) [START_REF] Kusnadi | Regulation of RDNA Transcription in Response to Growth Factors, Nutrients and Energy[END_REF]. Conditions that attenuate rDNA transcriptions include oxidative stress, nutrient starvation, and inhibition of protein synthesis and cell confluence (Grummt 2013). On the contrary, a growth factor might stimulate proliferation, and cell cycle activation will eventually upregulate rDNA transcription (figure 5,A). In most cases regulating rDNA transcription can occur by regulating RNApolI activity or other crucial players in the PIC complex as UBF. Several signaling pathways, such as the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin complex 1 (mTORC1) forms an intricate network with proteins that control rDNA transcription (UBF, RNApolI, SLI), such signaling pathways can be activated/inhibited under environmental challenges (nutrient availability, oxidative stress, heat shock) (figure 5,B) [START_REF] Bavelloni | Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster[END_REF][START_REF] Kusnadi | Regulation of RDNA Transcription in Response to Growth Factors, Nutrients and Energy[END_REF]. Part I The Nucleolus: Structure and Function 20 For example, increasing cell density and exhaustion of available nutrients inhibits the mTOR pathway in different cellular models. Inhibition of the mTOR pathway inactivated downstream ribosomal protein S6 [START_REF] Trajkovic | Fluctuations in Cell Density Alter Protein Markers of Multiple Cellular Compartments, Confounding Experimental Outcomes[END_REF], a component of the 40S ribosomal subunit and known to activate UBF, a crucial component of the PIC complex. In the absence of pS6 activity, rDNA transcription is attenuated [START_REF] Kusnadi | Regulation of RDNA Transcription in Response to Growth Factors, Nutrients and Energy[END_REF]; hence the overall protein production decreases. An essential protein of the PIC complex that I have addressed in my study is UBF (upstream binding factor).

UBF was discovered by Paule in 1986 (Bateman and Paule 1986) and is considered a critical factor in maintaining the rDNA chromatin state. UBF is about 764 amino acids with 4 HMG box DNA-binding motifs, a dimerization domain in its N-terminus, and a highly acidic C-terminal domain. Through these HMG boxes, UBF can bind and bend DNA, thus making it more accessible to other transcriptional factors. In this regard, a naturally occurring splice variant of UBF, UBF2, harbors a deletion in HMG box2, thus reducing its ability to bend the DNA and subsequently remodel chromatin (Sanij and Hannan 2009). The first logical explanation for the function of UBF came from various studies that showed enrichment of UBF across entire rDNA repeats to a much lesser extent with the non-transcribed spacer sequence. This was detected using UBF chromatin immunoprecipitation followed by large-scale parallel sequencing (ChIP-Seq). In 2008, Sanij and his colleagues showed further evidence for UBF in maintaining the euchromatin structure by depleting mouse fibroblast cells of UBF. Such depletion resulted in stable and reversible methylation of rRNA genes determined by psoralen accessibility (Sanij and Hannan 2009). Part I The Nucleolus: Structure and Function 21 Interestingly, the cellular amount of UBF has been shown to correlate strongly with the growth rate of non-dividing states in several cellular models, including cultured neonatal cardiomyocytes. The direct overexpression of recombinant UBF1 is sufficient to increase the activity of the reporter significantly for rRNA gene transcription in neonatal cardiomyocytes, even in the absence of hypertrophic stimuli [START_REF] Brandenburger | Chromatin Condensation[END_REF].

A protein that can regulate rDNA is not necessarily to be part of the pre-initiation complex. Examples on such proteins are nucleolin and fibrillarin, other nucleolar proteins that I studied their level of abundance and localization within the nucleolus under mechanical stress in my PhD project. Nucleolin is one of the most abundant nucleoproteins and localizes into the DFC and the GC sub-compartment. Escande-Géraud demonstrated nucleolin's role in regulating rDNA transcription in 1985, whereby inhibiting rDNA transcription, they could observe nucleolin release from the dense fibrillar zone (Escande-Géraud et al. 1985). Their findings suggested that nucleolin presence in the sub-nucleolar compartment depends on rDNA transcription. However, the mechanism by which nucleolin regulates rDNA transcription was still not fully described. Until 1998, Erard and his colleagues used circular dichroism coupled with binding assays between nucleolin and histone H1, and they showed that nucleolin interacts with histone H1 and induces chromatin decondensation. The explanation for their results was that the nucleolin N-terminal domain that is rich in acidic amino acids enables nucleolin to bind to the non-transcribed spacer between rDNA repeats and H1. This binding weakens the H1-nucleosome interaction, resulting in chromatin decondensation (Erard et al. 1988). The N-terminal region, which enables such protein-protein and nucleic acid interactions, is highly posttranslationally modified by several kinases, including casein Kinase II (CKII)* and cdc2*. This phosphorylation regulates nucleolin function and localization during the cell cycle. For example, CKII phosphorylates the acidic stretches at serine residues during interphase. This results in nucleolin activation and, by itself, triggers rDNA transcription. The basic stretches are phosphorylated at threonine by cyclin-dependent kinase (CDKII)* at the beginning of prophase, thus allowing the protein to localize into the cytoplasm before the nuclear membrane breaks down [START_REF] Ginisty | Structure and Functions of Nucleolin[END_REF].

On the other hand, fibrillarin, a protein described by Christensen initially in Physarum polycephalum as a highly abundant nucleolar protein associated with snoRNA of C/D box (Christensen et al. 1977). Fibrillarin's main role is to direct rRNA post-transcriptional modifications. More recently, Tessarz et al. have shown that human FBL is also a histone methyl-transferase, where they identified a single glutamine (Q105) in histone 2A (H2A) as the site of methylation and showed that FBL is responsible for this enzymatic activity. The methylation catalyzed by fibrillarin is restricted to histone H2A within the nucleolus. It acts as a platform to recruit and regulate other chromatin-regulatory factors and chromatin remodeling complexes to the rDNA thereby regulating rRNA genes transcription [START_REF] Tessarz | Glutamine Methylation in Histone H2A Is an RNA-Polymerase-I-Dedicated Modification[END_REF].

Introduction

Part I The Nucleolus: Structure and Function

22

II.2-rRNA maturation: Cleavages and post-transcriptional modifications

The transcription of rDNA produces the nascent pre-mature rRNA (47S) that contains the sequence of 18S, 5.8S, and 28S rRNA separated by ITSs and flanked by 5' and 3' ETSs (figure 3). The maturation of the pre-rRNA occurs in the DFC while RNA polI is still transcriptionally active at the FC/DFC border. The maturation step includes pre-rRNA (1) cleavage and (2) post-transcriptional modifications, achieved by snoRNAs, ribosomal proteins, assembly factors, and nucleolar proteins. The transient interaction between pre-rRNA with the assembly factors and ribosomal proteins leads to the formation of the pre-ribosomal particles.

Indeed, the pre-rRNA contains cleavage sites that are precisely located and strictly ordered. Several rRNA modifying enzymes and factors, such as endo-and exonucleases and putative RNA helicases, are required during the cleavage processes. The cleavage of the 5'-ETS necessitates ribosomal proteins (Ferreira-Cerca et al. 2005), snoRNAs, and small subunit (SSU) processome U3 (Dragon et al. 2002). On the other hand, ITSs processing is achieved by factors such as BOP1, NOL12, and ribosomal proteins of the large subunits (Lapik et al. 2004). A default in the location or the progression of these cleavages and modifications can lead to aberrant mature rRNAs and, therefore, defective ribosomes.

Briefly, in human cells, the maturation steps start in the nucleolus, where the 47S rRNA is first cleaved at the extremities giving rise to the pre-mature 45S rRNA. The formed 45S rRNA can be further matured through alternative pathways that vary in the location of the cleavage site, with variable kinetics and orders producing different intermediates but leading to the same outcome, 18S, 5.8S, and 28S rRNAs, assembled into pre-40S and pre-60S ribosomal particles [START_REF] Mullineux | Mapping the Cleavage Sites on Mammalian Pre-RRNAs: Where Do We Stand?[END_REF]. 5S rRNA that RNA polIII transcribe in the nucleoplasm is incorporated into the mature large ribosomal subunit, the 60S. The pre-ribosomal particles pre-40S and pre-60S are exported into the cytoplasm. The addition of a large number of post-transcriptional modifications accompanies the maturation of the rRNA. Such modifications include 2'-0-ribose methylation, pseudouridylation, and rDNA base methylation. The major chemical modifications are mostly guided to the pre-rRNA by snoRNAs within the core of a complex referred to as the ribonucleoprotein (RNP) complex. Many snoRNAs are processed from intronic sequences of genes coding nucleolar proteins related to biogenesis as nucleolin and fibrillarin. However, other snoRNA are synthesized from their promoter by polII or polIII [START_REF] Bratkovič | The Many Faces of Small Nucleolar RNAs[END_REF]. The role of these snoRNAs is to provide a scaffold by which partner proteins assemble and thus act as a guide to recognizing specific rRNA sequences.

Introduction

Part I The Nucleolus: Structure and Function

23

Interestingly, methylation of the rRNA is carried out on more than a hundred sites on the pre-rRNA. On the other hand, the H/ACA box catalyzes the pseudouridylation, and H/ACA snoRNA is associated with core proteins such as Nhp2p, Nop10p, and dyskerin (Kiss, Fayet-Lebaron, and Jády 2010). The association of snoRNAs with different sets of proteins allows them to form small nucleolar ribonucleoprotein (snoRNP) complexes. Strikingly, the snoRNA-guided modifications are not evenly distributed all over the ribosome, but instead, they are clustered on important functional regions, thus allowing ribosomal activity. These regions include the decoding and tRNA binding sites (A-, P-, E-sites), the peptidyl transferase center (PTC), and the intersubunit interface [START_REF] Sloan | Tuning the Ribosome: The Influence of RRNA Modification on Eukaryotic Ribosome Biogenesis and Function[END_REF]. Indeed, snoRNA-mediated rRNA modification is emerging as a significant source of ribosome heterogeneity [START_REF] Sloan | Tuning the Ribosome: The Influence of RRNA Modification on Eukaryotic Ribosome Biogenesis and Function[END_REF][START_REF] Marcel | Ribosome Heterogeneity in Tumorigenesis: The RRNA Point of View[END_REF].

Notably, snoRNA also directs modifications that are not limited to RNA within the nucleolus but also many mRNAs within the nucleoplasm and cytoplasm (Holley et al. 2015). These mRNA modifications provide an essential post-transcriptional regulatory mechanism to regulate the translational processes and protein production. For example, it has been shown that the abundance of Peroxidasin [PXdn], which has been reported to function in extracellular matrix consolidation and collagen IV crosslinking, is regulated through mRNA methylation catalyzed by fibrillarin and guided by box C/D snoRNAs (Elliott et al. 2019). As I have mentioned in the rDNA transcription section, fibrillarin is one of the crucial proteins in the snoRNAs complex that is directly involved in different posttranscriptional processes like pre-rRNA cleavages, rRNA modifications, and ribosome assembly. Within the nucleolus, fibrillarin plays several roles. Through its C-terminal domain that contains methyltransferase region (MD), fibrillarin methylates several sites on the pre-rRNA. Despite the variation of rRNA methylated sites among several organisms, fibrillarin is the main candidate for such modifications [START_REF] Shubina | Nucleolar Methyltransferase Fibrillarin: Evolution of Structure and Functions[END_REF]. In addition, fibrillarin directs the 5' end of 47S pre-rRNA sorting through the DFC (figure 7). At first, fibrillarin binds to the 5' ends of nascent pre-rRNA through its N-terminal domain (MD), and then the formed FBL-RNA complex moves outward thereby creating the DFC [START_REF] Yao | Nascent Pre-RRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus[END_REF].

The directional movement of the pre-rRNA by fibrillarin from the site of transcription (FC/DFC) border into the site of processing (DFC) contributes to the formation of the nucleolar tripartite organization. This sorting also prevents individual pre-rRNA from unproductive inter-and intra-molecular interactions that hamper nucleolus compartmentalization. Fibrillarin methylation activity is not limited to the rRNA within the nucleolus; several analyses have demonstrated that 2'-O-methylation by fibrillarin can also be directed on distinct RNA types such as small nuclear RNA (snRNAs) and mRNAs [START_REF] Shubina | Nucleolar Methyltransferase Fibrillarin: Evolution of Structure and Functions[END_REF]. Besides fibrillarin and its role in rRNA processing, nucleolin was demonstrated to play a role in the correct folding of rRNA and its processing (reviewed in [START_REF] Srivastava | Molecular Dissection of Nucleolin's Role in Growth and Cell Proliferation: New Insights[END_REF]. A precise experiment supported this in 1998, where HEP-2 cells were electroporated with an anti-nucleolin antibody, which resulted in increased nucleolar size. The nucleolar morphological changes were accompanied by an enlargement in the dense fibrillar center, where pre-RNA processing occurs. It was speculated to be an accumulation of the pre-rRNAs that are not matured yet, thus suggesting a pivotal role for nucleolin in pre-rRNA processing [START_REF] Abadía-Molina | Inhibition of Nucleolar Protein Nucleolin by Electroporation with Anti-Nucleolin AntibodiAdam[END_REF].

II.3-Ribosome formation and quality control

During the pre-rRNA maturation, much different ribosomal proteins and assembly factors are coupled to the rRNA, thus forming pre-ribosomal subunits. While still undergoing maturation, these pre-ribosome subunits roam in the nucleolus and into the nucleoplasm. Within the nucleoplasm, they still undergo maturation, where many of the already-added ribosomal proteins and assembly factors are removed. Thus, it seems that after the initial assembly stages, the ribosome subunits' biogenesis pathway becomes a disassembly pathway (Ulbrich et al. 2009). These disassembly steps are crucial for the maturation step, and any failure in removing one of the RBs or AFs might lead to non-mature ribosome subunits and, consequently, a default in ribosome activity.

Therefore, the main role of the nucleolus is the synthesis of small and large ribosome subunits. This sequential and highly coordinated process requires several proteins and noncoding RNAs and is regulated at different levels. III) Liquid-liquid phase separation: a mechanism that drives the nucleolus formation III.1-Definition of liquid-liquid phase separation Within a cell, the bio-molecular condensates such as proteins and nucleic acids can form liquid-liquid membrane-less condensates via liquid-liquid phase separation, also referred to as liquid-liquid demixing. This process provides another means for concentrating and segregating cellular compartments into a spatiotemporally defined manner, thereby allowing diverse functional processes to occur.

Several reports have ascertained the requirements for LLPS to occur and the mechanisms that tightly regulate such phenomenon, including temperature, pH, ionic strength, proteins regions, nucleic acids, and post-translational modifications (PTMs) [START_REF] Banani | Compositional Control of Phase-Separated Cellular Bodies[END_REF]2017). The mechanism that drives LLPS is multivalent and often weak interactions between proteins (different and similar species) and between protein and nucleic acids (RNA and DNA). The bio-molecular components that drive LLPS contain intrinsically disordered regions (IDRs) and low complexity regions (LCRs) [START_REF] Gonçalves-Kulik | Low Complexity Induces Structure in Protein Regions Predicted as Intrinsically Disordered[END_REF]).

The IDRs/LCRs are distinctly different from the molecular domains in that their amino acid compositions and distributions cannot fulfill compact folding requirements. These regions lack the tertiary structure and often exhibit flexible, versatile conformations and highly biased amino acid distribution. Importantly, IDRs-phase separation occurs among single species of proteins, and such interactions are often weak and transient [START_REF] Posey | Phase Separation of Intrinsically Disordered Proteins[END_REF]. The multivalent interaction that drives LLPS can also occur between two or more bio-molecular which contain multiple interaction domains and/or motifs; such interactions are relatively strong [START_REF] Banani | Compositional Control of Phase-Separated Cellular Bodies[END_REF]. Indeed, bio-molecular phase separation is not limited to proteins. RNAs participate extensively in forming RNA/proteins-rich membrane-less organelles by contributing to the phase separation mechanisms [START_REF] Shin | Liquid Phase Condensation in Cell Physiology and Disease[END_REF][START_REF] Banani | Biomolecular Condensates: Organizers of Cellular Biochemistry[END_REF].

LLPS allows cellular compartmentalization by forming condensates as mentioned earlier. These condensates include nucleolus, nuclear speckles, Cajal bodies, Pgranules, and others (reviewed in [START_REF] Zhang | Liquid-Liquid Phase Separation in Biology: Mechanisms, Physiological Functions and Human Diseases[END_REF]). Moreover, LLPS allows rapid concentration of components in one cell region, facilitating cellular reaction and increased rates [START_REF] Li | Phase Transitions in the Assembly of Multivalent Signalling Proteins[END_REF]). In addition, LLPS allows selective sequestration or exclusion of specific bio-macromolecules that affect gene expression, chromatin condensation, and translational processes (reviewed in [START_REF] Zhang | Liquid-Liquid Phase Separation in Biology: Mechanisms, Physiological Functions and Human Diseases[END_REF]).

The nucleolus, whose structure and function have been explained at the beginning of Part I, is assembled, and disassembled through liquid-liquid phase separation. Nucleolar protein's ability to interact together and with nucleic acids (RNA and/or DNA) contributes to the phase separation of the nucleolus from the liquid nucleoplasm and its tripartite organization formation. Here, I explain below how the nucleolus assembly and disassembly during the cell cycle as an example of LLPS.

III.2-LLPS drive nucleolar assembly and disassembly.

The formation of condensates in vivo likely occurs through nucleation and growth processes. Where the components that were evenly distributed start to associate together and form clusters that are large enough to overcome surface tension (Kashchiev, 2000); beyond that, they continue to grow and generate large condensates. An example is nucleoli assembly and disassembly during the cell cycle, which is driven by LLPS and it serves as a model to describe the steps of nuclear disorganization. Nucleolar proteins and complex machinery are inherited through mitosis, thereby allowing the nucleoli to re-assemble by the end of telophase (figure 8).

Nucleolar disassembly is a sequential process that starts at the beginning of prophase (Gautier et al. 1992). The process starts with releasing nucleolar complexes (proteins and rRNA), followed by the repression of RNApolI activity [START_REF] Falahati | Nucleation by RRNA Dictates the Precision of Nucleolus Assembly[END_REF]. Inhibiting rRNA transcriptions disrupts the surface tension and the interaction between nucleolar protein and the produced rRNA, this process is considered the early step leading to asynchronous nucleation and the starting point of disassembly [START_REF] Berry | RNA Transcription Modulates Phase Transition-Driven Nuclear Body Assembly[END_REF]. In addition to inhibiting rDNA transcription, post-translational modifications impact the phase separation where they drive the release of nucleolar proteins at the beginning of disassembly. For example, in living cells, the phosphorylation of NPM/B23 by CDK1-Cyclin B alters the binding affinity of NPM/B23 to the rRNA and thus causes nucleolar B23 to exit (Negi and Olson 2006;Okuwaki, Tsujimoto, and Nagata 2002). Interestingly, the rDNA transcriptional machinery, proteins for the rRNA processing, and other components are inherited through mitosis. This complex machinery assembles into foci, referred to as peri-nucleolar bodies (PNBs) (Ochs et al. 1985), which can be observed around the chromosomes during mitosis. Indeed, the sequential event that initiates the disassembly process, where B23 is released while rDNA transcription is still ongoing, is to ensure the formation of 45S rRNA (Dousset et al. 2000). The formed 45S and the inherited machinery allow the rapid nucleolar assembly by the end of telophase.

Nucleolar assembly is an early event process that starts at the end of telophase (Roussel et al. 1996). This process depends on activating rDNA transcription and the recruitment of RNA processing machinery. Activating rRNA transcription (active NORs) provides the seed for protein components to condense [START_REF] Shevtsov | Nucleation of Nuclear Bodies by RNA[END_REF]. As mentioned above, the activity of polI is repressed by CDK1-cyclinB kinase during mitosis. At the end of telophase, to start the assembly of nucleoli, CDK1-cyclinB activity is inhibited through phosphatases PP1 and PP2, thus activating RNApolI (Trinkle-Mulcahy and Lamond 2006). This step is followed by recruiting protein and processing complexes from the PNBs to the newly transcribed rDNA. The liquid-like state allows the concentration of high components within the condensates and maintains the mobility of these components, thereby enabling the repetitive cycles of assembly and disassembly on each mitosis. Telophase, rDNA transcription is activated (white octagons), whereas the early and late processing proteins are located within the pre-nucleolar bodies (PNBs). The PNBs appears in yellow structures, contains the DFC (D: green) and GC (G: red) processing complexes. In early -G1, the nuclear envelope is assembled at the periphery of the nucleus (double black rings), transcription and early processing take place and thus generate the DFC, at this stage, the late processing complexes are still in the PNBs (G: red). In early-G1 b, the late complexes are recruited around the DFC forming the granular components, and the incipient nucleoli form a single nucleolus. In Hela cells the duration of the telophase is ≈ 10 min, early-G1 a is ≈20 min and early-G1 b is ≈90 min. Adapted from (Danièle Hernandez-Verdun 2011).

III.3-Active ribosome biogenesis generates the surface tension required to form the nucleolar tripartite organization.

As the nucleolus assembles by the end of telophase, it is phase-separated into three sub-compartments. Interestingly, the macroscale structure of these three subcompartments is dictated by emerging properties of the phase-separated liquids, including miscibility and surface tension [START_REF] Zarzar | Dynamically Reconfigurable Complex Emulsions via Tunable Interfacial Tensions[END_REF].

As described in beginning of Part I, the nucleolus is a tripartite organelle. Each of the nucleolar sub-compartments has a unique complement of proteins and RNAs that executes its function: rDNA is transcribed to rRNA at the FC/DFC border, the rRNA is then processed into mature rRNA at the DFC, and finally, the rRNA is assembled into mature pre-ribosomal particles in the granular components GC [START_REF] Schwarzacher | The Nucleolus[END_REF]. The relative surface tension of these sub-compartments dictates whether and how they contact one another and which droplet will be embedded or partially contained with the other [START_REF] Zarzar | Dynamically Reconfigurable Complex Emulsions via Tunable Interfacial Tensions[END_REF]; this explains why the nucleolus is a membrane-less organelle formed by the act of building ribosome subunits [START_REF] Mélèse | The Nucleolus: An Organelle Formed by the Act of Building a Ribosome[END_REF].

As ribosome biogenesis is ongoing, the interaction between the existing components (RPs, RAFs, snoRNA, rRNA genes, RNApolI, transcriptional factors) and the newly formed one (pre-rRNA, mature-rRNA) determines the tripartite structure. This can be observed following the disruption of specific and crucial steps of ribosome biogenesis. For example, mammalian cells actively engaged in rDNA transcription upon treating them with low doses of Act-D for (1-3hr), and thus RNApolI transcription activity is inhibited, it triggers the segregation of nucleolar compartments, particularly the FC from the DFC regions. The structure formed upon segregation is referred to as the nucleolar caps (figure 9,B). These structures are highly dynamic and contain rDNA, transcription machinery, and other proteins required for processing [START_REF] Shav-Tal | Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition[END_REF]. On the other hand, treating active nucleoli by DRB (5,6-dichloro-1-ribo-furanosylbenzimidazole), thus the RNApolI transcriptional activity remains intact, but rRNA processing is impaired, resulting in nucleolar necklaces formation (figure 9,C). These nucleolar necklaces are a reversible disconnection of the FC/DFC from the GC (Louvet et al. 2005).

Moreover, it has been considered that the synthesis of 47S pre-RNAs and their binding affinity to snoRNPs and other processing proteins produces a flux that led to the formation of the three mentioned compartments. This flux is known as the vectorial transport of rRNA that appears as a Christmas tree by electron microscopy. The importance of nucleolar protein and rRNA interaction in maintaining the nucleolar tripartite organization can be observed in the examples mentioned in section II.2. Whereby using super-resolution microscopy, Yao and colleagues revealed that the fibrillarin and other tested proteins form spherically shaped beads that assemble within the DFC around the FC. They also showed that fibrillarin binds to the 5'ETS through its amino-terminal region that is glycine-arginine-rich (GAR) and contains IDRs [START_REF] Yao | Nascent Pre-RRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus[END_REF]. Such bindings enable phase separation of the RNA and thereby lead to DFC nucleation and initiate the first steps of pre-rRNA processing. Part I The Nucleolus: Structure and Function

29

Another example of such driven phase separation processes in maintaining nucleolar structure can be observed in nucleophosmin (NPM/B23) and its interaction with components of pre-ribosomal proteins in the granular center. Briefly, nucleolar protein NPM1 /B23.1 is a member of the nucleoplasmin family that is involved in diverse cellular processes such as ribosome biogenesis and RNA polymerase-dependent transcriptional activity [START_REF] Box | Nucleophosmin: From Structure References 130 and Function to Disease Development[END_REF].

It has been suggested that B23.1 contributes to maintaining the nucleolar structure, particularly the formation of the GC region, by its ability to phase separate through multivalent interactions with arginine-rich proteins and rRNAs. Using in vitro binding assays, Okuwaki and colleagues confirmed the interaction between B23.1 and pre-60S ribosomal units during maturation steps in the nucleoli. It was not revealed which rRNA in the pre-60S B23.1 interacts with. However, such interaction was shown to be crucial in accumulating the rRNA (components of the 60S preribosomal particles) and other assembly factors in the nucleoli, thereby allowing the maturation of the ribosomal subunit. More precisely, the knockdown of B23.1 has been shown to induce the release of the pre-60S ribosomal subunit into the nucleoplasm, therefore, confirming its pivotal role in such pre-ribosomal particle assembly [START_REF] Okuwaki | The Interaction between Nucleophosmin/NPM1 and the Large Ribosomal Subunit Precursors Contribute to Maintaining the Nucleolar Structure[END_REF]. III.4-LLPS and Ribosome biogenesis impact the nucleolar number and size.

LLPS that form the basic mechanism of nucleolar formation can also drive nucleolar fusion, thereby impacting nucleolar number and the shape within the nucleus. More recently, Brangwynne and colleagues used germinal vesicles (amphibian oocytes) to demonstrate that the nucleolus behaves like a liquid droplet-like. The authors could visualize nucleoli clustering and fusion on a time scale of seconds [START_REF] Brangwynne | Active Liquid-like Behavior of Nucleoli Determines Their Size and Shape in Xenopus Laevis Oocytes[END_REF]. The factors contributing to the change in nucleolar number have yet to be fully disclosed. To date, few factors have been identified to regulate nucleolar number and size, including RNApol1 activity, where its depletion in vivo causes nucleolar fusion to form a single large nucleolus in mouse embryonic fibroblasts (Hamdane et al. 2014).

As ribosome biogenesis was described initially in the manuscript, it's important to mention that nucleolar number and size are not only regulated by the nucleolar liquidlike behavior. There is increasing evidence linking the efficiency of ribosome biogenesis to the number and size of nucleoli [START_REF] Xie | Regulation of the MDM2-P53 Pathway by the Nucleolar Protein CSIG in Response to Nucleolar Stress[END_REF]. Several experiments have been conducted to understand better the interrelationship between environmental changes and its consequence on nucleolar morphology and function. For example, in 1976, Bailey and his coworkers showed that regulating mice's diet increases nucleolar size and activity, but they did not confirm the mechanism behind this. Still, it was speculated that it is due to increased DNA synthesis [START_REF] Bailey | Amino Acids and Control of Nucleolar Size, the Activity of RNA Polymerase I, and DNA Synthesis in Liver[END_REF]). We could also observe an increase in nucleolar size in a few types of cancer, which is mainly linked to increased RNApolI activity and increased UBF, DNA topoisomerase I, and fibrillarin expression [START_REF] Derenzini | Nucleolar Size Indicates the Rapidity of Cell Proliferation in Cancer Tissues[END_REF].

Increasing the transcription machinery allows cells to proliferate more (Derenzini et al. 1998). However, it is essential to mention that only a few types of cancer proliferation justify the large nucleolar area. For example, fast-growing small-cell anaplastic lung carcinomas have a small nucleolar area per nucleus; in contrast, slowgrowing large-cell carcinomas have much more prominent nucleoli per nucleus [START_REF] Zink | Nuclear Structure in Cancer Cells[END_REF].

Since nucleolar morphological changes are mostly linked to environmental changes and may indicate a change in cellular state, some pathologists use nucleolar number, morphology, and size in some cases to indicate certain severity or onset of disease. For example, an increase in nucleolar size and the number indicates hypertrophy of the heart myocardium, which is justified as an increase in nucleolar activity [START_REF] Neuburger | Change in Size, Number and Morphology of the Nucleoli in Human Hearts as a Result of Hyperfunction[END_REF].

Therefore, nucleolus organization and tripartite compartmentalization are driven by liquid-liquid phase separation. Each of the nucleolar sub-compartments has a unique complement of proteins and RNAs that execute its function and determines the sub-compartments relative surface tension, thereby determining which droplets contact one another and which droplets will be embedded or partially contained with the other.

IV) Role of nucleolus in regulating ribosome biogenesis and stress response

Ribosome biogenesis, as described earlier, is a highly energy-demanding process in which ribosomal RNA synthesis and ribosome production consume enormous cellular energy. In addition, ribosome biogenesis and the cell cycle are mutually regulated-by cyclin-CDK complexes (figure 10). Because of this link, the cell tends to regulate ribosome biogenesis when it experiences environmental challenges such as nutrient deprivation, viral infection, and genotoxic stress (figure 5;A).

This regulation allows the cell to preserve its internal cellular energy and balance ribosome production with the availability of resources and environmental conditions, most often, this regulation affects cellular fate.

Many external environmental changes regulate signal pathways that directly or indirectly interact with the transcriptional factors and components of the PIC complex, such as RNApolI, UBF, and others (Grummt 2013).

An example of such exerted external signal, ribosome biogenesis regulation, and functional cell change can be observed upon mitogenic stimuli. In 1997, Voit and his colleagues determined the effect of mitogenic stimulation by serum on quiescent NIH3T3 fibroblast cells. By analyzing the cell cycle and monitoring the expression at each phase, followed by correlating these changes with UBF phosphorylation and rDNA transcription. They revealed that, upon cell cycle entry due to serum activation, UBF shows a pattern of phosphorylation at different sites, which targets regulators of cell cycle. This shows how cells regulate rDNA transcription and cell cycle under a particular stimulus, thus allowing cell adaptation to certain conditions (Voit, Hoffmann, and Grummt 1999). Attenuating ribosome biogenesis could be regulated at the level of PIC complex and rDNA transcription.

However, ribosome biogenesis regulation can occur at different levels, including the distribution of the nucleolar proteins in the nucleolus, shuttling or sequestering proteins (nucleolar or non-nucleolar proteins) within the nucleolus (Andersen et al. 2005), down-regulating of RNApolI transcriptional activity, changes in the expression of pre-rRNA processing machinery and chromatin remodeling (Strohner et al. 2001).

Interestingly, not only the nucleolus senses the stimulus and regulates ribosome biogenesis, depending on the environmental requirement. However, also, the nucleolus responds as feedback to such changes by regulating downstream signaling pathways. One of the most documented stress response pathways downstream of nucleolar stress is the Mdm2-P53 signaling pathway (Xie et al. 2016, 2). Tumor suppressor protein (p53) is a transcriptional factor able to induce cell cycle arrest and apoptosis by activating the transcription of several target genes. Under normal conditions, p53 is found in the nucleoplasm at a steady-state level under the Mouse double minute 2 homolog (Mdm2) control. Mdm2 is the ubiquitin E3 ligase that negatively regulates p53 by marking it for ubiquitin-mediated proteasomal degradation. In response to events that induce nucleolar stress, several ribosomal proteins and nucleolar proteins translocate to the nucleoplasm and bind Mdm2, thus promoting p53 stabilization and subsequent activation of checkpoints (such as the p21 gene), DNA repair factors, and proapoptotic factors. This results in cell cycle arrest, apoptosis, or cellular senescence. Indeed, nucleolar stress can activate other signaling pathways independent of p53 (Lindström 2009).

Overall, because of the nucleolar ability to regulate ribosome biosynthesis that allows the cell to adapt to specific challenges, it is conceivable that the nucleolus participates in maintaining cellular homeostasis and determining cell fate. Besides the environmental stresses that are mentioned in Part I, section II and IV, cells can also experience mechanical cues. Mechanical cues from internal (actomyosin cell contractility) and external (that arise from ECM remodeling, neighboring cells, tensile forces, shear stress, and compressive forces...) exerted forces on/within the cells. These forces have been shown to contribute to forming crucial aspects of organs, tissue regeneration, and disease initiation and progression. Since the thesis project aims to study the impact of biophysical cues on nucleolar structure and function, Part II focuses on describing mechanical forces that cells experience within tissue and how these forces are transmitted, thereby altering cellular function.

Part 2 Cell Mechanics

Simple multicellular organisms hold together through cell-cell adhesion. As it becomes more complex, cells tend to organize into tissues and organs to perform a particular function. To facilitate tissue organization and integrity, cells must synthesize, organize, and remodel a three-dimensional network of extracellular lattice and glycoproteins referred to as extracellular matrix.

Cells are constantly subjected to mechanical stimuli originating from the extracellular matrix, neighboring cells, and surrounding fluids. Under these conditions, cells must integrate these numerous mechanical signals and properly regulate their function to form organs and maintain tissue homeostasis. The mechanism by which cells translate the mechanical information from their environment into a biological response that alters their behavior is referred to as 'Mechanotransduction'.

In the second part of my manuscript, I aim to describe the organization of epithelial cells, including anchor junctions and their connection to the intracellular cytoskeleton. I will also describe the coupling of the cytoskeleton and nuclear structure due to their importance in transmitting the signal into the nucleus. Moreover, I will mention the sources of mechanical signals that cells experience in their environment, with few evidence of how cells sense these forces, transmit them to the nucleus, and might result in regulating nuclear function and determining cell fate. At the end of Part 2, I will give examples of in vitro systems used to apply mechanical force, particularly compressive forces and highlight the results obtained.

Cell/Cell and Cell/ECM adhesions and forces acting on epithelial cells within the mammary gland.

I) Epithelial cell architecture and organization

The Epithelium is the most common cell type in all animals. They form the sheets and tubes that makeup organs and tissues. Some epithelia cover the outer surface of the organism, while others line the internal organs. Epithelial cells can perform different functions, some cells only play the role of a mechanical barrier, while others have a specific biochemical role, such as the secretion of hormones or the absorption of nutrients. Despite all differences in function, epithelial cells share standard features concerning their polarity. Epithelial cells are polarized along their apical-basal axis and consist of at least four distinct membranous domains: supra-apical, lateral, and basal. Each domain contains different proteins that attach to the underlying cytoskeleton (St Johnston and Sanson 2011). From the apical side, cells mainly face either the outer part of the tissue or the luminal space of the internal organs and can harbor specialized structures such as microvilli and cilia. From the basal side, cells rest on the basement membrane, part of the extracellular matrix. ECM is a threedimensional molecular network composed of collagen, proteoglycans, glycosaminoglycans, fibronectin, laminins and many other glycoproteins.

Cells adhere to the extracellular matrix through focal adhesions and to neighboring cells via complexes collectively called anchoring junctions. These adhesions are a group of multiprotein complexes that stabilize cell position, provide stability and rigidity, and support tissue integrity by holding cell sheets together. There are several types of cell/cell anchoring junctions, including: (1) Adherens junctions: which link one cell to another cell through the actin filament network; (2) Desmosomes: which link cells together through intermediate filaments;

(3) Hemidesmosomes: which link cells to the matrix through intermediate filaments.

Moreover, Cells adhere to ECM through, cell-matrix adhesion complexes (CMACs) (figure11). These adhesions, whether it is cell-cell or cell-ECM, are characterized by the presence of the following essential components:

1. Transmembrane cell-adhesion molecules (CAMs) and/or adhesion receptors that are located in the plasma membrane link the lateral surfaces of one cell to another or the basal surfaces of the cell to the ECM. Examples of adhesion receptors include cadherins (cell-cell adhesions), integrins, and syndecans (cell-ECM adhesions) (reviewed in [START_REF] Schwartz | Cell Adhesion Receptors in Mechanotransduction[END_REF]).

Adaptor proteins connect the adhesion molecules to the cytoskeleton and signaling

molecules-examples: catenins, talin, vinculin, and α-actinin [START_REF] Thompson | Cadherin Clusters Stabilized by a Combination of Specific and Nonspecific Cis-Interactions[END_REF].

3. The cytoskeleton whose fundamental structural unit is a filamentous network of microfilaments (MFs), microtubules (MTs) that are formed of actin and tubulin respectively; and intermediate filaments (IFs) divided into six major subtypes composed of different proteins. This cytoskeleton network maintains the cellular shape and acts as a force-sensing device (reviewed in [START_REF] Pegoraro | Mechanical Properties of the Cytoskeleton and Cells[END_REF]). I.1-Cell/ECM and Cell/Cell adhesions and their interaction with the cytoskeleton:

This part aims to describe how the cell-cell and cell-ECM adhesions form a molecular connection between the outside and the cytoskeletal filament from inside the cell. Thus, it provides a pathway for transmitting mechanical force and is also involved in the bidirectional signaling from the outside to the inside of the cell and vice versa (examples of such signaling pathways transmission will be given in section II). Indeed, there are several cell/ECM and cell/cell adhesions, here I only describe adherence junction and focal adhesions as an example to describe this relation.

I.1.1 Focal adhesions:

The outermost component of focal adhesions is transmembrane receptors of the integrin family, a large family of heterodimeric glycoproteins consisting of α and βsubunits [START_REF] Hynes | Integrins: Versatility, Modulation, and Signaling in Cell Adhesion[END_REF]. Interestingly most cells express the type of integrin that can interact with the matrix components they encounter. For example, on fibronectin, cells usually express and assemble α5β1 integrin, the fibronectin receptor. Following integrin clustering, a hierarchy of protein recruitment is initiated to the adhesion sites, including focal adhesive kinase (FAK), Paxillin, Talin, Vinculin, α-actinin, and several other signaling molecules (figure12) (Fath, Edgell, and Burridge 1989).

Interestingly, focal adhesions are the major site for the localization of phosphotyrosinated proteins in the cell. It is believed that tyrosine phosphorylation is crucial for focal adhesion assembly and the propagation of signals [START_REF] Volinsky | Complexity of Receptor Tyrosine Kinase Signal Processing[END_REF]. Building the protein complexes at the focal adhesion sites allows its maturation, which would meet cellular demands and elicit different functions, such as motility, adherence, migration, and others.

An essential protein in the signal transduction at the focal adhesions is focal adhesion Kinase pp125fak (FAK). Its activation by auto-phosphorylation triggers intracellular mechanotransduction by activating downstream adhesion proteins and mechanotransducer in the cytoplasm [START_REF] Schwartz | Cell Adhesion Receptors in Mechanotransduction[END_REF][START_REF] Geiger | Environmental Sensing through Focal Adhesions[END_REF]. Moreover, vinculin is one of the main components of the FA inner core, and its recruitment to the focal adhesion sites requires talin activation. In addition to talin binding sites, vinculin is characterized by actin binding sites, thus allowing vinculin to bind to the actin cytoskeleton. Therefore, linking the FA to the cytoskeletal filament (Dumbauld et al. 2013). Indeed, activating the focal adhesion induces a structural change in cytoskeletal filament organization and can activate several downstream-signaling pathways (Wada et al. 2011;[START_REF] Nobes | Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement[END_REF].

I.1.2 Adherens junctions:

The most direct effect of cell adhesion between neighboring cells is observed in morphogenesis. For example, the assembly of individual cells into highly ordered tissues and organs is achieved through the cell-cell junctions (Takeichi 1995). In some cells, these junctions are found in small patches, while in epithelial cells, adherens junctions form a complete belt known as zonula occludens. The specific adhesion interaction between cells involves transmembrane cell adhesion receptors of the cadherin family. Indeed, the adhesion receptor is not enough for effective adhesion and junction formation. Still, recruiting junctional plaque proteins that link the cadherin receptor to the cytoskeleton is essential.

Cell-Cell junctions and focal adhesions share several structural components, for example, the vinculin sub-membrane plaque and the α-actinin that allow cross-linking of F-actin fibers. The major difference between the two adhesions is the transmembrane adhesion receptors. In the adherens junctions, the transmembrane adhesion receptor is the cadherin family that mediated the ca2+ dependent homophilic cell-cell interaction (Geiger, Yehuda-Levenberg, and Bershadsky 1995). Cadherin molecules are distributed lateral side and bind to the neighboring cad receptor, forming a cis linkage, a feature of cadherin that strengthens intercellular adhesion. Intracellularly, the highly conserved tail of cadherin binds catenin (beta, alpha, and P120 catenin) (Takeichi 1995). Once β-catenin is activated, it recruits alpha-catenin (αcatenin), which has a structural similarity to vinculin and actin-binding properties. Suggesting that α-catenin allows the cadherins complex to bind to the actin cytoskeletal filaments (Ishiyama et al. 2018). Notably, even though β-catenin is a component of the cadherin adhesions system, the protein act as a mechano-actuated shuttling protein that has been shown to translocate from the cell membrane into the nucleus and act as a transcriptional factor (A et al. 2012). As for now, I described few of the proteins that assembles at focal adhesion sites and adherens junctions and how some of these proteins play an essential role in linking these adhesions to the cytoskeletal filaments. It is important to mention that this connection does not stop in the cytoplasm but extends to the nucleus, including its surface and interior. In the following part, I will describe the link between the cytoskeleton and the nuclear membrane.

I.2 Linking the cytoskeleton to the nucleoskeleton

In a landmark paper in 1997, It was shown that tugging on the integrin receptor by using bound microbeads or micropipette induces cytoskeletal filament orientation and distortion of the nucleus [START_REF] Maniotis | Demonstration of Mechanical Connections between Integrins, Cytoskeletal Filaments, and Nucleoplasm That Stabilize Nuclear Structure[END_REF]. This study paves the way for understanding the link between the cytoskeleton and the nucleus. In recent years, a clear view of the nuclear membrane architecture and its link with the cytoskeleton has emerged. The nuclear membrane of the cells is determined by the nuclear envelope (NE). This nuclear envelope is a highly dynamic structure that separates the cytoplasm from the nucleoplasm (Cain and Starr 2015). The nuclear envelope comprises the outer nuclear membrane (ONM) and the inner nuclear membrane (INM); the LINC complex links these two membranes. LINC complex consists of nesprin (nuclear envelope spectrin-repeat proteins) protein (in the ONM), where four nesprin have been identified, and the SUN (Sad1p-UNC-84) proteins (SUN1 and SUN2) in the inner nuclear membrane.

The four identified nesprin protein contains a C-terminal domain that consists of a highly conserved KASH (Klarischt, ANC-1, and SYNE homology) domain. These domains are required for the localization of nesprin in the nuclear membrane and its interaction with SUN proteins within the perinuclear space. The nesprin protein interacts with the cytoskeleton (directly or through actin cross-linkers, plectin and microtubules-associated motors) from the cytoplasmic region. Nesprins 1 and 2 contain an N-terminal domain that interacts with the cytoplasmic actin filament, while nesprins 3 binds to plectin protein which crosslinks the intermediate filament. Interestingly, nesprin 4, which is only expressed in secretory epithelial cells, is a kinesin-binding protein that connects the nucleus to the microtubules [START_REF] Starr | Interactions Between Nuclei and the Cytoskeleton Are Mediated by SUN-KASH Nuclear-Envelope Bridges[END_REF]. The SUN protein interacts with the nuclear lamina, chromatin, and other nuclear membrane proteins from the nucleoplasmic space that constitute the nucleoskeleton (figure13).

Within the nucleus, Lamins are the main component of the nucleoskeleton. Lamins are classified as type V intermediate filament proteins and categorized into type A-or B-based on their amino acid and biochemical characteristics. In mammalian cells, the LMNA gene produces four types of Lamins by alternative splicing; the predominant isoforms are lamin A (LA) and C (LC). In addition, LAMNB1 and LAMNB2 genes produce laminB1(LB1) and laminB2 (LB2) respectively [START_REF] Adam | Insights into the Differences between the A-and B-Type Nuclear Lamins[END_REF].

Even though Lamins have a unique mechanism of assembly to build a mature apolar Lamin meshwork, they share a common three-domain structural organization, and feature of post-translationally modifications that regulate their functions as other Ifs proteins within the cytoplasm [START_REF] Adam | Insights into the Differences between the A-and B-Type Nuclear Lamins[END_REF][START_REF] Snider | Post-Translational Modifications of Intermediate Filament Proteins: Mechanisms and Functions[END_REF].

Interestingly, Lamins are not the only component of the nucleoskeleton but also nuclear actin, motor proteins and nuclear membrane proteins are involved [START_REF] Liem | Cytoskeletal Integrators: The Spectrin Superfamily[END_REF]. Notably, actin is found in all eukaryotic cells; several roles have been ascribed to them, including facilitating the transcription, processing, and export of messenger RNA (mRNA), chromatin remodeling, and stimulating the activity of all three RNA polymerases (de Lanerolle and Serebryannyy 2011). Few reports have suggested that nuclear actin and myosin I cooperate to interact with both ribosomal and polymerase II transcribed genes and regulate their expression, with the possibility of positioning the chromosomes in the nucleus (de [START_REF] Lanerolle | Nuclear Actin and Myosins: Life without Filaments[END_REF]. This connection between the cytoskeleton and the nuclear components through the LINC complex and other nuclear membrane proteins creates mechanical tethers, thus connecting the cell membrane and the nucleus.

The mechanical landscape within a tissue is very complex, as cells within tissues are exposed to internal (actomyosin cell contractility) and external (that arise from ECM remodeling, neighboring cells, tensile forces, shear stress, compressive forces...) cues that often interrelate and cannot be decoupled. Therefore, in the next section of Part II, I will focus on (1) how mechanical force is transmitted from the cell's surface, mainly through the adhesions that I described, to the interior of the nucleus. Based on this, I will briefly mention (2) the importance of the cytoskeleton in transmitting mechanical force to the inside of the cell. And since the cytoskeleton is connected to the nucleus that contains the DNA and the transcriptional machinery, (3) I will give some examples that illustrate the effectiveness of mechanical forces in regulating cellular phenotype, which would determine the function of the cell.

Since the model I used in my project was epithelial cells, particularly mammary epithelial cells (MCF10A), I will discuss my aims by selecting the mammary gland as an example and supported by examples from other tissues and cellular models. The mammary gland forms a relevant model that shows how mechanical forces contribute to organ development and how dysregulation of mechano-reciprocity and/or heightened mechanical force can drive disease progression in the breast.

II) Mechanical signaling in mammary gland from development into tumor initiation and progression

"Cell and tissue, shell and bone, leaf and flower, are so many portions of matter, and it is in obedience to the laws of physics that their particles have been moved, molded and conformed. (…) Their problems of form are in the first instance mathematical problems, their problems of growth are essentially physical problems, and the morphologist is, ipso facto, a student of physical science." D'Arcy Thompson on "Growth and Form", 1917 Early biologists were always fascinated by how organisms developed different shapes. Nowadays, it has become increasingly evident that crucial aspects of organ formation, as well as tissue regeneration and repair, is a dynamic interaction between cells and their microenvironments, including hormones and neighboring cells, the extracellular matrix, and the forces applied to them.

Once mechanical cues are sensed by the cell, the cell propagate and amplify these physical cues and translate the signal into either a transient response or sustained cellular behavior. The force that is applied to the cell will be (1) transmitted to the inside of the cell through the integrin and cadherin coupling to the cytoskeleton [START_REF] Hynes | Integrins: Versatility, Modulation, and Signaling in Cell Adhesion[END_REF][START_REF] Thompson | Cadherin Clusters Stabilized by a Combination of Specific and Nonspecific Cis-Interactions[END_REF] or through mechanosensitive proteins and channels [START_REF] Hayakawa | Actin Stress Fibers Transmit and Focus Force to Activate Mechanosensitive Channels[END_REF], followed by (2) transmitting the force to the chromatin in the nucleus through the cytoskeleton-nucleoskeleton coupling [START_REF] Lombardi | Keeping the LINC: The Importance of Nucleo-Cytoskeletal Coupling in Intracellular Force Transmission and Cellular Function[END_REF] or shuttling proteins (Benham-Pyle, Pruitt, and Nelson 2015; [START_REF] Hsiao | Human Pluripotent Stem Cell Culture Density Modulates YAP Signaling[END_REF][START_REF] Sen | Mechanically Induced Nuclear Shuttling of β-Catenin Requires Co-Transfer of Actin[END_REF]) and ions, and then (3) the signal in the nucleus might modulate the gene expression by regulating chromatin state and organization [START_REF] Tajik | Transcription Upregulation via Force-Induced Direct Stretching of Chromatin[END_REF][START_REF] Nava | Heterochromatin-References 138 Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage[END_REF]). In addition, to the importance of cell signaling and force transmission into the inside of the cells (outside-inside), on the other hand, the forces exerted by the cell into the surrounding environment (inside-outside) play an essential role in defining the micro-rheology of the surrounding tissue, the organization of the ECM, that itself might trigger new signals, such as cell-generated forces that leads to new binding sites at the Cell/ECM adhesions. This is referred to "Mechanoreciprocity", and dysregulation in such feedback modifies the ECM components and generates forces exerted on surrounding cells, that could initiate diseases such as cancer [START_REF] Gilbert | Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage[END_REF][START_REF] Paszek | Tensional Homeostasis and the Malignant Phenotype[END_REF].

The advent of: (i) new microscopy techniques allow scientists to record cell dynamics in vivo; (ii) the ability to estimate forces using biophysical approaches; and (iii) the combined power of experimental manipulation and theoretical model all contributed to a significant advance in understanding how cells sense/produce the force and mechanisms used to convert the mechanical signal into the biological response, which is referred to Mechanotransduction (figure14).

II.1) Embryogenesis: Development of mammary gland

The development of mammary glands occurs in three defined stages: embryonic, pubertal, and reproductive (figure 15;A). Each set is closely associated with specific hormonal, growth factors, and mechanical cues that initiate and sustain the required morphological and cellular changes.

Briefly, development of the murine mammary gland begins around E10* with the establishment of milk lines, and around E11.5, ectoderm-derived structures named placodes appear from these milk lines. Further on during embryogenesis, placodes become epithelial mammary buds that later proliferate and elongate, invading the newly formed fat pad. This led to the formation of a rudimentary ductal tree that remains quiescent until the onset of puberty. During mammary gland development, mechanical cues direct branching morphogenesis [START_REF] Gjorevski | Endogenous Patterns of Mechanical Stress Are Required for Branching Morphogenesis[END_REF], a common feature with other tissue, including the salivary gland and lung. The mammary gland morphogenesis is characterized by differentiating mammary progenitor stem cells into luminal and myoepithelial cells.

The involvement of the cytoskeleton component in growth regulation and its ability to regulate gene expression in mammary epithelial cells was recognized in 1981 [START_REF] Bissell | How Does the Extracellular Matrix Direct Gene Expression?[END_REF]. However, acknowledging that ECM and, more particularly, the substratum compliance as a source of mechanical cues and the mechanism it regulates the mammary gland progenitor cells differentiation was not fully uncovered. In 2012, Lui et al. cultured immortalized human mammary progenitor cells on a synthetic substrate with variable elastic modulus. The modulus was regulated by adding different concentrations of cross-linkers into the polyacrylamide gels (PA).

And they showed that mammary progenitor cells seeded on soft substrate (those with Young's modulus <1kPa) differentiate into luminal epithelial cells. In contrast, cells seeded on a stiff substrate (Young's modulus >2kPa) differentiate into myoepithelial cells [START_REF] Lui | Matrix Compliance and RhoA Direct the Differentiation of Mammary Progenitor Cells[END_REF]. The authors also showed that only the appearance of myoepithelial phenotype is concomitant with an increased prevalence of focal adhesions and activation of RhoA pathways*, cytoskeletal reorganization, and actin stress fibers formation [START_REF] Lui | Matrix Compliance and RhoA Direct the Differentiation of Mammary Progenitor Cells[END_REF]. The appearance of two cellular phenotypes is, in fact, what we observe in-vivo, where luminal cells are in line with the mammary duct to the luminal region, while myoepithelial cells are in contact with the extracellular matrix (figure 15;B). This distribution shows us the importance of the external environment shaping the tissue and contributing significantly to regulating cellular function.

Here, I gave an example of how mechanical cues that arise from ECM compliance drive mammary progenitor stem cell differentiation; However, the concept that mechanical force regulates embryonic development gained momentum almost a century ago. In 1990, Carey et al. suggested that the growth of epithelium to form an esophageal lumen exposes the mesenchymal cells to tensional stress that induces their differentiation into smooth muscle cells (Carey 1920). In addition, the importance of muscle contraction in the development of bone was studied in 1990. Where the authors paralyzed the chicken embryo using decamethonium* which thus induces muscle paralysis at different embryonic developmental periods [START_REF] Banani | Compositional Control of Phase-Separated Cellular Bodies[END_REF]8, or 10 days); following that, the authors then examined the relative growth of individual bones and the muscles that act upon some of these bones. They showed that the lack of muscle contraction affects the growth of individual bones differentially. This study and several others using different approaches demonstrate that mechanical loads induce stimulation that is important for both the development and achieving proper tissue size [START_REF] Hall | Paralysis and Growth of the Musculoskeletal System in the Embryonic Chick[END_REF].

II.2) Branching morphogenesis and homeostasis.

For an organism to reach homeostasis through its body, there must be a balance between the absorption, trafficking, and expulsion of materials used in life. As the complexity of an organism increases, the mechanism to achieve this fundamental objective tend to scale in intricacy. Therefore, one mechanism to increase the transfer rate between an organism and its surroundings is expanding the area for exchange.

Branching, also referred to as the collective migration of groups of cells from the central lumen, is used by organs such as the mammary gland, lung vasculature network, and kidney to achieve several goals including material exchange with the external environment [START_REF] Varner | Cellular and Physical Mechanisms of Branching Morphogenesis[END_REF]. In the mammary gland, the rudimentary epithelium formed during development starts to invade the mammary fat pad until it is fully elaborated. The main drive of branching morphogenesis is the TEB (terminal end buds), located at the invading front of each branch. The TEB comprises a single layer of highly proliferative epithelium cap cells and underlying body cells that drive elongation and branching [START_REF] Inman | Mammary Gland Development: Cell Fate Specification, Stem Cells and the Microenvironment[END_REF]) (figure 15;B). This process is induced by cell-ECM interaction as well as cell-cell adhesion. Cell-ECM adhesion generates high mechanical stress at the front of TEB, where cells sense the tension through integrin receptors, followed by protein assemblies such as FAK, paxillin, and talin to form complexes. These complexes mature into focal adhesions linked to the actin cytoskeleton, thus inducing cell contractility and resulting in cell elongation and migration [START_REF] Sigaut | Live Cell Imaging Reveals Focal Adhesions Mechanoresponses in Mammary Epithelial Cells under Sustained Equibiaxial Stress[END_REF][START_REF] Nagy | Mammary Epithelial-Specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland[END_REF][START_REF] Raymond | Integrins in Mammary Development[END_REF].

Mechanical cues from cell-ECM adhesion contribute to branching and transmitting these forces through adhesion junctions to neighboring cells has provided a collective migratory cellular response [START_REF] Ewald | Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis[END_REF]. Notably, changes in the basement membrane or deletion of adhesion proteins disrupt the collective migration from the mammary gland, hence indicating the importance of the mechanical cues that arise from the surrounding environment during developmental processes [START_REF] Ewald | Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis[END_REF][START_REF] Da | RNA Polymerase I Activity Is Regulated at Multiple Steps in the Transcription Cycle: Recent Insights into Factors That Influence Transcription Elongation[END_REF][START_REF] Nguyen-Ngoc | ECM Microenvironment Regulates Collective Migration and Local Dissemination in Normal and Malignant Mammary Epithelium[END_REF]). In addition, collective cellular migration is also regulated by the fluid flow that cells experience from the apical part during development. For example, the collective migration of kidney epithelial cells in zebrafish pronephric duct is stimulated by fluid flow, and the obstruction of the ductal lumen, which blocks the fluid flow, inhibits cell migration, thereby disrupting the morphogenesis of kidney tissue [START_REF] Vasilyev | Collective Cell Migration Drives Morphogenesis of the Kidney Nephron[END_REF].

Interestingly, the branching of the mammary gland is preceded by lumen formation, which is essential for organogenesis and is driven by several cellular processes, including mechanical cues from the surrounding environment. One of the fundamental processes that occur during lumen morphogenesis is bending of the epithelium, which can initiate a new lumen by inducing the invagination of the sheet of cells and might extend new branches [START_REF] Schoenwolf | Quantitative Analyses of Changes in Cell Shapes during Bending of the Avian Neural Plate[END_REF]. The epithelial cells subjected to apical constrictions are characterized by high intracellular tension and actomyosin contractility [START_REF] Sherrard | Sequential Activation of Apical and Basolateral Contractility Drives Ascidian Endoderm Invagination[END_REF]) and patterned by specific signaling networks [START_REF] Myat | Fork Head Prevents Apoptosis and Promotes Cell Shape Change during Formation of the Drosophila Salivary Glands[END_REF][START_REF] Nobes | Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement[END_REF]. Part II Cell Mechanics

44

II.3) Lactation and involution

The processes of gland maturation and alveolar formation occur through the reproductive phase of mammary gland development. Under the influence of hormones during pregnancy, luminal epithelial cells proliferate and differentiate into structures called alveoli (figure 15; B), the milk-producing and secretory units of the mammary gland that mature fully upon lactation [START_REF] Macias | Mammary Gland Development[END_REF].

Upon lactation, alveoli cells produce and secrete the milk, thus resulting in the accumulation of milk in the duct and thereby inducing compressive forces on the lumen epithelial cells, myoepithelial cells, and the basement membrane. In addition to embryo suckling, these compressive forces induce inward projecting tensile stress on the luminal and myoepithelial cells contraction and force the milk out of the alveolar sacs into the larger ducts, thus enabling feeding of the young [START_REF] Biswas | The Mammary Gland: Basic Structure and Molecular Signaling during Development[END_REF].

One of the mammary gland features is the repetitive cycles of the formation of tertiary branches (alveoli acini) and their involution. In the absence of a suckling stimulus, lactation ceases, milk accumulates within the alveoli and the ducts continue to exert forces on the luminal and myoepithelial cells. It was proposed that these generated mechanical forces could be the earliest stimulus to trigger the expression and release of local factors that would initiate mammary gland involution. To prove that Quaglino and coworkers used an in vitro system that already existed and applied 20% of equibiaxial stretch to mammary epithelial cells (derived from pregnant mice) grown on silicone membranes. The authors showed that mechanical stretch activates downstream signaling pathways that trigger several molecular events implicated in initiating post-lactational involution events, mainly inducing cellular apoptosis. In addition, mechanically stretched mammary epithelial cells tend to produce several factors and cytokines that induce cell death in the neighboring cells and the mechanically-unstretched one [START_REF] Quaglino | Mechanical Strain Induces Involution-Associated Events in Mammary Epithelial Cells[END_REF]. How cells sense the mechanical forces during mammary involution events is unclear. However, it was speculated in a separate study that mechanically activated ion channels piezo1 might play a role in cellular sensing and transmitting the signals [START_REF] Stewart | Mammary Mechanobiology -Investigating Roles for Mechanically Activated Ion Channels in Lactation and Involution[END_REF]). These mechanosensitive ion channels are pore-forming membrane proteins that gate in response to mechanical stimuli and can switch between closed and open conformations, thereby allowing ions and other solutes to flow across the cellular membranes [START_REF] Martinac | Mechanosensory Transduction: Focus on Ion Channels[END_REF]. Also, within the prolonged milk stasis and continued gland distension, the compressive forces eventually compromise the integrity of the tight junctions between alveoli epithelial cells, hence the tissue undergoes involution [START_REF] Watson | Post-Lactational Mammary Gland Regression: Molecular Basis and Implications for Breast Cancer[END_REF]. Involution and the loss of the tertiary branches are accompanied by extensive extracellular remodeling and change in the surrounding tissues [START_REF] Mcdaniel | Remodeling of the Mammary Microenvironment after Lactation Promotes Breast Tumor Cell Metastasis[END_REF]). The remodeled stroma consequently alters the signals and the force encountered by the mammary epithelial cells. This allows the subsequent re-initiation of a new round of epithelial proliferation and differentiation and, thus, branch formation [START_REF] Watson | Remodeling Mechanisms of the Mammary Gland during Involution[END_REF].

II.4) Mechanical force in breast cancer

Just as mechanosensing and mechanotransduction plays an important role in mammary gland development, dysregulation of these regulators of tissue homeostasis is a feature of transformed cells. Epithelial cancer is characterized by an altered tissue tensional that reflects the difference in the rheology and an increased cell-generated force in transformed cells [START_REF] Suresh | Biomechanics and Biophysics of Cancer Cells[END_REF]. The tumor environment is characterized by increased compressive forces due to solid-state pressure exerted by the expanding tumor mass (figure 16;A). [START_REF] Paszek | The Tension Mounts: Mechanics Meets Morphogenesis and Malignancy[END_REF], matrix stiffening [START_REF] Paszek | Tensional Homeostasis and the Malignant Phenotype[END_REF] due to collagen deposition by variable cells, and an increase in interstitial pressure due to leaky vasculature and poor lymphatic drainage [START_REF] Netti | Role of Extracellular Matrix Assembly in Interstitial Transport in Solid Tumors[END_REF]. In addition, cancer cells show a compromised mechanorecprocity in a way where they often exert abnormally high force in response to a compliant matrix and therefore increase the force generated on the ECM, which disrupts cell-cell junction integrity, affecting tissue polarity and, therefore, enhances invasion of transformed cells into the surrounding environment (figure 16; B) [START_REF] Paszek | Tensional Homeostasis and the Malignant Phenotype[END_REF].

As the tumor mass expands, it exerts compressive stress on the surrounding extracellular matrix, vasculature, interstitial space, and lymphatic vessels. Reports have shown that compressive forces induce tumor angiogenesis and increase interstitial pressure up to 10× of that in normal tissue [START_REF] Rutkowski | A Driving Force for Change: Interstitial Flow as a Morphoregulator[END_REF]. At the subcellular level, compression in breast cancer cells induces significant changes to the cytoskeleton, including the altered composition of the actin, intermediate filament, and microtubule organization (Boyle et al., 2020). The induced changes in the cytoskeletal organization generate tension that activates downstream signaling pathways that have been shown to induce a multitude of cellular physiological effects in breast cancer cells, such as an acquisition of EMT phenotype, increase in proliferation, and enhanced cell motility and invasion [START_REF] Tse | Mechanical Compression Drives Cancer Cells toward Invasive Phenotype[END_REF]. Moreover, the generated stress in the tumor bulk has been shown to activate mechanosensing ion channels and downstream signaling pathways that lead to diverse arrays of cellular outcomes, including motility, proliferation, and invasion [START_REF] Broders-Bondon | Mechanotransduction in Tumor Progression: The Dark Side of the Force[END_REF].

The effect of compression on cancer cells and its ability to drive invasion was studied in vitro using several approaches. An example is a study in 2011, where the authors applied mechanical compression by pressing on the membrane where normal and cancer epithelial cells were seeded. The authors showed that applied mechanical compression induced invasive mammary epithelial cell migration in a scratch-wound assay; however, migration of normal and non-invasive mammary cell lines was suppressed [START_REF] Tse | Mechanical Compression Drives Cancer Cells toward Invasive Phenotype[END_REF]).

In addition, to the compressive stress, cells within the tumor bulk experience shear stress due to increased interstitial fluid flow. Through encapsulating breast cancer cells within an interpenetrating network (IPN) hydrogel and applied shear stress, Novak et al. showed that stimulated cells have higher proliferative capacity accompanied by chemoresistance toward paclitaxel (Novak et al. 2019). Here in Part II, I described briefly how the mammary gland is an active mechanical tissue. Through this model, I described the sources of mechanical cues exerted on cells; these forces could arise from the extracellular matrix, neighboring cells, and fluid. Cells sense such force and transform them into biochemical signals that could reach the nucleus and induce genotypic and phenotypic changes. These forces have been shown to contribute significantly to organ formation during the development and initiation of disease and progression. Indeed, most of the research findings were obtained using an in vitro system. These systems provided a better (1) mechanistic understanding of the role of individual mechanical forces in regulating cellular physiology, including gene expression and signaling, and (2) robust approaches for quantitative analysis.

At the end of Part II, I will describe a few systems that have been used to apply mechanical force in vitro and highlight the significant findings of each study. And since I applied only mechanical compression and stretch, with the focus on compression, in the plane of monolayer of epithelial cells, I will describe the methodology used to apply compressive forces and the relevant findings as a biological significance. III)-Technology leading the way ahead: recapitulating tissue mechanics in Vitro.

III.1) Applying mechanical compression by using Brick Strex

Brick strex device is an in vitro system that provides a robust solution for the mechanical manipulation of cells and allows the application of lateral compression and stretching with a given strain (figure 17,A. i). This device consists of a base PDMS membrane through which cells are seeded to achieve the desired confluency and attached membrane clamps and axes to adjust membrane stretching [START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF].

It has been studied that compressive forces that arise from intercellular contact in normal tissues stop cell growth, prevent high cellular density [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF], and thus preserve tissue/organ size [START_REF] Eder | Forces Controlling Organ Growth and Size[END_REF]. In order to study the molecular mechanism by which compression inhibits cellular proliferation in epithelial cells, a Brick strex device was used to apply lateral compression by seeding Madin-Darby canine Kidney cells (MDCKII) on a pre-stretched membrane (25% tensile stress). Cells were cultured to form 100% confluency, and then the membrane was released, thus resulting in lateral compression. Epithelial mechanical compressed cells show localization of YAP1 (Yes-associated protein 1) in the cytoplasm and an increase in β-catenin at the cell-cell junction. Notably, YAP1 and β-catenin are two mechanoresponsive proteins known to translocate into the nucleus under various mechanical signals (such as cyclic stretch, sparse growth, and substrate stiffness) and act as transcriptional factors that promote cellular proliferation and cell cycle entry [START_REF] Benham-Pyle | Cell Adhesion. Mechanical Strain Induces E-Cadherin-Dependent Yap1 and β-Catenin Activation to Drive Cell Cycle Entry[END_REF]. The observation in this study shows that lateral compression, which prevents the increase in cellular growth, is achieved by isolating these proteins in the cytosol and preventing their transfer to the nucleus. Interestingly, this is accompanied by low cytoskeletal tension.

Within a tissue, the exerted forces, whether mechanical stretch or compressive forces, can generate cellular processes and passive responses that enable epithelial sheets to fold. The mechanism by which cells release stress within a tissue or a monolayer is of great interest in the developmental biology field and mechanobiology. For example, Wyatt et al. showed that epithelial cell division along that axis of the exerted stretch helps redistribute the force within the monolayer and, thereby, stress relaxation. The results of rounds of division in a defined pattern/axis allow the cells to restore cell packing, thereby maintaining cellular homeostasis [START_REF] Wyatt | Emergence of Homeostatic Epithelial Packing and Stress Dissipation through Divisions Oriented along the Long Cell Axis[END_REF].

As for the compressive forces, Eisenhoffer et al. used a device similar to the Brick strex, which is formed of a flexible silicone membrane referred to as the Teflon chamber (figure 17,A. ii). The authors seeded kidney epithelial cells (MDCKII) followed by stretching the membrane up to 28% of their original length. After incubation and monolayer formation, the flexible membrane was released, thereby inducing compressive forces in the plane of the monolayer of epithelial cells MDCKII. The applied mechanical compression increased cell density over time. As a result, a significant percentage of epithelial cells were extruded from the monolayer concordantly with the increased cell density [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF]).

The triggered extrusion was sustained until the cell density decreased, thereby allowing the epithelial cells to maintain homeostasis within the monolayer. Interestingly, extrusion has also been observed in the mammary epithelial gland during involution (Monks et al. 2008). III.2) Applying mechanical compression on the cell surface by using a rigid flat plate.

Another simple in vitro method to apply mechanical compression is by applying compressive forces on the cell surface using a rigid flat (figure 17,B). This method was used to decipher how compressive forces control chromatin condensation, mainly in fibroblast cells exhibiting different geometries controlled by micropatterns [START_REF] Damodaran | Compressive Force Induces Reversible Chromatin Condensation and Cell Geometry-Dependent Transcriptional Response[END_REF]. The significant finding of this study was that mechanical compression induces chromatin condensation that appears as large foci observed through DAPI staining, mostly characterized by H3K9me3 methylation. Interestingly, the mechanism by which chromatin condensation is achieved is through the shuttling of protein histone deacetylase (HDAC3) from the cytoplasm into the nucleus, where the protein removes the acetyl group that are replaced then by the methyl group. The chromatin condensation is accompanied by reduced transcriptional activity and induced senescence.

Introduction

Part III Crosstalk between Nucleolus and Mechanobiology 52 I) Examples on the mechanosensitive components of the nucleolar environment: lamina is more than nuclear membrane meshwork, and chromatin remodels under stress.

Within the nucleus several components have been shown to be regulated by externally applied mechanical force, as the inner nuclear membrane protein : emerin [START_REF] Guilluy | Isolated Nuclei Adapt to Force and Reveal a Mechanotransduction Pathway in the Nucleus[END_REF][START_REF] Le | Mechanical Regulation of Transcription Controls Polycomb-Mediated Gene Silencing during Lineage Commitment[END_REF], Phospholipase A2 (PLA2) (Enyedi, Jelcic, and Niethammer 2016) and the nuclear pore complex [START_REF] Schuller | The Cellular Environment Shapes the Nuclear Pore Complex Architecture[END_REF]. Moreover, numerous reports showed that mechanical cues regulate chromatin organization and induce histones post-translational modifications [START_REF] Nava | Heterochromatin-References 138 Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage[END_REF][START_REF] Damodaran | Compressive Force Induces Reversible Chromatin Condensation and Cell Geometry-Dependent Transcriptional Response[END_REF][START_REF] Tajik | Transcription Upregulation via Force-Induced Direct Stretching of Chromatin[END_REF]. It is important here to mention, that nucleoli are spatially associated to chromatin in 1) Nucleolar associated domains that organize around the nucleolus and 2) Nucleoli are formed around the rDNA repeats that clusters in NORbearing chromosomes. Nucleolar-associated domains (NADs) are chromatin clusters in silent or inactive states, including the centromeres of the chromosomes that contain NORs, possibly centromeres of other chromosomes, telomeres, and the inactive X chromosomes (van [START_REF] Koningsbruggen | High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli[END_REF]). The localization of these genes near the nucleolus might be due to the specific concentration of proteins (e.g., transcriptional inhibitors or activators) (reviewed in [START_REF] Bizhanova | Close to the Edge: Heterochromatin at the Nucleolar and Nuclear Peripheries[END_REF]). Due to the connection between chromatin and the nucleolus, several questions arise; whether the mechanical force that could remodel chromatin might impact the nucleolus, and if such effect is universal to several mechanical cues or dependent on the chromatin compaction degree and histones modifications. On the other hand, it has been suggested that nucleolus influence chromatin organization and diverse aspects of gene expression. This suggestion comes from Act-D treated cells where following RNA transcription inhibition this results in an increase in the repressed gene that were found to be organized by the nuclear periphery (Ragoczy et al. 2014).

Interestingly, within the nucleus, nuclear lamina plays an essential role in maintaining nuclear structure [START_REF] Dechat | Nuclear Lamins: Major Factors in the Structural Organization and Function of the Nucleus and Chromatin[END_REF]. The molecular mechanism that connects the nuclear membrane, particularly lamins, to the nucleolus and whether they contribute to its plasticity was studied by Gupta et al., Buchwalter et al. 2017[START_REF] Martin | Lamin B1 Maintains the Functional Plasticity of Nucleoli[END_REF][START_REF] Martin | Lamin B1 Maintains the Functional Plasticity of Nucleoli[END_REF] observed that the depletion of LMNB1 coincided with nucleolar morphological changes that appear as elongated shapes near the nuclear periphery and continue to remodel over time, reaching nuclear caps that mimic the nucleolar shape observed in Act-D treated cells. In addition, the presence of lamin B1 in the nucleolus was confirmed by isolating intact nucleoli from Hela cells. Its nucleolar association is achieved through interactions with B23. The authors concluded that the lamin B1-nucleophosmin interaction contributes to the maintenance of the global organization of the nucleolus and participates in regulating the dynamics of its sub-compartments (Martin et al. 2009, 1). Moreover, Gupta et al. showed that lamin B2 knockdown disrupts nucleolar morphology. lamin B2 is associated with the nucleolus by interacting with nucleolin, thereby maintaining nucleolar structure (Sen Gupta and Sengupta 2017, 2).

Understanding the interplay between lamin A and the nucleolus was based on the studies conducted on fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome (HGPS). These fibroblast cells are characterized by a mutation in the nuclear scaffolding protein lamin A, referred to as progerin. Such a mutation in the lamin A network partially disrupts the global heterochromatin environment, leading to expansion of the nucleolus and an increased rRNA gene transcription, and thus an increase in 28S and 18S rRNAs abundance. Interestingly, increased rRNA gene transcription is associated with increased un-methylated marks on rDNA and RNApolI coupling to the rDNA promoter region [START_REF] Buchwalter | B2[END_REF].

Therefore, chromatin and Lamins that are associated with the nucleolus can be remodeled and post-translationally modified under an applied mechanical force. Based on that, we anticipate that applied force that reaches the nucleus could also change nucleolar structure and function through this connection.

II) Regulation of cellular homeostasis under an applied mechanical force: is the nucleolus associated?

Interestingly, cells experience diverse environmental cues that feed on the tight regulation of rRNA synthesis as part of quality cellular control, thereby placing the nucleolus at the center of the nuclear function. Cells use strategies to preserve internal cellular energy under stress to regulate ribosome production, which can be regulated at different levels: rDNA transcription, processing, and quality control. Indeed, I have also mentioned that some external stress favor ribosome biogenesis while others attenuate such processes.

Based on that, interesting fundamental questions need to be underscored: do mechanical cues favor ribosome biogenesis or not? And if the cell tends to regulate ribosome biogenesis, is that dependent on the type of mechanical cues, and would that be accompanied by nucleolar structural changes, and at what level could it be regulated?

Even though we don't have a clear, direct answer to these questions, it is important to mention that it has been reported that mechanical cues can regulate diverse signaling pathways that contribute significantly to regulating ribosome biogenesis at different levels. An example of such signaling pathways is the mTORC, which I have described in Part I; II.1 of the manuscript. The mammalian target of rapamycin or recently known as the mechanistic target of rapamycin (mTOR), is a serine/Threonine protein kinase that belongs to the phosphoinositide-3kinase (PI3K) related protein kinase (PIKK) family. mTOR is the catalytic subunit of two distinct protein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2); both complexes are associated with different protein substrates that respond to diverse signaling transduction networks and, therefore, play a different role in cellular homeostasis (Murugan 2019). mTOR signaling pathway has been shown to upregulate ribosome biogenesis at different levels, rDNA transcription (RNApolI, transcriptional proteins within the PIC complex) following serum activation (Hannan et al. 2003), and ribosome subunits translational machinery (activating eEF2 and eIF4B) in cancer (Gentilella, Kozma, and Thomas Part III Crosstalk between Nucleolus and Mechanobiology 54 2015). In addition, mTOR is considered at the center of many intrinsic signaling pathways, where cytoskeletal components and mechanosensitive proteins facilitate their activation (Lin and Liu 2019; Hornberger 2011). An interesting study in 2016 provided original evidence showing that RNA polymerase (I) activity is tightly controlled by integrin signaling. Their study showed that mechanical cues that arise from the ECM are sensed by fibroblast cells through integrin receptors and activate downstream signaling pathways [FAK/SRC/PI3K/mTOR]. This leads to the upregulation of rDNA transcription, verified by an increase in RNApolI occupancy on the rDNA gene loci, thereby increasing 47S premature-rRNA (Wu et al. 2016). Even though the authors did not investigate nucleolar structural changes and the distribution of critical nucleolar proteins within the nucleolus, this finding raises several possibilities for the mechanical cues to reach the nucleolus and induce structural and functional changes.

The motivation to understand the impact of biophysical cues on nucleolar structure and function comes from the rationale that 1) the nucleolus is closely associated with critical components that support nuclear architecture and are known to respond to mechanical forces; 2) mechanical signals can regulate diverse pathways that affect ribosome subunits formation and contribute to the maintenance of cellular homeostasis. In the next section, I will discuss recent studies investigating the interplay between nucleolar structure and function.

III) Direction of epithelial folding defines the impact of mechanical forces on the epithelial state.

Cell shape dynamics during development are tightly regulated and coordinated with cell fate determination. Triggered by an interplay between biochemical and mechanical signals, epithelia form complex tissues by undergoing coordinated cell shape changes, but how such spatiotemporal coordination is controlled remains an open question.

To dissect biochemical signaling from purely mechanical cues, we developed a microfluidic system that experimentally triggers epithelial folding to recapitulate stereotypic deformations observed in-vivo. Using this system, a controlled deformation (that resembles an epithelial fold) is applied to a monolayer and arising local compressive and tensional stresses acting on cells that were fully characterized. Our results showed that folding is associated with strains spreading away from the out-ofplane deformation and induces substantial changes in the local tissue tension. Combining the information of the mechanical state with the transcriptomic analysis we revealed that at the relatively short time scales (4 hours), cell compression led to the alteration of genes encoding transcripts involved in ribosome biogenesis and snoRNA [START_REF] Blonski | Direction of Epithelial Folding Defines Impact of Mechanical Forces on Epithelial State[END_REF].

Introduction

Part III Crosstalk between Nucleolus and Mechanobiology 55

Based on this research finding, we discovered that inducing changes in tension, particularly by applying compressive forces in the plane of the epithelial monolayer, regulates the transcriptional level of genes that encodes for snoRNAs and ribosomal proteins.

Therefore, there is a need to characterize and understand how global epithelial compression impacts nucleoli structure and function, which is the objective of my PhD study that I will address in the 'Thesis objectives'.

Here, it is worth mentioning that during the third year of my PhD and while investigating the influence of external compressive forces on the structure and function of the nucleolus, two studies emerged showing that the nucleolus responds to ECM-cell adhesion and ECM stiffness, which I will describe in the next section.

IV) Recent studies investigating the interplay between mechanical cues and nucleolar structure and function.

The first proofs of nucleolar sensitivity to mechanical cues have recently been published. The first study is by Pundel and his coworkers, which aims to investigate how the size of the cell adhesion area regulates the morphology and function of the nucleolus. In their study, the authors cultured human keratinocyte cells (HKs) on different micropatterns of 20 µm or 50 µm diameter to adopt round or spread morphology. First, they reveal that the limited ECM adhesion area (20 µm) promotes nucleolar fusion, with increased area and fewer nucleoli per nuclei, contrary to the larger adhesion area where more nucleoli appear.

Second, they showed that limited adhesion on small micropatterns significantly reduced both 47S rRNA expression and protein synthesis; these nucleolar morphological and functional changes were associated with the enrichment of genes that encode for terminal differentiation, contrary to the large adhesion micropatterns where proliferation genes were enriched [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF].

In this study, we observe that mechanical cues defined by cell-ECM adhesion impact nucleolar structure and function. However, the mechanism that induces such changes and if these observed changes impact the nucleolar tripartite organization still needs to be investigated.

Another report by Jaecker and colleagues shows that nucleolus is remodeled during collective cell migration of epithelial cells cultured on different extracellular matrix rigidity. It has been previously shown that a stiff or confined matrix triggers collective cell invasion (or migration) that is associated with the formation of leader cells that pull the follower cells. Moreover, leader cells are characterized by higher mechanosensitive proteins, spreading, and migration, thereby requiring more protein synthesis [START_REF] Ng | Substrate Stiffness Regulates Cadherin-Dependent Collective Migration through Myosin-II Contractility[END_REF]). However, it was not clear how the extracellular matrix stiffness impacts nucleolar structure. Therefore, to study that, the authors seeded healthy mammary epithelial cells (MCF10A) on fabricated polyacrylamide hydrogels of different elastic moduli coated with collagen type I. They showed that leader cells seeded on the stiff substrate have in average more than two nucleoli per nuclei compared to the leader cells on soft matrices. The authors also showed that ECM stiffness impacted the tripartite organization of the nucleolus, where leader cells on a stiff substrate show an increase in the number of fibrillar centers compared to the soft matrices [START_REF] Jaecker | Nucleoli in Epithelial Cell Collectives Respond to Tumorigenic, Spatial, and Mechanical Cues[END_REF].

By comparing these two studies, we could observe that as the cells adhere more to the ECM (adhesion areas from 20 µm to 50 µm or ECM rigidity from soft to stiff), this impacts the nucleolar morphology and function. These structural and functional changes are associated with cellular functional response, whether that is proliferation or differentiation.

The appearance of studies showing that adhesion cues define nucleolar structure and function, proves that nucleolus is part of the mechanoresponsive components in the nucleoplasm that is modulated structurally and functionally by biophysical cues.

Despite the appearance of studies showing that adhesion cues impact nucleolar structure and function. We still lack the precise spatio-temporal description of the effect of mechanical stress on nucleolar structure and function. Besides, it is of great importance to probe if other stresses than spatial constraints can equally participate in the regulation of the nucleolus.

The thesis aims to gain insight into the role of mechanical stress as a regulator of nucleolar structure and function.

As described in the introduction, several discoveries pointed to the importance of the mechanical environment in regulating cellular function. Mechanical forces are known to induce lamin remodeling at the subcellular level and to re-organize the chromatin. These two have proved to be in direct contact with nucleoli and regulate their structure and function. Because of these indirect links between biophysical signals and the nucleolar microenvironment, I hypothesize that the nucleolus responds to compressive forces.

Therefore, in my thesis, I had the following objectives:

⮚

The primary objective was to uncover the impact of compressive forces on the nucleolar structure.

Within this objective, my aim was to elaborate an experimental setup that would allow probing the effect of mechanical compression without the contribution of the continuous proliferation within the monolayer. Once achieved, I analyzed how mechanical compression impacted cells at the monolayer (cell-cell organization) and sub-cellular scale (nuclear morphology and nuclear envelope) at different time points after compression. Subsequently, I quantified the number and area of the nucleoli per nuclei as a first identification of morphological changes. Consequently, I probed if mechanical compression affects the nucleolar structure (tripartite organization) at the timescale at which the monolayer is affected.

In parallel with compressive forces, I also studied the effect of tensile stretch on nucleolar structure in MCF10A epithelial cells to know whether the nucleolus is sensitive to another type of induced tensional changes.

⮚

Second objective: was to determine the impact of compressive force on nucleolar protein localization and function.

Nucleolus has two important roles: 1) the initiation of ribosome biogenesis and 2) the response to environmental stress. Within this objective I aimed to explore if these two functions are affected by the compressive forces at the time scale of several hours post compression. I followed changes in localization and abundance of the main nucleolar proteins participating in nucleolar stress response and ribosome biogenesis, and measured changes in the amount of transcribed rRNA under mechanical stress.

. I) Cellular model and force application.

I.1-Cellular model:

MCF10A is an epithelial cell line isolated in 1984 from the mammary gland of a white, 36-years old female with fibrocystic breast. The cells were obtained from ATCC supplier with the following reference: (ATCC® CRL-10317™).

MCF10A fucci cells were also used in our project, kindly gifted by Alexis GAUTREAU (2021, Laboratoire de biologie structurale de la cellule, Ecole polytechnique, Paris). The cells are fluorescently labeled with cell-regulated proteins, Cdt1-tagRFP and Geminin-emerald GFP that are expressed during specific phase of the cell cycle, particularly G1 and S/G2/M respectively. Cells are cultured in DMEM/F12 (Gibco®) at 4.5g/l of glucose supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (EGF), 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 µg/ml insulin and 1% penicillin, in 37°C atmosphere, 5% CO2. The medium is changed 3 times per week. The cells are passed twice per week when reached 80% confluence. The cells are dissociated in trypsinethylenediaminetetraacetic acid (EDTA) at 0.05%, only 500 ul is added. Following cell detachment, we add 4ml of the medium followed by centrifugation at 1200 rpm for 6mins. The cell pellet is suspended in culture medium and then seeded in new culture dishes or T75 flask.

1.2-Methodology for the force application using in vitro system. 1.2.1-Flex-cell plate an in vitro system to apply uniaxial compressive forces.

In this system, MCF10A cells were seeded on a pre-coated PDMS silicone membrane with collagen type I and incubated at 37°C and 5% CO2 for 48 hours to form a monolayer. The plates used are designed for uniaxial stretch (Flex cell International Corporation); therefore, to apply mechanical compression, MC10A cells were seeded on the silicone membrane; once the cells were attached, a piston was inserted from beneath, thereby stretching the membrane. The cells on the stretched membrane were cultured at 37°C and 5% CO2 for 48 hours for a monolayer formation. After 48 hours, the piston was removed, inducing compressive forces in the plane of the epithelial cells. In order to generate mechanical stretched cells, MCF10A cells were seeded on the silicone membrane and incubated for 48 hours for a monolayer formation. After the incubation period, the piston was inserted, allowing the stretching of the membrane uniaxial (figure 18). 1.2.2-4D cell Dynamic confiner an in vitro system to apply confined cells between surface.

The 4D cell confinement enables the confinement of cells between two parallel surfaces to a defined micrometer height with sub-micrometer precision. The confinement height (distance between the substrate on which the cells are seeded and the device itself), is determined by the micropillars made of polydimethylsiloxane (PDMS), the kit provides a different height that ranges from 1-20µm. These micropillars are attached to a glass coverslip of 10 mm thickness. The coverslip containing the micropillars is attached to the PDMS piston so-called suction cup. The proximity of the coverslips toward the cell is controlled by the vacuum pump. In order to apply the confinement, first: the coverslips and the suction cup are prepared. This is carried out by cleaning the suction cup with 70% ethanol followed by drying it. Prior to 1 hour of conducting the experiment, the suction cup and the confinement coverslips are placed in the culture medium that will be used in the experiment. Secondly, following the dryness of the confinement slide and the suction cup from the cultural medium, the coverslips are placed on the cup, with micropillars faced up. The suction cup/coverslips are connected to a vacuum pump device and by aspirating the vacuum (-100mbar), the confiner is brought in close proximity with the cells (figure 19).

Figure 18: Scheme showing the in vitro flex-cell plate device used to apply uniaxial compressive and tensile forces. The left scheme shows the methodology followed to apply mechanical compression and tensile stretch. A monolayer is followed is formed seeding cells for 48 hours and at a density of 1.6x10 6 cell/ml. In order to apply compressive forces, once the cells are seeded, a piston is inserted for 48 hours, and then removed. To apply stretch, after 48 hours, of incubation and hence a monolayer formation, a piston is inserted thereby applying tensile stretch. The left scheme shows the system, in the upper part (disassembly) and the lower picture represents the assembled state.

II)-Electron microscopy

After mechanical force application, in order to prepare the samples for electron microscopy, 3ml of 4% glutaraldehyde was added to the culture medium, followed by incubation for 15 min at 4 ° C. After that, a new mixture of 1 Volume glutaraldehyde 4% and 1 Volume cacodylate 0.2 M pH 7. Then the previous solution was replaced with the glutaraldehyde/cacodylate mixture, and cells were preserved in the fixative at 4 ° C. Samples were embedded in resin and sectioned with a microtome. The sections were mounted on TEM grids and stained with uranyl acetate. For imaging, these sections were observed at various magnifications, ranging from 1500 to 25,000×, to cover different length scales-from cells to nuclei to nucleoli, along with details of the cytoskeleton-which was performed using a JEOL JEM-1400 120 kV Transmission Electron Microscope. Images were acquired with an AMT XR111 high-speed 4k × 2k pixel phosphorscintillated 12-bit charge-coupled device camera.

III)-Immunofluorescence of fixed samples

After applying the mechanical force, the control MCF10A epithelial cells and mechanically stimulated cells (compressed and stretched MCF10A cells) were stained against different nucleolar proteins in order to ascertain nucleolar morphological changes and probe the localization of nucleolar proteins.

Immunofluorescence labeling is processed as following for each condition: the cells on the pre-coated flex-cell plates were washed with 1X PBS (phosphate buffered saline) at pH 7.4 at 37°C then fixed with 4% paraformaldehyde for 15 min.

After fixation, samples were washed with 1X PBS pH 7.4 at room temperature each wash was done for 5 mins. The samples were then permeabilized for 15 mins with 0.1% Triton X-100 diluted in 1X PBS. The cells are then washed again for three times with 1X PBS, then unspecific sites are blocked for 30 mins with 3% BSA diluted in 1X PBS. The samples are then incubated with primary antibodies diluted in blocking solution at 4°C or at room temperature depending on the antibody (Table I). Following the incubation with primary antibody, 3-time washes with 1X PBS tween twenty at room temperature was carried out. Then the samples are incubated with secondary antibodies diluted in blocking solution for 1 hour (Table I). After that, three times washing was done, to remove the secondary antibody, followed by mounting the samples on lapteck with 4', 6'-diamidino-2-phenylindole (DAPI) to label nuclei, and sealed with aluminum foil. IV-Labeling of De-novo rRNA synthesis EU pulse labeling of RNA was performed according to the manufacturer's instructions (Click-IT EU RNA labeling kit, Life Technologies). After 48 hours and following the monolayer formation, the piston was removed to generate the compressed conditions, followed by directly adding the 5-ethynyl uridine into the medium for 4 hours. In step one, the 5-ethynyl uridine compound is actively incorporated into the nascent RNA. After 4 hours the medium is removed, and the samples are washed with PBS. After that 1 ml of 4% of PFA is added for 15 mins to fix the samples. Following that the fixative was removed and the samples were then washed twice with PBS, followed by adding 1 ml of 0.5% Triton X-100 in PBS and incubated for 15 mins. The samples are then washed, and a Click reaction cocktail buffer is added for 30 mins at room temperature. The cocktail contains the needed compounds that interact with the alkyne to produce the radioactive fluorescence. By the end, the samples were washed and incubated with the target antibody that was selected as a nucleolar marker, that is ncleophosmin B23.1. Immunofluorescence was carried out based on the methodology indicated in this section.

V)-Confocal microscope V.1-Confocal Biophoton microscope

The two-Biphoton excitation microscopy is a fluorescent imaging technique that allows imaging of life tissue up to one millimeter in thickness. The microscope is accompanied with an inverted motorized Zeiss Axiovert 200M. During all image acquisition, the objective used Plan-Apochromat 63×/1.4 Oil, images were obtained at zoom 1 and zoom 2. During image acquisition, the microscope laser was adjusted to prevent saturation and photobleaching. The settings were maintained: Laser used for the Far-red (600-700nm) =1%, Gain =650, pinhole=50 µm. Green laser (500-539 nm) =25%, Gain=800, pinhole=228 µm and DAPI/ Hoechst= 10%. The objective used Plan-Apochromat 63x/1,4 Oil.

In parallel with the two-Biphoton, the Dynascope a confocal microscope (Microscope confocal multiparametrique LSM710 NLO -LIVE7 -Confocor3) was also used. This microscope allows the acquisition of images in multiple planes. Lasers used on this confocal microscope, Green (500-539 nm) =3%, Far-red(600-700nm) =17%, and DAPI/ Hoechst (358-461 nm) =1%. The objective used Plan-Apochromat 63x/1,4 Oil.

VI) Image analysis VI.1-Analysis of nucleolar area

In order to analyze nucleolar area, nucleolin was selected as a nucleolar marker due to its high abundance within the nucleolus. Immunolabeled images with nucleolin in all conditions were obtained by confocal microscopy. First, from images of max projection, each nucleus was cropped to allow proper demarcation and detection of nucleoli. Second, for each cropped image, a median filter was applied in order to reduce the noise. Third, nucleoli were segmented to create a mask and added to the ROI manager. Fourth, from the selected ROI were overlaid on the corresponding nucleoli in order to obtain nucleolar size.

VI.2-Measurement of nucleolin concentration in the nucleolus over the nucleoplasm.

In order to verify that nucleolin is shuttling from the nucleolar core into the nucleoplasm, the ratio of nucleolin intensity was measured in the nucleolar core over the nucleoplasm. To do this, each nucleus was cropped and for each single nucleolus, five circles were drawn on the nucleolar core and in parallel 5 circles were drawn in the nucleoplasm, all the circles were the same size. The intensity in each circle was measured and then the average of the 5 circles in the nucleolar core was divided to the average of the intensity of the five circles in the nucleoplasm.

VI.3-Analysis of lamin A/C by the nuclear periphery

Measuring lamin A/C intensity by the nuclear rim was followed based on the already published data methodology by Miura et al [START_REF] Miura | Measurements of Intensity Dynamics at the Periphery of the Nucleus[END_REF]. Nuclei were cropped individually from the Z-projection of multitask images obtained by confocal microscope. First, each nucleus was blurred to eliminate the noise from the backgrounds, then the threshold was adjusted in order to generate the mask. Following that, the obtained mask was inverted and duplicated. The original form was eroded, and the duplicate form was dilated from the duplicated one. After that eroded was subtracted from the dilated in order to obtain the nuclei rim. Nuclei rim was added to the ROI and then overlaid on the nuclei with lamin A/C staining in order to measure the intensity.

In all conditions it is important to mention that the depth of the rim was approximately to 0.64 µm measured from the nuclear border.

VI.4-Analysis of 5'EU Signal in the nucleolus and the nucleus

Images with 5'EU intensity that indicated the newly synthesized RNA were acquired using confocal microscopy. To analyze the intensity of 5'EU in the nucleolus, Z stack images obtained from B23 staining, first median filter was applied to the images in order to remove the background. Seconds, by applying thresholding the B23 was segmented, and the generated nucleoli were added into the ROI. Third, from the generated images and the ROI, it was later overlaid into the images obtained from the EU click labeling, that how the signal in the nucleoli was estimated.

VII)-Analysis of expression of proteins by western blotting: VII.1-Preparation of cell lysates:

Cells that were previously cultured on the flex-cell pre-coated with collagen type I in all studied conditions were lysed in the following way to extract the proteins. Cells were rinsed with cold cell culture PBS low in magnesium with pH 7.4, at 37°C. Following that cell cellular lysates was collected by using 2x laemmli buffer formed of the following compositions: 4% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, 10% Glycerol, 0.004% Bromophenol Blue, 0.125M Tris HCL, the solution is prepared at a pH =6.8. Following that the samples were sonicated for few seconds~10sec, to rapture the cell membrane and fragment the DNA. Before each western blotting experiments, the samples were denatured for 5 mins at 95°C, then placed on ice before loading.

VII.2-Migration and transfer of Membrane

To separate the proteins by gel electrophoresis, proteins from the cell lysates were added into the wells of a mini format SDS-PAGE gel, along with molecular weight markers. In all the experiments, the used gel was of 4-10 % polyacrylamide. Following that, the gel was run for 2 hours at 40V, 400mA. After electrophoresis, the gel was placed in 1x transfer buffer for 3 mins. Followed by assembling the transfer sandwich and making sure no air bubbles are trapped in the sandwich. In all the experiments and transfers nitrocellulose membrane (Trans-Blot® TurboTM Bio Rad) was used, and membrane transfer was carried out for 10 mins.

VII.3-Immunobloting and revelation:

Once the membrane is obtained, it is saturated in 5% bovine serum albumin (BSA) (Sigma Aldrich®) diluted in tris-buffered saline (TBS)/Tween (TBS: 50 mM Tris at pH 7.5,150mM NaCl; Tween20 at 0.1%) for 45 minutes at room temperature while agitating. Following that the membrane was washed for 3 times with TBS/Tween, followed by incubation with primary antibodies diluted in BSA overnight at 4°C under continuous agitation. The membrane was then washed with TBS/Tween, for 3 times, followed by incubation with secondary antibodies for 45 mins at room temperature. The antibodies that were used in western blotting, are the same as used for IF, except to be diluted at 1/1000. With additional primary antibody fibrillarin that is indicated in Table2 with secondary antibodies used in western blotting.

Detection is performed by using chemiluminescence (SuperSignal® West Pico Chemiluminescent Substrate from Thermo Scientific). The revelation of the bands was done through the Vilber machine. The normalization is made based on the constitutively expressed genes housekeeping gene that encodes for Glyceraldehyde-3phosphate dehydrogenase (GAPDH) and Actin. The gene expression of UBF nucleolar protein was assessed by quantitative PCR in real time from extracted RNA in the several studied conditions. RNA extraction was performed by using RNeasy Plus Mini Kit (cat.nos.74134 and 74136). Once isolated the RNAs were finally assayed using a Nanodrop® microvolume spectrophotometer.

VIII.2-Reverse transcription of the RNA into cDNA

For the reverse transcription of RNA into cDNA, 1µg of RNA is used from the previously diluted RNA in ribonuclease (RNase) free water. The RNA samples are denatured for 5 mins at 70°C, then its immediately cooled for 2 mins. In parallel a transcriptional mixture is prepared that contains 4ul of iScript TM Reverse Transcriptase Supermix (contains 5x RT supermix with RNase H + Moloney murine leukemia virus (MMLV) reverse transcriptase, RNase inhibitor, dNTPs oligo random primers, buffer, MgCl2, and stabilizers reference 1708840, Bio-rad) that catalyzes the reverse transcription reaction, in addition to 14.25 µl of RNase free water. In parallel a negative control is prepared that contains the same mixture except the reverse transcriptase is replaced with no-RT (same components except it does not include reverse transcriptase). Once all the samples are prepared, they are incubated in a thermocycler for 1 hour initially at 42 (activation of the reverse transcriptase) then 3 mins at 65 (inactivation for the enzyme). Once the DNA complementary cDNA is formed it is stored at -20 to be used later for q-PCR.

VIII.3-Quantitative real time PCR:

SYBR ® Green technology was used to detect the level of cDNA in each condition. This technique is based on the intercalation of SYBR ® green to the dsDNA. As the amplification is processed, the amount of the DNA product increases, and hence the number of SYBR ® green molecules incorporated to the DNA increases. qPCR was performed by adding 5 ng of cDNA for a final volume of 10 µl per reaction, with 5 µl of SYBER green, 2.6 µl of RNase-free water and 0.11 µl of target primers.

The amplification was performed by using the C1000 Dx Thermal Cycler with CFX96 Dx ORM according to the following program: pre-denaturation at 95 °C for 5mins, denaturation for 15 seconds at 95 °C, followed by annealing at 60°C for 30 mins and extension at 72 °C.

Primers used for the: UBF1 (UBTF) Human qPCR Primer Pair: Forward Sequence: CCGAATGTGGAACGACCTGTCT Reverse Sequence: GATCTCCTCAGCTCTTTTGGGG 45S Human qPCR Primer Pair: Forward Sequence: GAA-CGG-TGG-TGT-GTC-GTT-C Reverse Sequence: GCG-TCT-CGT-CTC-GTC-TCA-CT

Materials and Methods

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IX)-Statistical studies

All results are expressed as means ±Standard Error of the MEAN (SEM). The statistical study is represented along with the number of replicates per group or samples (N) and the number of quantified variables (n or n'). The significance of the results is assessed by the statistical study indicated by one way ANOVA, Dunnett's test, and two-tailed test (comparing between two groups). Only probabilities less than 5% (p<5) are considered significant. Statistical tests were performed using GraphPad software prism.

Results

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5.Results

Results

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5.1-Introduction

Over the past few decades, there has been a wider recognition that mechanical cues arising from neighboring cells, extracellular matrix, and fluid flow contribute to the development of crucial aspects of organs, tissue regeneration, and disease initiation and progression [START_REF] Paszek | The Tension Mounts: Mechanics Meets Morphogenesis and Malignancy[END_REF]. Notably, these biophysical cues are received by specific receptors or molecular assemblies associated with cell membranes and subsequently converted into a biochemical signal to trigger cellular response and alter cellular behavior (reviewed in Maurer and Lammerding 2019). Among these mechanical forces, changes in tension, including tensile and compressive forces, are recognized to regulate the cells and surrounding tissue. In contrast to the tensile forces, which are far more widely and extensively studied in the mechanobiology field, the biological effect of compressive forces and the mechanism of its reception, transmission, and transduction in cells are less defined. However, at the cellular level, the significant role of compressive forces has been demonstrated in several biological scenarios. For example, in stem cell biology, compressive forces on mouse embryonic stem cells induce cellular differentiation [START_REF] Pagliara | Auxetic Nuclei in Embryonic Stem Cells Exiting Pluripotency[END_REF]. In oncology, compressive forces have been shown to induce cancer cell migration, proliferation (Dolega et al. 2017), and death [START_REF] Cheng | Micro-Environmental Mechanical Stress Controls Tumor Spheroid Size and Morphology by Suppressing Proliferation and Inducing Apoptosis in Cancer Cells[END_REF][START_REF] Tse | Mechanical Compression Drives Cancer Cells toward Invasive Phenotype[END_REF]. In developmental biology, compressive forces that arise from cell-cell collision have been shown to contribute to branch formation and involution of the mammary gland [START_REF] Ewald | Mammary Collective Cell Migration Involves Transient Loss of Epithelial Features and Individual Cell Migration within the Epithelium[END_REF][START_REF] Nguyen-Ngoc | ECM Microenvironment Regulates Collective Migration and Local Dissemination in Normal and Malignant Mammary Epithelium[END_REF][START_REF] Watson | Remodeling Mechanisms of the Mammary Gland during Involution[END_REF]. In vitro studies have shown that compressive forces inhibit cellular proliferation [START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF]) and induce extrusion [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF] when applied in the plane of the monolayer of epithelial cells.

Generally, biophysical cues are sensed by the cells through an intricate network of adhesive receptors (reviewed in Lim, Jang, and Kim 2018) membrane mechanosensitive channels, and cytoskeletal elements (reviewed in [START_REF] Martino | Cellular Mechanotransduction: From Tension to Function[END_REF], which regulate a wide range of cellular responses and signaling pathways. In addition, the cytoskeleton is connected to the nuclear membrane through the LINC complex, consisting of nesprins and SUN proteins. The LINC complex spans the nuclear membrane, it interacts with the underneath nucleoskeleton, an intermediate filament referred to as Lamins [START_REF] Lombardi | Keeping the LINC: The Importance of Nucleo-Cytoskeletal Coupling in Intracellular Force Transmission and Cellular Function[END_REF]. Exerted forces have been shown to regulate nuclear morphology and induce lamin A/C remodeling that could be associated with post-translational modifications. [START_REF] Buxboim | Matrix Elasticity Regulates Lamin-A,C Phosphorylation and Turnover with Feedback to Actomyosin[END_REF]. Moreover, mechanical compression has been shown to have an effect on chromatin state and chromatin condensation, hence altering gene expression [START_REF] Nava | Heterochromatin-References 138 Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage[END_REF][START_REF] Damodaran | Compressive Force Induces Reversible Chromatin Condensation and Cell Geometry-Dependent Transcriptional Response[END_REF].

Within the nucleus, the nucleolus is the most prominent membrane-less nuclear body, where the synthesis of the small and large ribosomal subunits for the later translational processes occurs. The nucleolus is formed of three building blocks, the fibrillar center (FC), surrounded by the dense fibrillar center (DFC). The FC/DFC is included in the largest compartment, the granular center (GC). Each of these nucleolar sub-compartments has a unique complement of proteins and rRNA that executes its function. When cells experience diverse environmental cues that feed on the tight regulation of rRNA synthesis, one strategy followed by the cells to preserve their Results 71 cellular energy under stress is to regulate ribosome biogenesis, thereby preserving their internal energy. This regulation can happen at different levels, including rDNA transcription and processing (reviewed in [START_REF] Grummt | Wisely Chosen Paths -Regulation of RRNA Synthesis[END_REF]2013). The nucleolus is considered the central hub of nuclear function, by which its role goes beyond ribosome subunits formation into cell cycle regulation (reviewed in [START_REF] Boisvert | The Multifunctional Nucleolus[END_REF]), stress response [START_REF] Xie | Regulation of the MDM2-P53 Pathway by the Nucleolar Protein CSIG in Response to Nucleolar Stress[END_REF], and maintaining cellular homeostasis (reviewed in (Grummt 2013)). Due to this multifunctionality, defining nucleolar structural organization, protein composition, and morphological changes becomes a prerequisite for understanding its role in cell physiology, pathology, and in response to mechanical cues.

Interestingly, mechanistically sensitive nuclear components such as chromatin and Lamins that are remodeled by mechanical forces are, in fact, directly or indirectly associated with the nucleolus. For example, in addition to the fact that the mammalian nucleolus contains specific regions called nucleolar organizing regions (NORs) and rDNA, nuclear chromatin within the nucleus is organized in regions near the nucleolus referred to as nucleolus-associated domains (NADs) (van [START_REF] Koningsbruggen | High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli[END_REF]. Moreover, the nucleolus is a bio-molecular condensate that generally occurs via multiple weak interactions between proteins and RNA; it has been reported that the formation of condensates is affected by the viscoelastic properties of chromatin where condensates form in chromatin-poor regions [START_REF] Hansen | The Solid and Liquid States of Chromatin[END_REF]. In addition, lamin A, lamin B1, and B2 have been shown to affect the nucleolar structure, while a mutation in lamin A results in nucleolar expansion and increases ribosome biogenesis in Hutchinson-Gilford-derived fibroblast cells (Buchwalter and Hetzer 2017a). Lamin B1 and B2 have been reported to contribute to the global organization of the nucleolus by interacting with a crucial nucleolar protein referred to as nucleophosmin (B23) and nucleolin (C23), respectively (Sen Gupta and Sengupta 2017, 1;Martin et al. 2009, 1).

The interplay between biomechanical cues and nucleolus has gained interest recently. As I have detailed earlier in Part III, It has been shown that external mechanical cues, particularly the cell-ECM adhesion and extracellular matrix stiffness, impact the nucleolar structure and function [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF][START_REF] Jaecker | Nucleoli in Epithelial Cell Collectives Respond to Tumorigenic, Spatial, and Mechanical Cues[END_REF]. The appearance of studies showing that adhesion cues define nucleolar structure and function, proves that nucleolus is part of the mechanoresponsive components in the nucleoplasm that is modulated structurally and functionally by biophysical cues. However, the impact of compressive forces that have been shown to regulate cell function, and contribute to diseases initiation and progression, on the nucleolar structure and function is still undefined.

In our recent results, we showed that compressive forces and not tensile stretch, when induced in the plane of a monolayer of epithelial cells at a short timescale for 4 hours, regulate the transcript level of genes encoding for adhesion proteins, ribosomal proteins and small non-coding RNAs (snoRNAs) [START_REF] Blonski | Direction of Epithelial Folding Defines Impact of Mechanical Forces on Epithelial State[END_REF]. Based on these findings, I asked several fundamental questions: could the observed changes in the transcription of the ribosomal proteins be associated with reorganization of nucleolar structure, and how could compressive forces impact nucleolar function? And if I observe Results 72 nucleolar structural and functional changes, would that contribute in maintaining epithelial homeostasis under the external exerted forces? Therefore, to understand and uncover the impact of compressive forces on the nucleolar structure, I optimized an in vitro system to induce in-plane compressive stress within the epithelial monolayer. We selected MCF10A cells since they are known to form polarized monolayers, which is important for proper force transmission through cell-cell junctions.

After inducing compressive forces, and in order to uncover the effect of the stress on nucleolar structure, I quantitatively described changes in the nucleolar tripartite organization, and morphology including area and number of nucleoli per nuclei. Following that, to know whether exerted force impacts nucleolar function, I probed the effect of force on the localization and abundance of nucleolar proteins that plays a crucial role in ribosome biogenesis, in addition to measuring the level of the newly synthesized rRNA.

The results are divided in the following pages into two chapters, depending on the objectives described before. In Chapter I, I will present the methodology used to create a semi-quiescent monolayer that will enable probing the effect of compressive stress independently to cell cycle activity within the monolayer. I will also provide evidence showing that external compressive forces applied for different periods in the plane of the MCF10A monolayer regulate nuclear morphology and induce lamin A/C remodeling. From structural and morphological studies, I will show that the nucleolus is mechanically sensitive and reorganize its structure under applied external compressive forces.

In Chapter II, I will present evidence showing the influence of compressive forces on rDNA transcription and on two nucleolar proteins involved in this process. In particularly, I will show that compressive forces induce dynamic and transient changes in UBF and NCL, abundance, and localization. Furthermore, I will show that applying compressive forces of 4 hours attenuates rRNA synthesis, and that the observed response to mechanical stress might be independent to p53.

5.2-Chapter 1

I) Uncovering the impact of the compressive stress on nucleolar structure I.1) Optimization of the in vitro system in order to apply in-plane epithelial compression and tensile stretch I.1.1-Methodology for force application using flex-cell plates Epithelia that naturally occur as monolayers can experience different tensile stresses, such as stretch and compression [START_REF] Chen | Tensile and Compressive Force Regulation on Cell Mechanosensing[END_REF]. To reproduce these physiological cues in vitro, I used a previously developed system referred to as flex-cell plates that are commercially available. The in vitro system is formed of an elastic membrane where cells are cultured to grow into a monolayer that can be mechanically stretched and compressed. This system was initially used for static stretching of the cells (33% strain application) [START_REF] Gudipaty | Mechanical Stretch Triggers Rapid Epithelial Cell Division through Piezo1[END_REF], but I established a simple method to cause compressive stress using the same system. Deformation of the membrane relies on inserting the piston, which causes direct stretching. However, if cells are seeded on the pre-stretched membrane, in that case, removing the piston will relax the membrane causing in-plane compression of the same value (33%) within the monolayer.

Briefly, MCF10A epithelial cells are seeded at a seeding density that corresponds to 1.6×10 6 cells/ml on the top of PDMS (in a 6-well plate format) and incubated for 48 hours until a monolayer is formed. In order to apply the uniaxial compression, the same density is seeded on the PDMS membrane. Once the cells adhere, a piston is inserted, applying up to 33% uniaxial tensile strain to the membrane ("Cell Stretching Bioreactors | Flex-cell International" n.d.). A scheme showing steps to apply mechanical compression and the assembly of the used system is illustrated in figure 18 in the methodology section. The cell seeding density and incubation period was optimized as described below.

I.1.2-Creating a semi-quiescent monolayer is a prerequisite to apply mechanical uniaxial force

In our system, I aim to create a semi-quiescent monolayer of epithelial MCF10A cells, in which the cells are not in a high proliferative state but rather in a semiquiescent state. That will allow me to distinguish the effect of external applied compressive forces from internal cellular changes resulting from proliferation. Therefore, the cell seeding density was optimized by seeding cells at different densities (0.6×10 6 cells/ml, 1.6×10 6 cells/ml, and 2.5×10 6 cells/ml), followed by DAPI staining to observe the nucleus and immunostaining against nucleolin a nucleolar marker.

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Through this I could observe that increased cell seeding density results in overcrowding of epithelial cells observed by DAPI staining, as observed in 2.5×10 6 cells/ml (figure 20;A). Crowding due to increased cell density, has been shown to inhibit diverse signaling pathways that form an intricate network with the ribosome biogenesis in the nucleolus such as mTOR signaling pathway. Moreover, increased cellular density has been shown to increase the expression of Histone deacetylase 1 (HDAC1), which removes acetylation marks, thereby allowing chromatin methylation and its condensation [START_REF] Trajkovic | Fluctuations in Cell Density Alter Protein Markers of Multiple Cellular Compartments, Confounding Experimental Outcomes[END_REF]. Therefore, in order to avoid the overlapping between cellular crowding and the crosstalk with the applied compressive forces, I selected 1.6×10 6 cells/ml as the cell seeding density through all the experiments.

To further optimize conditions for epithelial monolayer culture in the used system, I seeded MCF10A cells at an equal cell seeding density (1.6×10 6 cells/ml) and observed formed epithelium at different incubation periods, i.e., 24 hours and 48 hours. Since adhesion provides spatial cues that initiate cell polarization via the recruitment of cadherin-associated cytosolic proteins, including β-catenin, an essential component of the adherent junction, and α-actinin that binds to the F-actin within the cytoplasmic region [START_REF] Clevers | Wnt/β-Catenin Signaling in Development and Disease[END_REF]); MCF10A cells were stained for β-catenin at both incubation period. After 24 hours, β-catenin showed nuclear localization (figure 20;B). However, after 48 hours, β-catenin re-localize to the cell-cell junction. The presence of β-catenin in the nucleus after 24 hours of incubation indicates that cells are still undergoing proliferation. β-catenin is a component of the cell-cell anchoring junctions and also act as a transcriptional factor, where it translocates into the nucleus and transcribes genes that induce proliferation [START_REF] Monga | Role and Regulation of β-Catenin Signaling During Physiological Liver Growth[END_REF]). On the contrary, the observed localization of β -catenin by the cell junction after 48 hours of incubation (figure 20;B). indicates that the monolayer is in a polarized state. In order to know whether MCF10A cells seeded at 1.6×10 6 cells/ml and incubated for 48 hours form a monolayer that is in a semiquiescent state. I further ascertained the MCF10A cell-cycle by using MCF10A fucci cells, that is considered an efficient tool for cell cycle analysis. MCF10A fucci cells are fluorescently labeled with cell cycle-regulated proteins, Cdt1-tagRFP and Gemininemerald GFP that are expressed during specific phase of the cell cycle, particularly G1 and S/G2/M respectively. I quantified the percentage of cells in G1 and G2 cell cycle phase in epithelial monolayers (N= 3 independent experiments, with n= 882 representing the total number of nuclei in the three independent experiments). Selected cell culture conditions for the monolayers of MCF10A cells resulted in a high percentage of cells in G1 phase (mean percentage of nuclei with, Cdt1 fluorescently labeling=79%), and significantly smaller population of cells in S/G2/M (mean percentage of nuclei with Geminin fluorescently labeling =20%) (figure 20;C). This quantification confirmed that epithelial MCF10A cells created a monolayer that is at semi-quiescent state.

Despite the overall monolayers stalling in the cell cycle, it is important to underline that there is a significant spatial heterogeneity in cell density (figure 20;D). With images of a large field of view, I could observe regions of lower and higher density in both control and compressed monolayers. Such heterogeneity is common for

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numerous models of epithelial monolayers cultured in vitro and can trigger locally different responses upon compression.

To avoid ambiguity in image analysis I always took images at least in 5 randomly chosen zones within the monolayer. Moreover, to verify whether the applied compressive forces for 4 hours, impacts MCF10A cell cycle, I analyzed the percentage of cells in G1 and S/G2/M. (N=3 total number of independent experiments, n'= 971 total number of counted nuclei from the three experiments). Through this analysis I could not observe a significant change compared to the control (figure 20;C). This indicates, compressive forces applied for 4 hours does not induce cellular proliferation.

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I.1.3-Compressive stress reinforces β-catenin at cell junctions

Following protocol optimization, I wanted to probe the effect of applied compressive forces on the epithelial MCF10A cells by observing localization of crucial protein at the cell-cell junction. Compression is known to increase cellular packing (density per area) [START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF][START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF]) and to induce extrusion as a mean of epithelial tissue homeostatic regulation [START_REF] Rosenblatt | An Epithelial Cell Destined for Apoptosis Signals Its Neighbors to Extrude It by an Actin-and Myosin-Dependent Mechanism[END_REF]. While stretching of epithelial tissues proved to be quickly dissipated by tissue contractility and cell division [START_REF] Wyatt | Emergence of Homeostatic Epithelial Packing and Stress Dissipation through Divisions Oriented along the Long Cell Axis[END_REF][START_REF] Gudipaty | Mechanical Stretch Triggers Rapid Epithelial Cell Division through Piezo1[END_REF], the effect of in-plane compression is too date much less understood. It has been shown that the time required for a monolayer to dissipate the mechanical compression and to return to a homeostatic cell density takes about 6 hours and is based on cell extrusion [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF]. At the same time, monolayers exposed to stretching or compressive stress are known to regulate cell-cell adhesion and cell-ECM adhesion (Charras and Yap 2018). Cell-cell junctions such as cadherin adhesions proved to be mechanosensitive with the reinforcement upon tension [START_REF] Borghi | E-Cadherin Is under Constitutive Actomyosin-Generated Tension That Is Increased at Cell-Cell Contacts upon Externally Applied Stretch[END_REF].

To observe the time scale at which mechanical stress affects epithelial organization in MCF10A monolayers, I followed the organization at cell-cell junctions in time up to 6 hours. To study the effect of the mechanical compression on the monolayer organization I stained the control epithelial MCF10A monolayers and mechanically stimulated monolayers at the different time points against β-catenin. The membrane bound β-catenin that associates with E-cadherin was reported to require inhibition of proteolytic cleavages. This results in its stabilization and accumulation of heavy multiubiquitinated forms at the cell-cell junction [START_REF] Aberle | Beta-Catenin Is a Target for the Ubiquitin-Proteasome Pathway[END_REF]. It has been shown that when cells are in contact with each other following lateral compression, β-catenin accumulates at the cell-cell junctions [START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF]).

In our system, I found that in the control epithelium, β-catenin was positioned at the cell-cell junctions with a measurable signal in the cytoplasm. In contrast, following compression β-catenin significantly accumulated at the cell-cell junction after 2 hours and decreased to control levels after 4 and 6 hours post compression (figure 21A,i). This observation was quantified by calculating the ratio of β-catenin intensity at the celljunction /nucleus. Analysis confirmed that β-catenin intensity increased significantly after 2 hours of compression as compared to the control (figure21A; ii) and stabilized at the control level for 4 hours and 6 hours post compression. These findings indicate that compressive forces regulated the organization of the epithelial monolayer particularly with the strongest effect at 2 hours after mechanical stress was applied. Our observations are confirmed by another report showing that both, in-plane stretch and compression result in the increased signal of catenin at the cell-cell junctions [START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF]. This confirmed that a static compression in our system imposed significant changes in tissue tension that are maintained at the scale of hours.

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I.1.4-Compressive stress impacts nuclear morphology and induce lamin A/C remodeling

Numerous microenvironmental cues (ECM, rigidity, confinement and tensional stresses) have proved to impact not only cellular but also nuclear shape and volume [START_REF] Alisafaei | Regulation of Nuclear Architecture, Mechanics, and Nucleocytoplasmic Shuttling of Epigenetic Factors by Cell Geometric Constraints[END_REF]). These in turn, showed to impact nuclear lamina and chromatin organization [START_REF] Buxboim | Matrix Elasticity Regulates Lamin-A,C Phosphorylation and Turnover with Feedback to Actomyosin[END_REF]. Therefore, I investigated the nuclear morphological changes upon mechanical compression. To do so, MCF10A monolayer in control and under compression were stained with DAPI (figure 22A). Using automatic thresholding in ImageJ I quantified the nuclear projected area which shows significant reduction in cells under mechanical compression as compared to control (figure 22A, ii). Notably, this reduction in nuclear area is significant at 4 hours post compression. Furthermore, mechanical compression affects the nuclear shape through time, where nuclear circularity was significantly higher at 4 hours (N=2 total number of independent experiments and n=57 total number of analyzed nuclei per condition) (figure 22A, iii). To further evaluate the significance of observed morphological changes after 4 hours, I performed a more profound analysis (N=6 total number of independent experiments, n=719 total number of analyzed nuclei per condition for area, n'=216 total number of analyzed nuclei for circularity) (figure 22A, iv and v). Overall, these findings indicate that mechanical compression has an impact on nuclear morphology up to 6 hours.

The architectural component and regulator of nuclear morphology and rigidity is nuclear lamina (luv Kishore Srivastava et al. JCS 2021). Composed of a meshwork of lamin B and lamin A/C, located beneath the nuclear membrane and provides the structural stability for the nucleus [START_REF] Dechat | Nuclear Lamins: Major Factors in the Structural Organization and Function of the Nucleus and Chromatin[END_REF]. It has been shown that Lamin A/C can be post-translationally modified and remodeled under various mechanical cues [START_REF] Ihalainen | Differential Basal-to-Apical Accessibility of Lamin A/C Epitopes in the Nuclear Lamina Regulated by Changes in Cytoskeletal Tension[END_REF][START_REF] Swift | Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation[END_REF]. Moreover, nuclear lamina is in a direct proximity and linked through numerous nuclear membrane proteins with the chromatin and nucleolus [START_REF] Gupta | Lamin B2 Modulates Nucleolar Morphology, Dynamics, and Function[END_REF]Buchwalter and Hetzer 2017b). Therefore, knowing that nuclear morphology was affected by compressive forces and to further probe the response of lamin A/C upon compression, MCF10A control and mechanically compressed monolayers were stained against Lamin A/C (specific to an epitope located between amino acids 2-29 at the N-terminus). Interestingly, at 2 hours post compression when β-catenin was increased at cell-cell junctions I observed loss of lamin-A/C signal and only few nuclei showed complete lamin A/C staining by the nuclear membrane (figure 22B;i). This behavior was transient and re-occurring in time, after mechanical compression as at 4 hours typical lamin A/C staining was observed which then abolished again at 6 hours (figure 22B;i). Based on lamin A/C image analysis of intensity at the nuclear rim, the levels were non significantly different between the control and monolayers under compression of 4 hours (N=3 independent experiments, n= 35 total number of analyzed nuclei) (figure 22B; ii). Analyzing lamin A/C at 2 and 6 hours post compression, was not possible due to the observed lamin A/C changes.

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The absence of lamin A/C staining in nuclei clearly observed by DAPI in addition to our results showing that the total level of lamin A/C measured by WB did not change over time) (figure 22B; iii). suggest that compressive forces induce lamin A/C remodeling. This remodeling might prevent the accessibility of the antibody for the target epitope which is a likely hypothesis as it has been recently shown [START_REF] Wallace | The Lamin A/C Ig-Fold Undergoes Cell Density-Dependent Changes That Alter Epitope Accessibility[END_REF][START_REF] Ihalainen | Differential Basal-to-Apical Accessibility of Lamin A/C Epitopes in the Nuclear Lamina Regulated by Changes in Cytoskeletal Tension[END_REF].

Altogether these results showed that global epithelial mechanical compression not only affects epithelial organization at the cellular level but also at the sub-cellular scale by causing significant changes in the nuclear morphology and by affecting nuclear lamina.

Interestingly, the effect of mechanical compression extends beyond expected short time-scale response and its influence is visible even at 6 hours post compression.

Based on the previous reports showing that nucleoli are sensitive to changes in nuclear morphology and lamina composition, my hypothesis is that nucleoli adapt and respond to mechanical stress.

……………………………………………………Figures are presented on the next page.

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I.2) Unraveling the effect of compressive stress on nucleolar structure and morphology I.2.1-Mechanical compression induces a dynamic change in nucleolar tripartite organization Following our findings that compressive forces induce i) remodeling of nuclear lamin A/C at 2 hours post-compression, ii) impact nuclear morphology observed by the decrease in area and increase in circularity, and iii) alteration of genes encoding transcripts involved in ribosome biogenesis and snoRNAs [START_REF] Blonski | Direction of Epithelial Folding Defines Impact of Mechanical Forces on Epithelial State[END_REF], I investigated the effect of compressive forces on nuclear components particularly the nucleolus.

The three building blocks of nucleolus correspond to the fibrillar center (FC), which appears as low-contrast areas surrounded by a highly contrasted dense fibrillar center (DFC). The FC/DFC is included in the granular center (GC), the largest compartment [START_REF] Goessens | Nucleolar Structure[END_REF][START_REF] Hernandez-Verdun | Structural Organization of the Nucleolus in Mammalian Cells[END_REF]. The function of each compartment generates surface tension that dictates whether these sub-compartments will contact one another, and which sub-compartment will be embedded or partially contained with the other [START_REF] Zarzar | Dynamically Reconfigurable Complex Emulsions via Tunable Interfacial Tensions[END_REF]. Based on that, I wanted to investigate whether compressive forces impact the nucleolar tripartite organization, therefore, I observed the nucleolar sub-compartments in MCF10A control and mechanically compressed monolayers over time by electron microscopy.

Through this technique, I could observe the structural organization in control MCF10A cells, where all three sub-compartments were identified; the fibrillar center is indicated by an asterisk, is further surrounded by DFC indicated by the arrowhead, and all are maintained within the GC (figure 23A,control). On the other hand, compressed cells showed a dynamic change in the structural organization, mainly observed in the FC compartment (figure 23,A). After 2 hours of mechanical compression, I observed an increase in the mean number of FC which is reduced at 4 hours and then increased back at 6 hours of compression (figure 23C, ii) (N=2 total number of experiments at different time points, total number of analyzed nuclei= 36). Moreover, at 4 hours I could observe cells in which FC was hard to distinguish from the DFC and GC suggesting a strong internal reorganization of the nucleoli (figure 23,A). I then estimated the percentage of cells showing less than or equal than 4 FCs (≤4 FC) (where 4 is the mean number of FC in control). As shown in representative graphs (figure 23C,i), after 4 hours of mechanical compression, there is an increase in the percentage of cells having ≤4 FC (70%) compared to the control, which is (54.5%) (N=3 total number of experiments, total number of analyzed nuclei= 54).

As a positive control, I used Act-D, a known agent to disrupt the nucleolar tripartite organization by inhibiting RNApolI activity and inducing nucleolar stress [START_REF] Shav-Tal | Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition[END_REF]. MCF10A cells were treated with Act-D at a high dose (80ug/ml) for 4 hours. As a result of Act-D treatment, I observed the complete disruption of Results 89 nucleolar structure as compared to the control, indicating nucleolar sensitivity (figure 23,B).

The observed effect of nucleolar structural disruption and the time scale at which it occurs are in agreement with the strong changes in the nuclear morphology. Interestingly, this indicates that an in-plane compression causes long-term (up to several hours) changes in the organization of the epithelium at the tissue and subcellular scale level.

I.2.2-Mechanical compression impacts nucleolar area and number

The nucleolus is a bio-molecular condensate, where its formation is driven by liquid-liquid phase separation of its components from the surrounding liquid nucleoplasm. Due to this liquid-like behavior, several nucleoli could fuse on a time scale of seconds or hours [START_REF] Brangwynne | Active Liquid-like Behavior of Nucleoli Determines Their Size and Shape in Xenopus Laevis Oocytes[END_REF][START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF]. In addition, few evidence link the efficiency of ribosome subunit synthesis to the nucleolar size and number [START_REF] Bailey | Amino Acids and Control of Nucleolar Size, the Activity of RNA Polymerase I, and DNA Synthesis in Liver[END_REF]). Based on that, quantifying nucleolar number becomes a prerequisite to identify possible nucleolar functional changes in several pathological conditions and under numerous external environmental challenges [START_REF] Farley-Barnes | Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number[END_REF][START_REF] Tiku | Small Nucleoli Are a Cellular Hallmark of Longevity[END_REF][START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF]. Therefore, to evaluate how mechanical stress and consequent changes in nuclear morphology and nuclear lamina affect the nucleolar structure, I decided to use two nucleolar parameters: the nucleolar size (a projected area) and the nucleolar number (number of nucleoli per nucleus). To visualize nucleoli and extract morphological parameters, the control and mechanically stimulated monolayers were stained for nucleolin; the highly abundant nucleolar protein localized in the DFC and the GC sub-compartment [START_REF] Biggiogera | Simultaneous Immunoelectron Microscopic Visualization of Protein B23 and C23 Distribution in the HeLa Cell Nucleolus[END_REF][START_REF] Ginisty | Structure and Functions of Nucleolin[END_REF]. Mechanical compression over 6 hours had a significant impact on the mean nucleolar area as compared to the control. Initially, 2 hours after compression the area decreased from 10 µm 2 in control to 8 µm 2 and then increased up to 18 µm 2 after 4 hours, to decrease again and reach 6 µm 2 . (N= 3 independent experiments, with n= 471 representing the total number of analyzed nucleoli per condition) (Figure 24,A). When results were normalized to the control, I could observe the highest impact at 4 hours postcompression where nucleolar area increased by 1.8-fold. (Figure24, C).

Since changes in nucleolar number could suggest changes in nucleolar activity in different pathological conditions and under diverse external challenges [START_REF] Zink | Nuclear Structure in Cancer Cells[END_REF][START_REF] Bailey | Amino Acids and Control of Nucleolar Size, the Activity of RNA Polymerase I, and DNA Synthesis in Liver[END_REF][START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF], I have manually counted all nucleoli per nuclei in control and compressed cells (N= 6 independent experiments, with n= 120 total number of analyzed nuclei per condition/experiment). Through this quantification, I have observed similar (as for area) fluctuations of the mean nucleolar number during the first 6 hours of compression. At 2 hours there was an increase in the cells with more than two nucleoli, followed by an increase in the percentage of cells with one nucleolus at 4 hours.

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Therefore, following these findings, I anticipated that mechanical compression induces structural remodeling in the nucleoli in mammary epithelial cells. Unfortunately, despite several attempts, I was unable to probe (live experiments were unsuccessful due to the thickness of the elastic membrane) if the observed changes in the nucleolar area are due to nucleolar fusion or expansion/shrinkage and this has to be further investigated through time-lapse imaging of cells expressing a fluorescent marker of the nucleolus.

I.2.3-Compressive forces triggers an increase in the abundance of proteins known to regulate nucleolar phase separation

Nucleolar size has been reported to be influenced by diverse environmental cues [START_REF] Kusnadi | Regulation of RDNA Transcription in Response to Growth Factors, Nutrients and Energy[END_REF]. Intracellularly, different mechanisms including protein concentration have been shown to regulate nucleolar size. An example of such proteins is the methyltransferase fibrillarin [START_REF] Yi | A Genetic Cascade of Let-7-Ncl-1-Fib-1 Modulates Nucleolar Size and RRNA Pool in Caenorhabditis Elegans[END_REF]. Moreover, fibrillarin together with nucleophosmin (B23) are also known to dissociate from the soluble nucleoplasm into the nucleolus in an rRNA-dependent manner, thus preserving the relatively spherical morphology of the nucleolus [START_REF] Feric | Coexisting Liquid Phases Underlie Nucleolar Sub-Compartments[END_REF]. Notably, the alternative splicing generates three B23 isoforms, however only the major isoform B23.1 contains a Cterminal NOLS (nucleolar targeting sequence), that localizes the protein to the nucleolus [START_REF] Lindström | NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling[END_REF]).

To understand if the observed significant changes in nucleolar size, particularly after 4 hours of applied compressive forces, are associated with changes in B23.1 and fibrillarin abundance. Therefore, I measured the changes in concentration of B23.1 and fibrillarin. Knowing the dynamic nature of B23.1 protein, I performed immunolabeling in mechanically compressed cells and control. Microscopy observations revealed nucleolar B23.1 positioning in control and under compression. Image analysis showed significant changes in the intensity of B23.1 in the nucleoli (Figure 25A), with the strongest changes after 2 hours of compression and milder increase at 4 and 6 hours. From the obtained results, it is not clear how much changes in B23.1 intensity contribute to the structural changes of nucleoli (size), but they are again maintained over a period of at least 6 hours.

Because it was previously shown that changes in fibrillarin concentration are directly correlated with the size of the nucleoli, I extracted the proteins from control and mechanically stimulated cells and performed a western-blot analysis of the fibrillarin abundance. Interestingly, at 4 hours post compression, when the nucleoli area increased by 1.8-fold, fibrillarin was also significantly increased (N=6 of independent experiments). Following changes in protein abundance (fibrillarin, B23) in nucleoli in parallel with nucleolar size in live microscopy would bring insights into the role and impact of the observed changes on the regulation of nucleolar structure/size/number. …………………………………………………… Figures are presented on the next page.

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I.2.4-Mechanical stretch has no impact on the nucleolus tripartite organization, morphology, and number Following our observation that at 4 hours post compression, nucleoli show reduced number of fibrillar centers. I wanted to investigate whether nucleolar tripartite organization would also be impacted by another type of mechanical stress. While applying compressive forces using our system have been shown to induce cellular extrusion [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF], tensile stretch showed to induce cellular division [START_REF] Gudipaty | Mechanical Stretch Triggers Rapid Epithelial Cell Division through Piezo1[END_REF].

Mechanical stretch was applied in the plane of MCF10A monolayer for 4 hours Following force application nucleolar tripartite structure was observed using electron microscopy (figure26A). Through that I could observe that mechanical stretch did not impact the nucleolar sub-compartment where I could still observe the fibrillar center that appears in bright areas, surrounded by the highly dense fibrillar center.

In parallel I investigated whether mechanical stretch would impact nucleolar morphology. Therefore, MCF10A mechanically stretched after 4 hours, and control were stained for nucleolin (as it was done for mechanical compression). Quantitative analysis did not show any significant change in nucleolar area in mechanically stretched epithelial cells compared to the control. The mean nucleolar area in both conditions was 10 µm 2 (N=3 independent experiments, n=471 analyzed nucleoli per condition) (figure26B). Finally, as a morphological parameter I further processed to count the number of nucleoli per nuclei in both conditions. As shown in the stacking graph I could not observe a significant difference in nuclei percentage that contains 1, 2, 3, 4, 5 and >5 nucleoli per nuclei (N=6 independent experiments, total number of analyzed nuclei n=120 for the control and n=99 for the stretched per each experiment) (figure26C).

Therefore, these results showed that mechanical stretch for 4 hours in the plane of epithelial monolayer does not impact nucleolar tripartite organization, neither nucleolar morphology (the area and the number). There are three potential hypothesis for such difference between mechanical stretch and mechanical compression: (1) the epithelial cells have released the tensile strain before 4 hours of stretch (could be confirmed further by quantifying nucleolar structure at different time points); (2) higher tensile stretch is required to cause nucleolar modifications; and (3) nucleolar structural changes are dependent on the externally applied mechanical cues and the resulting internal induced modifications (changes in chromatin organization, Lamin remodeling, fluidization of cytoplasm and nucleoplasm).

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5.3-Discussion

Cells within living organisms are subjected to different varieties of mechanical cues, including shear, tension, compression, and rheological properties of the proximal environment. Several reports have identified the nucleus as a well-established mechanosensory unit that converts biophysical stimuli into cellular and tissue responses [START_REF] Miroshnikova | Emerging Roles of Mechanical Forces in Chromatin Regulation[END_REF]. Recently, the interplay between biomechanical cues and the nucleolus, which is the most prominent organelle within the nucleus, has just emerged. Biomechanical cues that arise from cell-ECM adhesion have been shown to define the nucleolar structure and regulate its function [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF][START_REF] Jaecker | Nucleoli in Epithelial Cell Collectives Respond to Tumorigenic, Spatial, and Mechanical Cues[END_REF]). However, how physiological type of mechanical stresses of epithelial tissues such as compression or stretch regulate nucleoli and their function in epithelia is unknown. Compressive forces and tensile stretch have been shown to impact the nucleus and regulate cellular function in health conditions and contribute to disease initiation and progression [START_REF] Chen | Tensile and Compressive Force Regulation on Cell Mechanosensing[END_REF].

To study that, I optimized the experimental setup in which MCF10A creates semiquiescent monolayers that are exposed to a controlled mechanical stimulation. This ensures that observed effects on nucleolar structure are independent from cellular proliferation and come from mechanical compression. By performing β-catenin and lamin A/C quantification at different time points after compression I identified the time scale at which mechanical compression continues to have its effect. My results showed that 2 hours after applying compression there is an increase in β-catenin by the cell junctions and significant remodeling of the lamin A/C. Melanie Wallace et al. have recently reported that lamin A/C Ig-fold, which are the immunoglobulin-like (Ig)-fold, undergo remodeling in a cell-density dependent manner [START_REF] Wallace | The Lamin A/C Ig-Fold Undergoes Cell Density-Dependent Changes That Alter Epitope Accessibility[END_REF]. In parallel, the lamin A/C Ig-folds have also been shown to respond to shear stress through conformational changes and exposing several cryptic sites [START_REF] Swift | Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation[END_REF]).

The time scale at which tissues dissipate stress is not precisely known. Several strategies are employed by cells to release the excess of stress including cellular proliferation [START_REF] Wyatt | Emergence of Homeostatic Epithelial Packing and Stress Dissipation through Divisions Oriented along the Long Cell Axis[END_REF], extrusion [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF], death [START_REF] Rosenblatt | An Epithelial Cell Destined for Apoptosis Signals Its Neighbors to Extrude It by an Actin-and Myosin-Dependent Mechanism[END_REF], and active rearrangement of actomyosin and contractility [START_REF] Khalilgharibi | Stress Relaxation in Epithelial Monolayers Is Controlled by the Actomyosin Cortex[END_REF]. Whereas former processes typically take several hours the latter can occur rapidly in the scale of seconds/minutes. Mechanical stretching of MDCK monolayers showed to trigger rapid cell divisions of cells residing in G2 cell cycle within the first 50 minutes post stimulation [START_REF] Gudipaty | Mechanical Stretch Triggers Rapid Epithelial Cell Division through Piezo1[END_REF]. On the other hand, suspended monolayers of MDCK cells dissipate excess of tension through contractility in less than 1 minute [START_REF] Khalilgharibi | Stress Relaxation in Epithelial Monolayers Is Controlled by the Actomyosin Cortex[END_REF]. The same suspended monolayers, when compressed they initially fold (out of plane deformation) to then return to flat conformation within approximately 7 minutes [START_REF] Wyatt | Actomyosin Controls Planarity and Folding of Epithelia in Response to Compression[END_REF]). Yet, suspended monolayers are devoid of substrate and cell-ECM interactions and therefore the dynamics of dissipation can vary significantly as compared to monolayers grown on the substrate. From the experiences in my team, we have learned also that the mechanism of response and its dynamics depends on the initial tensional state of the epithelium (confirmed also by the work of Wyatt et al.). Therefore, it is not surprising that it takes about 6 hours to dissipate increased monolayer's cell density through extrusion as

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shown in another report from Rosenblatt team [START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF]). In our system, the characteristic time of response is about 6 hours as well if taking together the results from catenin, Lamin and nucleolar structure analysis described in this chapter.

Interestingly, our observation with β-catenin and lamin A/C at 2 hours post compression is accompanied by an increase in the number of fibrillar centers (FCs) within the nucleolus and the percentage of nuclei having two and three nucleoli, associated with a decrease in the nucleolar area marked by nucleolin in MCF10A epithelial cells. It has been reported that the fibrillar center, which contains the rDNA transcriptional machinery, can reflect the degree of ribosome biogenesis efficiency, where cells with a high rate of ribosome biogenesis have numerous FCs, possibly by separating NORs from each other (Pavel Hozák, Novák, and Smetana 1989). Therefore, I speculate from this observation that mechanical compression for 2 hours, induced rDNA transcription and hence ribosomal subunit production that is needed for protein synthesis.

Interestingly, after a significant increase in FCs number at 2 hours post compression, the situation changed drastically at 4 hours post compression where I observed a decrease in the number of FCs, associated with an increase in nucleolar area and an increase in the percentage of cells having one nucleolus. These observed nucleolar structural changes are accompanied by decrease in nuclear area and an increase in isotropy. The interplay between nucleus and nucleolar size have been determined in several cellular model and under various conditions, where it was shown that as keratinocyte cells detach from the supra-basal layers of the epidermis and differentiate, they undergo well-defined changes in nuclear architecture associated with a reduction in nuclear volume and a decrease in nucleolar number (Perdigoto et al. 2014). My results are in agreement with the report published by Pundel, as in planecompression causes the reduction of the cell area associated with an increase in the cell density per unit area within the epithelium [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF]. Moreover, the observed changes in the structure of the nucleolus including the number of FCs, could be related to transient changes in rDNA transcription, which I have investigated in part II of the presented results.

It was reported that the dynamics of nuclear condensates, including nucleolus, are impacted by the chromatin state in the surrounding microenvironment. Nucleoli assemble on active rDNA in chromatin-poor regions (Style et al. 2018). From this, we may anticipate that the increased nucleoli number after 2 hours of mechanical compression could result from induced chromatin decondensation that renders chromatin more available in the open, accessible state, thereby allowing rDNA transcription and nucleoli assembly. However, following the idea of chromatin response, I also observed a decrease in nucleolar number at 4 hours post compression, that in that case could be related to an increase in chromatin condensation, which creates a free region thereby allowing nucleoli fusion. This hypothesis is supported by the observation of Damodaran et al., who reported that compressive forces (weight positioned on top of the cells) applied on fibroblast for 1hr induce shuttling of histone deacetylase (HDAC3) into the nucleus, which triggers an increase in heterochromatin content by initiating the removal of acetylation markers from histone tails [START_REF] Damodaran | Compressive Force Induces Reversible Chromatin Condensation and Cell Geometry-Dependent Transcriptional Response[END_REF]. In parallel, Pundel et al showed that reducing the area of the cell adhesion has slightly but significantly increased peripheral heterochromatin [START_REF] Pundel | Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function[END_REF]. However, Nava et al., showed that uniaxial cyclic stretch in the plane of the monolayer, but also compression from top, of skin epidermal stem/progenitor cells triggers transient reduction in lamina associated H3K9me3 heterochromatin that protect (through nuclear softening) genome from DNA damage [START_REF] Nava | Heterochromatin-References 138 Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage[END_REF]. These opposing works indicate that chromatin state is strongly regulated by mechanical cues but the determinants of chromatin compaction or decompaction under mechanical stress are not known, and therefore the role of chromatin state in regulating nucleolar number in our cellular model and used system, needs further investigations.

In addition, I showed that fibrillarin abundance increases at 4 hours post compression. Fibrillarin has been shown to regulate nucleolar size, where the knock down of fibrillarin decreases nucleolar size in C. elegans model [START_REF] Tiku | Small Nucleoli Are a Cellular Hallmark of Longevity[END_REF]. Based on that, we might also speculate that the observed increase in nucleolar area might be due to inside-out nucleolar compositional changes (from the nucleolus to nucleoplasm) rather than outside-in (from the nucleoplasm-nucleolus). Where compressive forces may regulate the abundance of crucial nucleolar proteins, as fibrillarin that was significantly induced upon compressive forces, or sequester other proteins, thereby resulting in an increase in nucleolar area. In addition, the perturbation of the nucleocytoplasmic transportation showed to impact the size of the nucleus and the nucleolus [START_REF] Jevtić | The Nucleoporin ELYS Regulates Nuclear Size by Controlling NPC Number and Nuclear Import Capacity[END_REF]. Based on that we can hypothesize that compressive forces might impact the import system thereby resulting in the nuclear and nucleolar observed changes [START_REF] Ganguly | Perturbation of Nucleo-Cytoplasmic Transport Affects Size of Nucleus and Nucleolus in Human Cells[END_REF].

Interestingly, contrary to the compressive stress that showed to induced nucleolar structural and functional changes, MCF10A monolayers stretched for 4 hours did not show any significant changes in nucleolar tripartite organization or morphology (nucleolar area and number per nuclei). The difference between compressive and tensile stress can arise from the different dynamics of stress dissipation (discussed above) but also from the differential cellular response to force including known regulators of nucleus/nucleolus cytoskeletal organization, actomyosin contractility, and chromatin organization. Only more precise analysis at the short time scale could bring insights on the lack of nucleolar response to stretch.

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5.4-Summary and outlook

In the first part of the results, I showed that compressive forces applied in the monolayer of epithelial cells, induced dynamic changes in monolayer organization and impacted nuclear morphology. The forces are also transmitted to the nucleoplasm and induce nucleolar remodeling as observed by changes in the tripartite organization and morphology. Moreover, in contrast to compressive forces, tensile stretch applied for 4 hours did not induce nucleolar structural changes.

Since nucleolus is built by the act of ribosome subunits formation, and significant changes in the structure indicate possible nucleolar functional changes I proceeded to investigate possible functional changes, by probing the localization of nucleolar proteins that plays a crucial role in different steps during ribosome biogenesis and ascertain the level of the produced rRNA.

………………………………………………… Chapter II is presented on the next page. Nucleolus has been shown to be an indispensable player in stress response via the initiation of signaling pathways (reviewed in (Yang et al. 2018)) that lead to regulation of cell cycle arrest, apoptosis, and senescence [START_REF] Lindström | P53 at the Crossroad of DNA Replication and Ribosome Biogenesis Stress Pathways[END_REF]. At the same time mechanical stress such as in-plane compression or stretch is known to perturb the tensional and cell-density homeostasis. As described in the previous chapter, tissues exposed to mechanical stress recover the homeostatic state by numerous mechanisms including the regulation of the cell cycle or cell elimination processes. Therefore, I verified first, if the standard signaling pathway associated with the nucleolar stress response is involved in the regulation of monolayer response to the applied mechanical compression. The MDM2/P53 is one of the principal signaling pathways activated following nucleolar stress and plays a pivotal role in cell cycle regulation. Under physiological conditions, the level of p53, is maintained low mainly by the E3 ubiquitin ligase MDM2 (Mouse double minute homolog) that targets it for degradation [START_REF] Lindström | P53 at the Crossroad of DNA Replication and Ribosome Biogenesis Stress Pathways[END_REF]. On the contrary, in the stress conditions (oxidation, serum starvation, heat shock) MDM2 is sequestered resulting in p53 stabilization in the nucleoplasm, hence causing cell cycle arrest.

To determine if mechanical stress activates the downstream p53 signaling pathway MCF10A cells compressed for 4 hours, and the control were stained for p53 and co-stained with nucleolar protein (nucleolin) and DAPI for spatial quantification (figure 27A,i). After 4 hours of mechanical compression, quantitative image analyses based on measuring the total signal intensity in the nucleus (based on DAPI mask) did not show any increase in p53 (figure 27A, ii). Moreover, the expression of the cellular p53 protein level was the same between both conditions analyzed by western blotting (figure 27A,iii). In addition, we obtained new preliminary results that shows no significant change over the 6 hours post compression (results not shown). As a positive control, MCF10A cells were treated with high doses of Act-D to induce nucleolar stress and p53 activation. Act-D is known to inhibit rDNA transcription, and the release of the ribosomal and nucleolar proteins into the nucleoplasm, that sequester MDM2 and might enhance the mRNA transcription and/or protein translation of p53. As another positive control, I quantified changes of P53 in monolayers that were stretched for 4 hours and observed the stabilization of P53 protein in the nucleus. MCF10A cells that were mechanically stretched for 4 hours, showed a significant increase in nuclear p53 intensity compared to the control (figure 27B, ii). Mechanical stretch applied by our experimental system, showed previously to trigger rapid epithelial cell division [START_REF] Gudipaty | Mechanical Stretch Triggers Rapid Epithelial Cell Division through Piezo1[END_REF].

Our preliminary results suggest that a stereotypical nucleolar stress response might be unlikely to be involved in the regulation of the response to mechanical compression. The principal function of nucleoli, besides the stress response, is transcription of rDNA and early stages of the ribosome biogenesis process. How mechanical stress affects rDNA transcription remains to be elucidated. From the structural studies using immunofluorescence and electron microscopy, I observed that upon mechanical compression there is a transient response in the regulation of the number of FCs. To study precisely the essential transcription process occurring in FC under mechanical stress, I selected the Upstream binding factor protein (UBF). UBF is a component of the pre-initiation complex (PIC) where it binds at the promoter region, thereby initiating the transcription of the rRNA genes at the FC/DFC border. Following the binding of UBF to the promoter region, it loops approximately 140 base pairs (bp) of DNA into a single turn, thus maintaining the rDNA in the open chromatin-accessible state [START_REF] Kuhn | The Nucleolar Transcription Activator UBF Relieves Ku Antigen-Mediated Repression of Mouse Ribosomal Gene Transcription[END_REF]. Control and mechanically compressed MCF10A monolayers were stained for UBF together with nucleolin for spatial quantification of the UBF in the nucleoli figure28A. UBF intensity quantification revealed that compressive stress induced a dynamic and transient change in UBF (figure28, B). At 2 hours of mechanical compression, I measured a significant increase in nucleolar UBF intensity as compared to the control that was also accompanied by an increase in the protein distribution within the nucleoplasm (figure 28C indicated by star). This result is in accordance with the increased number of FC upon mechanical compression suggesting an increase in the transcriptional activity. To further confirm this transient behavior, I examined the abundance of UBF by western blotting. There was an almost 2-fold increase in total UBF protein at 2 hours but not at 4 hours as compared to mechanically unstimulated cells (figure28D, i and iii). The increase in protein abundance is most likely due to increase in translational level, rather than gene expression level, as I confirmed by measuring the gene expression level by quantitative real-time PCR [RT-qPCR] (figure28D, iii). Interestingly after 4 hours, there was no increase in UBF as measured by WB or by IF intensity image analysis. Even though there was no difference in protein abundance or total signal intensity between control and 4 hours after compression (figure28D, i) the UBF localization within the nucleoli was altered. Surprisingly mechanically compressed MCF10A cells at 4 hours post compression showed UBF localization by the nucleolar border, (figure 28C indicated by arrowhead). After 6 hours of applied compression, I could observe that UBF is redistributed within the nucleolar core again, with a slight reappearance of UBF within the nucleoplasm (figure 28C indicated by arrow).

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Altogether I showed that mechanical compression triggers a transient increase in the UBF protein abundance and localization. This is in accordance with previously obtained results presented in the Chapter I which showed alteration in FCs quantity per nucleolus and nucleoli number. Since UBF is a crucial protein in rDNA transcription, this suggests that nucleolar function particularly rDNA transcription and rRNA production is impacted dynamically by the applied compressive forces. Transcription of rDNA is essential for the function of the nucleoli but also for their structural stability [START_REF] Shevtsov | Nucleation of Nuclear Bodies by RNA[END_REF]. From the general point of view mechanical compression is known to trigger the extrusion of cells, cause apoptosis, and slow down cell proliferation and mobility until the mechanical constraint is released. Prolonged, growth-associated compression also showed that yeast cells lose volume and down-regulate the process of ribosomal biogenesis in order to overcome molecular crowding (M. [START_REF] Delarue | MTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding[END_REF]. By mechanical compression of epithelial monolayer, I expected therefore to observe a downregulation of rDNA transcription which would be also expected regarding the decrease in the number of FCs at 4 hours. In addition, to the observed UBF transient change, where mechanical compression in our system caused an increase in the expression of UBF in the nucleoli at 2 hours and affected the nucleolar organization at 4 hours (Chapter I). (Figure28C).

Ribosome biogenesis is an oriented process that starts with the transcription of rRNA genes (18S, 5.8S, and 28S) at the border of FC/DFC. Where UBF accompanied with other proteins binds to the rDNA promoter region and recruit RNApolI for transcription initiation (Hadjiolov 1985). This step is essential in determining the rRNA output that counts to 80% of cellular RNA. To ascertain the rRNA abundance upon mechanical compression for 4 hours, I pulse-labeled nascent RNA using 5'ethynyl uridine (EU) and analyzed levels of nascent rRNA within nucleoli (Figure29A).. Because 5'-ethynyl uridine (EU) incorporates into the globally synthesized RNA in the nucleus; I selectively measured rRNA quantity either in nucleolus or in the nucleus. To analyze RNA production within the nucleolus I used a spatial mask based on B23 staining whereas for global RNA synthesis I used the mask from the DAPI staining. Quantification showed a significant decrease of the total RNA synthesis after 4 hours of mechanical compression (as compared to control) (N=3 total number of independent experiments, n=573 total number of analyzed nuclei per condition). By quantifying 5'EU in the nucleolus (based on B23 mask), I also observed, that applied compressive stress for 4 hours significantly attenuated rDNA transcription compared to the control (N=3 total number of independent experiments, n'=942 total number of analyzed nucleoli per condition) (Figure29B, i and ii).

Altogether, these results confirmed that after a transient increase in nucleolar number and UBF nucleolar expression at 2 hours, at 4 hours the global transcriptional activity decreased. Not only the total freshly synthetized RNA was decreased but also, the rRNA synthetized in the nucleoli.

While the 5'EU click allows the detection of the 47S rRNA, that will later be cleaved at the extremities to give rise to the pre-mature 45S rRNA. I wanted to further verify the expression level of 45S in control and mechanically compressed cells (N=3 total number of independent experiments). Through this I can observe a transient change in the expression level of 45S in the compression compared to the control, where it decreases at 4 hours post compression. This confirms that at 4 hours of applied Results 111 compressive forces, and following the reduction in the rRNA synthesis, this is accompanied by a decrease in the level of cleaved 45S (figure 29C).

To further verify my observations particularly at 4 hours, post compression, I used low doses of Act-D treatment (10 ug/ml). Act-D is a known inhibitor of RNA polI and thus inhibitor of rDNA transcription. In control cells, nucleoli display the homogenous distribution of UBF within the nucleolus that has been co-stained with nucleolin (figure 29D). When RNA polymerase activity was inhibited, cells showed (1) smaller and hollow nucleoli observed by DAPI staining. (2) a small number of dense UBF aggregates at the nucleolar periphery, (3) significant decrease of nucleolin inside the nucleoli and increased appearance of nucleolin staining in the nucleoplasm (figure 29D I.4-Compressive forces induce nucleolin release from the nucleolar core.

Nucleolin, which is a bona fide nucleolar marker that was selected to quantify nucleolar morphology and co-stained with UBF was also analyzed for its localization and abundance under compressive forces. Besides the high abundance of nucleolin within the nucleolus, it is considered a multifunctional nucleolar protein. It has been shown that nucleolin could interact with other proteins and chromatin through the Nterminal domain, to recognize the substrate for pre-RNA through its central domain, and to interact with RNA through the C-terminal domain [START_REF] Ginisty | Structure and Functions of Nucleolin[END_REF]. Therefore, nucleolin participates in regulating rDNA transcription and rRNA processing [START_REF] Abadía-Molina | Inhibition of Nucleolar Protein Nucleolin by Electroporation with Anti-Nucleolin AntibodiAdam[END_REF]. To ascertain the impact of compressive forces on nucleolin localization and abundance, MCF10A control and compressed cells were stained against nucleolin figure 30A. From the immunostaining images and nucleolin inverted images (figure 30A), I could observe the enrichment of nucleolin in the nucleolar core at 2 hours post compression (figure 30A, indicated by asterisk). At 4 hours post compression, I observed nucleolin release from the nucleolar core that became more enriched in the nucleoplasm (figure 30A, indicated by arrow). This observed behavior was transient, since at 6 hours post compression, I could observe nucleolin to be enriched again in the nucleolar core (figure 30A, indicated by arrowhead).

To verify the observed phenotype, the ratio of nucleolin intensity within the nucleolus/nucleoplasm was quantified in all studied conditions and plotted, as shown in (N=3 independent experiments, n=276 total number of analyzed nucleoli), (figure30B). Through this, I could observe a pattern in the quantified ratio under compression, where the ratio increased after 2 hours, then decreased significantly after 4 hours, followed by a slight increase again at 6 hours. To understand if the decrease at 4 hours was due to degradation of nucleolin I performed a western blot analysis to show that the total amount of nucleolin did not change after mechanical compression (N=6 total number of independent experiments), (figure 30,C). That indicates that nucleolin at 4 hours post compression is released from the nucleolus. Because mechanical in-plane compression leads to the increased cellular density within the monolayer, I verified if increased monolayer density (by increased cell seeding density) would recreate the observed phenotype in mechanically compressed cells (Figure 20A). Through that, I could not observe a change in nucleolin localization as cell seeding density increased. Based on that we anticipated that nucleolin release from the nucleolar core, is not a result of high density and cannot be mimicked by only inducing cellular packing, therefore indicating a unique effect of compressive forces on nucleolin.

After I have observed nucleolin release from the nucleolar core at 4 hours post compression, I wanted to further investigate whether nucleolin shuttling from nucleolus into the nucleoplasm could be mimicked by applying another type of applied compressive forces in epithelial MCF10A cells monolayer. To tackle this question, I used the 4D cell device (system assembly and utilization is described in the methodology part) were we selected coverslips with pillars height 4 µm to apply compression on the top of the monolayers. Mammary epithelial cells MC10FA were seeded at the same density 1.6×10 6 cells/ml on the coverslips pre-coated manually with collagen type I, followed by incubating the coverslips for 48 hours. After a monolayer formation, cells were apically compressed for 4 hours, followed by staining MCF10A cells against nucleolin and DAPI to visualize the nucleolus and nuclei respectively (figure 31A). Through this we could observe that the total intensity of nucleolin within the nucleus is the same in both conditions (N=1, total number experiments, n=158 total number of analyzed nuclei). Interestingly, nucleolin intensity significantly decreased from nucleolar core, in apically compressed MCF10A cells compared to the control (N=1, total number experiments, n=99 total number of analyzed nucleoli) (figure 31B) as I previously observed for in-plane compression phenotype.

Altogether our results indicated that in response to compression there is a transient response in UBF increase, followed by decreased rDNA transcription associated with the nucleolin translocation to the nucleoplasm. Because UBF remained at the same level in control and compressed cells for 4 hours, it is likely that nucleolin is a ON/OFF switch to regulate transcription under mechanically challenging conditions. Results 120

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6-Discussion

Mass spectrometry analysis of isolated nucleoli identified approximately 4,500 nucleolar proteins that modulate ribosome biosynthesis, cell cycle control, and stress response (Ahmad et al., 2009). Although the nucleolus is an organelle without a membrane, it achieves an impressive level of segmentation by phase separation through multiple weak interactions between proteins and/or rRNAs. The nucleolar proteins nucleolin and upstream binding factor (UBF) are well-characterized nucleolar proteins essential for nucleolar function, particularly, the rRNA synthesis and rRNA modifications (Ma et al., 2007).

Here I studied the impact of compressive forces on the main function of the nucleolus by quantifying the localization and the abundance of the proteins involved in nucleolar stress response and rDNA transcription. I showed that applying compressive force for 2 hours increases the whole cellular UBF protein abundance, where it appears to be distributed within the whole nucleus and enriched in the nucleolus. The upstream binding factor (UBF) is a component of the pre-initiation complex (PIC complex) that binds to the promoter region of rRNA genes, thereby initiating rDNA transcription at the FC/DFC border. UBF binding is essential for maintaining rRNA genes in a euchromatin state through the displacement of repressive histone H1 and supports rDNA structure into NORs. Brandenburger et al. reported that the cellular amount of UBF (mRNA and protein level) correlates strongly with the elevation of rDNA transcription [START_REF] Brandenburger | Chromatin Condensation[END_REF]. Therefore, these findings suggest that compressive forces at 2 hours induce the rRNA gene transcription, and thereby increase the synthesis of rRNA. This is further supported by the fact that the increase in rDNA transcription has also been shown to be linked to the number of fibrillar centers. Hozák et al. have shown that in resting lymphocytes (G0), the nucleolar organizing regions (NORs) are in close contact, following the activation of cell proliferation and hence rDNA transcription the number of fibrillar centers increases. This increase has been anticipated to result from separating NORs from each other, thereby creating an individual fibrillar center [START_REF] Hozák | Three-Dimensional Reconstructions of Nucleolus-Organizing Regions in PHA-Stimulated Human Lymphocytes[END_REF]. Therefore, the increase in UBF justifies the appearance of several FCs in a single nucleolus observed by electron microscopy (Chapter 1 of results). The observed increase in UBF was also accompanied with an enrichment in nucleolin and B23.1(Chapter 1 of the presented results) within the nucleolar core. While NCL is involved in rRNA gene transcription regulation and rRNA maturation [START_REF] Ginisty | Structure and Functions of Nucleolin[END_REF], B23 have been shown to play a pivotal role in accumulating pre-60S ribosomal subunit within the nucleolus, for later maturation steps, particularly by interacting with rRNA [START_REF] Okuwaki | The Interaction between Nucleophosmin/NPM1 and the Large Ribosomal Subunit Precursors Contribute to Maintaining the Nucleolar Structure[END_REF]). These two studied proteins are important in regulating the initial and last steps in ribosome subunits formation. Therefore, I suggest that the increase in rDNA transcription might be accompanied by an increase in final products as mature rRNA. Further experiments using 5'EU would allow us to measure the newly synthesized rRNA and verify this hypothesis. In addition, to measuring the total abundance of mature rRNA (18S, 5.8S, and 28S) will help us in verifying whether the differences in the level of 45S pre-rRNA at 2 hours post compression is due to an increase in the processing of the 45S into mature rRNA.

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Our finding that the percentage of nuclei with three and four nucleoli increase in epithelial cells at 2 hours post compression compared to the control (Chapter 1 of results), further suggested the nucleolar activity increase. Indeed, this suggestion can only be valid if these observed nucleoli contain active NORs. Interestingly, after the observed increase in UBF and enrichment of nucleolin within the nucleolar core at 2 hours post compression, the situation changes strongly for 4 hours post compression. I showed that the level of UBF decreased to the level of control, yet the number of FC and the structural change in nucleoli were drastically different. I further observed that UBF was positioned more by the nucleolar border, and this was accompanied by the global reduction in the rRNA synthesis. Yet, how this nucleolar decrease in transcriptional activity is regulated remains to be uncovered. Upon mechanical compression, over time nucleolin showed significant change in the spatial distribution in nuclei and nucleoli. At 4 hours post-compression the observed phenotype was associated with the release of nucleolin into the nucleoplasm. These results open a possibility that nucleolin spatial distribution induced by mechanical compression is involved in the transient regulation of the rDNA transcription. Moreover, another experimental setup in which epithelial cells were compressed from top, has shown release as well of the nucleolin from the nucleolar core. In my thesis project, I did not investigate the mechanism by which nucleolin is released from the nucleolus; and it is not clear yet, whether nucleolin release is a consequence of attenuating rDNA transcription, or nucleolin is the reason for such attenuation. Here, I could speculate few reasons that could justify nucleolin release. First, since I showed that compressive forces for 4 hours attenuate rDNA transcription, nucleolin release might be a consequence of weakening interaction with rRNA in.the nucleolus. Second, compressive forces might be impacting the expression/localization of proteins that regulates chromatin organization and state within the nucleus and the nucleolus in particular, and as a consequence disrupt nucleolar protein localization, thereby, impacting rDNA transcription. For example, Smith et al. showed a novel role for p150, in regulating nucleolar protein distribution and macromolecular interactions. P150 is a subunit of the chromatin assembly factor (CAF1), besides its role in depositing histones during DNA synthesis, it could also interact with rDNA and nucleolar protein through the N-terminal domain. Following the depletion of p150, the authors could observe a change in nucleolar proteins localization that shuttles into the nucleoplasm [START_REF] Smith | A Separable Domain of the P150 Subunit of Human Chromatin Assembly Factor-1 Promotes Protein and Chromosome Associations with Nucleoli[END_REF]. Third, nucleolin release might be associated by the release and shuttling of other proteins and transcriptional factors. Fourth, since nucleolin localize into the nucleolus through post-translational modifications, nucleolin release at 4 hours of compression might be due to regulating these modifications.

Moreover, it's not clear yet what is the role that nucleolin could contribute to in the nucleoplasm, thereby justifying the reason for its release. While nucleolin is considered a multifunctional nucleolar protein, it has been shown also to play several roles in the nucleoplasm. Nucleolin have been shown to inhibit nucleotide excision repair and due to its multifunctionality it can inhibit replication by binding to the Replication protein A (RPA) under various stress conditions (K. Kim et al. 2005). Through the sequestration and the release of nucleolin and other factors, the nucleolus plays a significant role in maintaining genome stability after induced stress by Results 122 transiently delaying proliferation in favor of activating the DNA repair machinery. Therefore, nucleolin release could be to prevent DNA damage that is induced by compressive forces. Determining nucleolin targets at different time points post compression would allow enlightening the role of its translocation upon mechanical stimulation. In addition, nucleolin translocation might be to attenuate rDNA transcription; it has been shown that transcriptionally competent rRNA genes could switch between ON and OFF states depending on cellular conditions (reviewed in (Durut and Sáez-Vásquez 2015)). It has also been shown that nucleolin level impacts rDNA state and hence the transcription of the rRNA gene. Under compressive forces, I do not observe a change in nucleolin protein abundance. However, besides the cellular amount of nucleolin, it has been suggested that post-translational modifications such as glycosylation (Carpentier et al. 2005), methylation (Pellar and DiMario 2003), phosphorylations (Tuteja and Tuteja 1998), that regulate nucleolin localization, can also regulate nucleolin function in terms of rDNA transcription. Interestingly, regulating rDNA transcription is achieved by recruiting chromatin remodelers that could activate or repress RNApolI transcriptional activity (Durut and Sáez-Vásquez 2015). From that, and if the induced nucleolin release from the nucleolar core into the nucleoplasm is required for controlling the transcriptional ON/OFF states of the rDNA chromatin in epithelial cells after 4 hours of applied compressive forces, then we might be able to underline the potential involvement of nucleolin in mechano-adaptation of epithelia to compressive stresses. Down-regulation of rDNA transcription might function as a strategy to maintain cellular homeostasis under compressive forces.

Another interesting result of my work was to show that mechanically compressed MCF10A cells for 4 hours, despite the observed nucleolar changes, did not trigger activation of the classical stress response pathway, the p53 pathway. This is especially interesting because changes in nucleolar protein localization and rDNA attenuation, have been shown to induce nucleolar stress that is dependent on p53 (Yang, Yang, and Yi, 2018.). Our findings suggest that nucleolar stress response under compressive forces is independent of p53. To further confirm this observation, it is still relevant to the level of MDM2 and other downstream proteins as p21 (regulatory of cell cycle). Altogether, the observed changes in nucleolar activity upon mechanical compression show the need of cellular machinery to adapt to mechanically challenging conditions. It is generally accepted that cells experiencing diverse environmental challenges, attenuate ribosome biogenesis as a strategy to preserve the cellular energy (Grummt 2013). Based on that, the observed nucleolar structural and functional changes, can be important in maintaining cellular homeostasis, and intracellular balance.

…………………………………Conclusion and perspectives are presented on the next page.

.

Conclusion and Perspectives

7-Conclusion and Perspectives

The purpose of determining the impact of compressive forces on nucleolar structure and function may have significant implications for diseases such as cancer and in different developmental processes. While compressive forces contribute significantly to maintaining the cellular number and organ size, in the initiation and progression of cancer disease, and in regulating different developmental processes such as branch formation and involution of the mammary gland, the nucleolus is considered the central hub of the nucleus. Several processes regulated by compressive forces necessitate proper regulation of nucleolar function to achieve the general biological outcome. For example, for a cancer cell to migrate and invade the surrounding tissue under compressive forces requires an increase in protein synthesis that is regulated by the abundance of synthesized ribosomes controlled by nucleolus. For the branching glands to undergo involution and lumen formation, which is regulated by compressive forces, the epithelial cells need to undergo apoptosis. Interestingly, apoptosis is one of the downstream processes that is generated upon nucleolar stress. Yet, the effect of compressive forces on nucleolar structure and function is not determined until now. To determine the effect of compressive forces on nucleolar structure and function, an in vitro system was optimized, and several techniques, including microscopy and immunofluorescent labeling, were used to probe the effect of the exerted force on the nucleolus, as described in the manuscript.

MCF10A mammary epithelial cells were used as a model to determine the effect of compressive forces on nucleolar structure and function. After optimizing the MCF10A monolayer culture, I have shown that compressive forces dynamically impact the epithelial monolayer organization over time. This was accompanied by force transmission inside the cells, inducing nuclear morphological changes and impacting lamin A/C organization.

In the manuscript, I provided evidence that compressive forces transmitted to the nucleus impact the nucleolar tripartite organization, particularly the fibrillar centers (FCs), that houses several rDNA transcriptional factors. Alongside, I have shown that morphological changes accompany nucleolar structural changes, particularly an increase in nucleolar area and a decrease in number of nucleoli at 4 hours post compression.

Measurements and observation reveal that after 4 hours of applied compressive forces, the induced structural changes are accompanied by changes in UBF localization by the nucleolar border and nucleolin shuttling into the nucleoplasm. Moreover, I have shown that a reduction in rRNA synthesis accompanies these changes in protein localization.

Notably, the induced nucleolar structural and morphological changes and the enrichment of nucleolar protein within the nucleolus over time are dynamic and occur after a relatively long period from the application of the compressive stress, indicating the importance of such changes in regulating cellular function, which is not yet identified in the presented work. Here I note that the mechanism by which compressive forces induce such observed changes has yet to be discovered. Moreover, whether the increasing number of FCs observed at 2 and 6 hours post compression indicates an increase in ribosomal subunits formation that allows translating the needed proteins is also yet to be determined. Future studies will be conducted to probe chromatin organization and state over time to determine whether it's the reason behind such observed structural and morphological changes and measure the rRNA synthesis and protein synthesis.

Despite the current absence of understanding for the mechanism behind such observed changes, our findings suggest that the nucleolus is mechanosensitive to external compressive forces, which could be an avenue for cell mechanobiology study that covers different areas, including cancer and development.

One of the intriguing questions that I didn't experimentally address was to test if other epithelial cell models would respond in the same way. Despite many attempts it was hard to obtain quiescent epithelial monolayers of cell lines such as RWPE1 or MCF7 cells. Due to the time limitations, I couldn't either give the answer if other than epithelial cells would present the same nucleolar response. This is especially interesting because cells from different organs and tissues show different functions and differ in the organization of the internal cytoskeleton, the state/rheology of chromatin, and the abundance of proteins. Having an insight on universality of nucleolar alterations on such diverse cell models would bring insights on the mechanisms of response and better understand the importance of nucleolus remodeling from a physiological point of view.

In the present report, I have focused on the role of compression on the 2D epithelial monolayer. In the long term, it would be interesting to expand the study and to include 3D models, such as organoids and tumor spheroids. The effect of compressive forces on such models has been studied before. Delarue et al. and by applying mechanoosmotic compression showed that the cells stopped proliferating within the 3D structure. Changes in the abundance of proliferative inhibitor p27 kip1 protein accompanied this observed phenotype and a decrease in cell volume that was significantly higher in the center of the spheroids (Morgan [START_REF] Delarue | Compressive Stress Inhibits Proliferation in Tumor Spheroids through a Volume Limitation[END_REF]. Determining the effect of compressive forces on nucleolar structure and morphology in such models will allow us to relate nucleolar changes to stress distribution. This is especially important for living organisms, where forces exerted on tissues and organs are not uniformly distributed.

One of the intriguing questions to answer besides studying the mechanism by which compressive forces induce nucleolar structural and functional changes, is to investigate the feedback loop from the nucleolus back to the nucleus. Particularly, to understand how the induced nucleolar structural and functional changes are further impacting the nucleus (nucleus stiffness, genome organization, and gene expression). This will allow us to further characterize the importance of the nucleolus in the nuclear organization under external forces.

. Limitations in the study 125

8-Limitations in the study

As a limitation is my study, is the lack of live observations. Throughout my PhD, I tried to generate fluorescently labeled MCF10A for nucleolar proteins, specifically the nucleolin, which would allow me to further prove the hypothesis on dynamic shuttling and to precisely follow nucleolar changes over a period of time in the same cells. Unfortunately, MCF10A cells are hard to be transfected and after several optimizations and trials, it was still unsuccessful. Additionally, observations of nucleoli using phase contrast microscopy were at first impossible due to the limitations in the working distance of available objectives.

To overcome these limitations, we decided to try a commercially available system Strex, however, after many attempts and changes in the protocol (protein concentration, oxygen plasma activation of PDMS) the protein coating on the PDMS membrane was inefficient and MCF10A monolayers were detaching after 24 hours post seeding.

With the help of Jacque Mazega from the microscopy platform, we managed to 3D print a special support to enable live imaging compressed monolayers. However, due to the lack of time I could not precisely explore this path and further optimization is necessary. In my opinion, live imaging is the most important part to be further explored as it would enable us to observe nucleolar changes over a precise period of time, to better identify the time points where such changes occur and determine whether compressive forces would induce nucleoli fusion or expansion.

……………………………………Supplementary data are presented on the next page. Supplementary Data 126 9.Supplementary results

Effect of tensile stretch on nucleolus function and protein localization

While mechanical compression is known to induce cellular packing and induce extrusion, tensile stretch has been shown to induce cellular proliferation. After I have observed that applied tensile forces for 4 hours does not impact nucleolar tripartite organization or nucleolar morphology, I wanted to determine whether this would indicate that there is no impact on nucleolar protein localization. Therefore, MCF10A mechanically stretched for 4 hours, and control cells were stained for UBF and nucleolin (major proteins that change in terms of their localization following applied compressive forces for 4 hours). Interestingly, I could not observe a change in UBF protein localization, after 4 hours of applied tensile stretch, where I could observe that the protein is dispersed in the nucleolus as in control condition (figure 32,A). However, there was a slight increase in UBF enrichment in the nucleolar core, compared to the control (figure 32B,i). In addition, applied tensile stretch for 4 hours, did not impact nucleolin localization neither enrichment within the nucleolar core observed by immunofluorescence (figure 32,A), and confirmed by quantifying the ratio of nucleolin in the nucleolar core/nucleoplasm (figure 32C,i). In addition, I could not observe an increase in protein abundance analyzed by western blotting (figure 32C, ii).

Following these obtained results, we cannot rule out the possibility the tensile stretch is impacting the function of the nucleolus in terms of rRNA synthesis since I could observe an enrichment of UBF in the nucleolus. Also, to conclude that compressive forces and not tensile stretch is inducing major nucleolar structural changes, it is important to investigate the effect of tensile stretch on epithelial cells at different time points. We could speculate that the time points by which our observation is done in stretched condition (4 hours), is in fact dissipated, and we are observing the downstream effect of the stress.

In the future studies, it will be interesting to answer these raised hypotheses, through that we might be able to determine certain types of biophysical cues that define nucleolar structure and justify the absence of changes in other mechanical cues. Moreover, determining the mechanism, which induce such changes, whether that is chromatin organization, alteration of viscosity of different cellular compartments, and potentially nucleo/cytoplasmic shuttling system, is of great relevance for future studies.

…………………………………………………Figures are presented on the next page.

Résumé'

Les cellules des organismes vivants sont soumises à différents signaux mécaniques, notamment le cisaillement, l'étirement en traction, la compression et les propriétés rhéologiques de l'environnement proximal. Parmi ces signaux mécaniques, les changements de tension, y compris les forces de compression, sont reconnus pour réguler le comportement des cellules et des tissus environnants. Cependant, l'effet des forces de compression sur les composants nucléaires internes, en particulier le nucléole, reste mal compris. Cette étude étudie l'impact direct des forces de compression sur la structure nucléolaire et la distribution des protéines dans les cellules épithéliales mammaires à l'aide d'un système in vitro optimisé. Ici, nous avons appliqué des forces de compression dans le plan de la monocouche MCF10A à différents moments et montré que la compression induisait un remodelage dynamique et transitoire dans l'organisation tripartite nucléolaire, en particulier le centre fibrillaire (FC) qui abrite plusieurs facteurs transcriptionnels d'ADNr. Parallèlement, nous avons montré que les changements morphologiques accompagnent les changements structurels nucléolaires. De plus, les mesures et l'observation ont révélé que le remodelage nucléolaire induit s'accompagne de changements nucléolaires fonctionnels. Nos résultats suggèrent que le nucléole est mécanosensible aux forces de compression externes, ce qui pourrait être une piste d'étude de la mécanobiologie cellulaire couvrant différents domaines, notamment le cancer et le développement, où les forces de compression régulent plusieurs fonctions cellulaires.

Mots Cles: cellules épithéliales mammaires, forces de compression, noyau, nucléole, protéines nucléolaires.

  the Nucleolus as a Consequence of the Dynamics of Ribosome Biogenesis I) Building blocks of nucleolar compartmentation II) Role of the nucleolus in ribosome biogenesis II.1-rDNA organization, transcription, and regulation II.2-rRNA maturation: Cleavages and post-transcriptional modifications II.3-Ribosome formation and quality control III) Liquid-liquid phase separation: a mechanism that drives the nucleolus formation III.1-Definition of liquid-liquid phase separation III.2-LLPS drive nucleolar assembly and disassembly. III.3-Active ribosome biogenesis generates the surface tension required to form the nucleolar tripartite organization. III.4-LLPS and Ribosome biogenesis impact the nucleolar number and size. IV) Role of nucleolus in regulating ribosome biogenesis and stress response Part 2 Cell/Cell and Cell/ECM adhesions and forces acting on epithelial cells within the mammary gland I) Epithelial cell architecture and organization I.1-Cell/ECM and Cell/Cell adhesions and their interaction with the cytoskeleton: I.1.1 Focal adhesions: I.1.2 Adherens junctions: I.2 Linking the cytoskeleton to the nucleoskeleton II) Mechanical signaling in mammary gland from development into tumor initiation and progression II.1) Embryogenesis: Development of mammary gland II.2) Branching morphogenesis and homeostasis. II.3) Lactation and involution II.4) Mechanical force in breast cancer III)-Technology leading the way ahead: recapitulating tissue mechanics in Vitro. III.1) Applying mechanical compression by using Brick Strex III.2) Applying mechanical compression on the cell surface by using a rigid flat plate. Part 3 Crosstalk between Nucleolus and Mechanobiology I) Examples on the mechanosensitive components of the nucleolar environment:

  III)-Immunofluorescence of fixed samples IV) Labeling of De novo RNA synthesis V) Confocal microscopy VI)Image analysis VII)-Analysis of expression of proteins by western blotting: VII.1-Preparation of cell lysates: VII.2-Migration and transfer of Membrane VII.3-Immunoblotting and revelation: VIII)-Analysis of RNA expression: VIII.1-Extraction of the RNA: VIII.2-Reverse transcription of the RNA into cDNA VIII.3-Quantitative real time PCR: XIthe impact of the compressive stress on nucleolar structure I.1) Optimization of the in vitro system in order to apply in-plane epithelial compression and tensile stretch I.1.1-Methodology for force application using flex-cell plates I.1.2-Creating a semi-quiescent monolayer is a pre-requisite to apply mechanical uniaxial force I.1.3-Compressive stress reinforces β-catenin at cell junctions I.1.4-Compressive stress impacts nuclear morphology and induce lamin A/C remodeling I.2) Unraveling the effect of compressive stress on nucleolar structure and morphology I.2.1-Mechanical compression induces a dynamic change in nucleolar tripartite organization I.2.2-Mechanical compression impacts nucleolar area and number I.2.3-Compressive forces triggers an increase in the abundance of proteins known to regulate nucleolar phase separation I.2.4-Mechanical stretch has no impact on the nucleolus tripartite organization, morphology, and number 5.3-Discussion 5.4-Summary and outlook 5.5.-Chapter 2 I) Uncovering the impact of compressive stress on nucleolar function I.1 Compressive forces do not activate p53 nucleolar stress response. I.2-Mechanical compression induces a dynamic change in UBF abundance and localization. I.3-Compressive forces decrease nucleolar activity. I.4-Compressive forces induces nucleolin release from nucleolar core

Figure 1 :

 1 Figure 1: Nucleolar organization in eukaryotic cells. A) Differential interference contrast (DIC) images of Hela cells highlight the prominent nucleoli within the nuclei (white arrows). Adapted from (Boisvert et al. 2007). B) Standard Electron microscopy preparation of Hela cells. The images show the three nucleolar compartments due to their different contrast: the fibrillar center (FC; asterisks), the dense fibrillar center (white arrows), and the granular center (GC). Scale bar: (a) = 0.5 µm and (b) = 0.1 µm. Adapted from (Danièle Hernandez-Verdun et al. 2010). C) rDNA repeats visualized by the Miller spread technique. Transmission electron micrograph of transcription of ribosomal RNA genes from the extrachromosomal nucleoli of a newt oocyte. Active transcription is seen by the tree-like structures "Christmas trees", in which each branch represents a nascent transcript. Adapted from (Miller and Beatty 1969).

Figure 2 :

 2 Figure 2: Localization of nucleolar proteins markers in the three nucleolar subcompartments in the human Hela cell. The top panel shows the nucleoli of Hela Cells by phase contrast. The bottom panel shows the distribution of nucleolar markers that corresponds to each sub-compartment. Antibodies against UBF decorate several foci in the nucleolar interior corresponding to FCs. Nopp140-GFP shows dotted labeling that corresponds to the DFC. NPM/B23-GFP fusion decorates the GC. Scale bar: 5 µm. Adapted from (Danièle Hernandez-Verdun et al. 2010).

Figure 3 :

 3 Figure 3: Scheme of human ribosomal gene and their transcripts. A) Ribosomal genes organized in tandem repeats, flanked by external nontranscribed spacers. B) rRNA genes are transcribed to give rise to polycistronic rRNA. C) Maturation of rRNA to produce mature 18S, 5.8S, and 28S. Adapted from (Raska et al. 2004).

Figure 4 :

 4 Figure 4: Scheme showing factors required for transcription initiation by RNA polI in mammals. Pre-initiation complex formation in mammals, consists of four components (SL1, UBF, Rn3, and TTF-I) are key for initiating complex formation and recruitment of RNApolI. Adapted from(Da 2012).

Figure 5 :

 5 Figure 5: Short-term regulation of rDNA transcription under different environmental cues. A) Schematic representation of different extracellular signals on rDNA transcription (upregulation in green, downregulation in red). Adapted from (Grummt 2013). B) Scheme showing different signaling pathways that regulates rDNA transcription depending on the availability of growth factors, glucose and nutrients. Growth factor-dependent signaling via the PI3K/AKT/mTORC1 and RAS/RAF/ERK pathways forms an intricate control network with the transcription factor MYC to regulate the rate of rDNA transcription. Adapted from (Kusnadi et al. 2015).

Figure 6 :

 6 Figure 6: Eukaryotic C/D box snoRNA and snoRNPs organization. A) Schematic representation of a C/D box snoRNA. SnoRNAs are represented in black with box C and D consensus sequences, shown in white boxes, respectively, close to 5' and 3' ends. Substrate rRNAs are indicated in red. Methylated residues are highlighted in yellow. B) Schematic representation of eukaryotic C/D box snoRNPs. 15.5kD (red) is recruited to the C/D box motif. Two copies of FIB1 (green), each is interacting with either one copy of Nop56 (orange) or one copy of Nop58 (yellow). Nop56 and Nop58 heterodimerize. Adapted from (Therizols et al. 2015).

Figure 7 :

 7 Figure 7: A model of FC/DFC organization and the 47S pre-rRNA sorting in the human nucleolus. The RNApolI occupies the rRNA genes at the FC/DFC border, allowing their transcription to form the nascent pre-rRNA 47S. The interaction of the 47S pre-rRNA with Fibrillarin facilitates its flow and the sorting of pre-rRNA from the FC/DFC border into the DFC region. Adapted from (Yao et al. 2019).

Figure 8 :

 8 Figure 8: Schematic illustration of nucleolar assembly by end of telophase. During

Figure 9 :

 9 Figure 9: Nucleolar reorganization upon stress. A) A normal nucleolus during interphase in PtK1 cells, showing intact structure. The button scheme shows a tripartite structure composed around rDNA. Adapted from (Louvet et al. 2005; Latonen 2019). B) Electron microscopy of Hela Cell nucleoli upon treatment with low doses of actinomycin D. The Ag-NOR staining (black dots) revealed the Ag-NOR proteins in FC. The scheme illustrates nucleolar segregation, or nucleolar caps, due to RNApolI transcriptional inactivation. Adapted from (Danièle Hernandez-Verdun et al. 2010; Latonen 2019). C) Electron microscopy of Ptk1 cells nucleoli, upon treatment with DRP for 1h. The scheme shows nucleolar necklace formation when RNApolI transcription remains active but rRNA processing is impaired. Adapted from (Louvet et al. 2005; Latonen 2019).

Figure 10 :

 10 Figure 10: rDNA transcription and cell cycle are mutually regulated. Regulation of RNApolI-mediated transcription during cell cycle progression. UBF is activated during interphase by phosphorylation of serine 484 (S484) by Cdk4/cyclin D and phosphorylation of serine 388 (S388) by Cdk2/cyclin E and A. At the entry into mitosis, phosphorylation of TAFI110 at threonine 852 (T852) by Cdk1/cyclin B inactivates TIF-IB/SL1. At the exit from mitosis, Cdc14B dephosphorylates T852, leading to the recovery of TIF-IB/SL1 activity. Activating phosphorylations are marked in green, inhibiting ones in red. Adapted from (Drygin, Rice, and Grummt 2010).

Figure 11 :

 11 Figure 11: The mammary duct has a bilayered organization of luminal (epithelial) and myoepithelial cells. The microvilli occupy the apical surface of most luminal epithelial cells. These luminal epithelial cells are connected by extensive intercellular junctions (Adherent Junctions, Desmosomes, and Tight junctions) and basally located Myoepithelial cells connected to the ECM through focal adhesions (Adhesion receptor: Integrin) and hemidesmosomes (not shown). Adapted from (Shamir and Ewald 2015).

Figure 12 :

 12 Figure 12: Main molecular players acting at Integrin (green) and Cadherin (red) adhesion sites. The authors proposed that both adhesions organize into a signaling layer (SL), a force transduction layer (FTL) and an Actin regulatory layer (ARL). A key feature shared between Integrin and Cadherin complexes is the tensiondependent conformational switch in Talin (purple) and α-Catenin (blue), respectively, which mediates binding of Vinculin (light brown), thereby resulting in adhesion strengthening. Adapted from (Han and de Rooij 2016).

Figure 13 :

 13 Figure13: Molecules contributing in the nuclear-cytoskeletal coupling. Lamins localize beneath the inner nuclear membrane and also form stable structures within the nuclear interior. Membrane proteins such as emerin, LBR, and SUN1/2 are retained at the inner nuclear membrane through their interactions with lamins, chromatin, or other nuclear proteins. Nesprins bind to SUN proteins across the perinuclear space and can directly interact with actin filaments (nesprins-1 and -2) or with intermediate filaments via plectin (nesprin-3). Nesprins-1 and -2 have been proposed to bind to microtubules via kinesin or dynein. Adapted from (Friedl, Wolf, and Lammerding 2011).

Figure 14 :

 14 Figure 14: Schematic illustration of Mechanotransduction processes.Mechanotransduction refers to converting a mechanical stimulus, such as ECM stiffness, stretching (substrate deformation or neighboring cells), compression (neighboring cells...), and shear stress (fluid) into chemical signals to regulate cell behavior and function. The pathway involves mechanical force sensors that are activated within seconds or even milliseconds, followed by altered gene expression after several hours, which will change the behavior and cellular functions after several days or even weeks at the tissue level. Adapted from (Iskratsch, Wolfenson, and Sheetz 2014).

Figure 15 :

 15 Figure 15: Development of mammary gland and the mechanical forces exerted on the cells during different developmental stages. A) Mammary gland development begins in the mouse embryo at E11.5 and results in a rudimentary gland at E18.5. The gland remains quiescent until puberty, when it undergoes recursive rounds of extension and bifurcation to produce a treelike epithelial tissue. The tissue undergoes further morphogenesis during pregnancy, resulting in alveoli formation. Adapted from (Nerger and Nelson 2019). B) Mechanical forces are exerted upon tissues during branching morphogenesis (pubertal stage) and lactation (pregnancy stage). The left scheme shows the terminal end bud (TEB), which is formed of a simple layer of luminal epithelial cells and myoepithelial cells in contact with the ECM. In addition, to the body and cap epithelial cells at the terminal end. The right scheme shows the formation of tertiary branches in mammary epithelial cells and alveolar cells during pregnancy. The schemes show the forces acting on cells in different directions. Adapted from (Boyle, Poltavets, and Samuel 2021).

Figure 16 :

 16 Figure 16: Mechanical forces during cancer initiation and progression. A) forces generated during the early stages of breast cancer growth within mammary ducts. B) additional mechanical forces generated during local invasion, and C) forces exerted during cancer cell invasion (an early stage of metastasis) and upon circulating tumor cells. Adapted from (Boyle, Poltavets, and Samuel 2021).

Figure 17 :

 17 Figure17: In vitro systems used to apply mechanical compression, with significant findings. A) Uniaxial compression i) Brick strex system used to apply lateral mechanical compression, through these system users showed that lateral mechanical compression inhibits cellular proliferation observed by retention of phospho-YAP1 in the cytoplasm. Adapted from[START_REF] Mäntylä | Brick Strex: A Robust Device Built of LEGO Bricks for Mechanical Manipulation of Cells[END_REF] ii) A custom-design Teflon chamber fabricated to culture cells on flexible silicone membranes. Applied compression in the plane of epithelial cells MDCKII induces extrusion of epithelial cells. Adapted from[START_REF] Eisenhoffer | Crowding Induces Live Cell Extrusion to Maintain Homeostatic Cell Numbers in Epithelia[END_REF]. B) Methodology used to apply mechanical compression on the top of the cells. The approach of mechanical compression induces chromatin condensation represented by an increase in chromatin foci observed by DAPI staining. Adapted from[START_REF] Damodaran | Compressive Force Induces Reversible Chromatin Condensation and Cell Geometry-Dependent Transcriptional Response[END_REF]).

Figure 19 :

 19 Figure 19: Scheme showing steps followed to use the 4D in vitro dynamic device. Step1: The suction cup and the confinement coverslips, are prepared by incubation in the culture medium for 1hr. Step2: prior to usage, the confinement coverslip is attached to the suction cub with micropillars faced up and then flipped on the cup where cells are seeded. Step 3: the suction cup/coverslip are attached to a vacuum pump device.

Figure 20 :

 20 Figure 20 : Creating a semi-quiescent monolayer of MCF10A epithelial cells. A) Optimizing the cells seeding density, representative images of MCF10A cells seeded at different densities and incubated for 48 hours. B) Representative immunofluorescence images of MCF10A cells grown on the silicon surface for 24 hours and 48 hours, stained for nucleus (DAPI, Blue), βcatenin (magenta). Scale bars = 8μm. C) Cell cycle of MCF10A cells seeded at 1.6×10 6 cells/ml and incubated for 48 hours and cells compressed for 4 hours. Images represent green cells fluorescently labeled by cell-regulated Geminin-emerald GFP proteins that represent cells in S/G2/M phase. The representative graphs show the mean percentage of cells in G1 and S/G2/M phase (N= 3 independent experiments for each condition, with n= 882 representing the total number of nuclei in the three independent experiments in control, n'=971 total number of nuclei in the three independent experiments in the compressed condition. D) Large field observation of MCF10A epithelial monolayer in both condition control and mechanically compressed for 4 hours, observed using 20× objective. Scale 10µm.

Figure 21 :

 21 Figure 21 : In-plane mechanical compression reinforce β -catenin at the cell-cell junctions A) i-Representative images of β -catenin staining in one plane of MCF10A epithelial cells monolayer, nuclei in blue and β -catenin in green scale bar=8μm, ii-Graph represent the ratio of intensity of β -catenin signal at the cell junction over the nucleus (N=2 independent experiments, n=100 total number of analyzed nuclei per condition), each point represents mean cell junction to nucleus ratio per image acquired, ****p<0.0001, **p=0.0101; (ordinary one-way ANOVA, Dunnett's test).

Figure 22 :

 22 Figure 22 : Compressive forces impact nuclear morphology and induce lamin A/C remodeling. A) i-Representative confocal images of MCF10A epithelial cells stained against the nucleus (DAPI, blue). Scale bar=8μm, ii-Representative graph of quantified nuclear area (µm 2 ) in control and compression over time, N=2 total number of independent experiments and n=57 total number of analyzed nuclei per condition, ****p<0.0001, ***p=0.0005 (ordinary one-way ANOVA, Dunnett's test), iii) Representative graph of quantified nuclear circularity in control and compression over time, N=2 total number of independent experiments and n=57 total number of analyzed nuclei per condition, ****p<0.0001, **p=0.0201, *p=0.0320 (ordinary one-way ANOVA, Dunnett's test). IV-and V-Representative graphs of nuclear area (µm 2 ) and circularity in control and at 4 hours post compression respectively, N=6 total number of independent experiments, n=719 total number of analyzed nuclei per condition for the area, n'=216 total number of analyzed nuclei for circularity. ****p< 0.0001 (tailed two t-test). B) i-Representative confocal images of MCF10A epithelial cells stained against nucleus (DAPI, blue) and lamin A/C in control and compression over time. Scale bar=8μm, ii-Representative graph of quantified lamin A/C intensity by the nuclear rim in control and at 4 hours post compression (N=3 independent experiments, n= 35 total number of analyzed nuclei), two-tail t-test, iii-Western blotting of whole cellular lamin A/C lysates in control and compression over time (N=4 total number of independent experiments). Bands normalized to the housekeeping gene, GAPDH.

Figure 23 :Figure 24 :

 2324 Figure 23 : Mechanical compression regulates the nucleolar tripartite organization particularly the fibrillar center. A) Electron Microscopy images of nuclei (left) and nucleoli (right) in MCF10A control and mechanically compressed overtime after 48 hours of culture on pre-coated collagen type I plates. In the left panel, the asterisk symbol represents the fibrillar center (FC), the arrowhead represents the dense fibrillar center (DFC), and the granular center (GC) is the largest compartment. B) Electron microscopy images of the whole nuclei in control MCF10A cells control and cells treated with Act-D 80 ug/ml. C) Left graph represents quantitative data of the mean percentage of MCF10A cells in control and mechanically compressed for 4 hours, showing the percentage of nuclei with less than or equal to 4 FC (FC≤4); total number of experiments=3, the total number of nuclei n=51. The right graph represents the mean of the fibrillar center in control and mechanically compressed over time, the total number of experiments=2, the total number of nuclei n=36.

Figure 25 :

 25 Figure 25: Compressive forces trigger an increase in the abundance of proteins known to regulate nucleolar phase separation. A) i-Representative confocal images MCF10A mechanically compressed overtimes and control stained for B23 (Green) and nucleus (DAPI, Blue). Images are presented as merged. Scale bar=8um, ii-Graph represents quantified nucleolar B23 intensity. N=3 The total number of independent experiments, n=640 total number of quantified nucleoli per condition, ****p< 0.0001, ** p=0.0012 (oneway ANOVA multiple comparisons, Dunnett's test). And iii-Western blot analysis of total fibrillarin abundance in the whole cell lysates, Actin (Housekeeping control). Semiquantification of total fibrillarin protein intensities to Actin by densitometry analysis of Western blots. Error bars represent the standard deviation-the total number of used lysates 6= six independent experiments, ****p<0.0001, ***p=0.003 (two tail t-test).

Figure 26 :

 26 Figure 26: Mechanically stretched epithelial cells for 4 hours do not show a significant change in nucleolar structure and morphology. A) Representative confocal images of MCF10A in control and mechanically stretched cells for 4 hours, stained for nucleus (DAPI, Blue) and Nucleolin (Magenta). Scale bar=8μm; Zoom=2. In addition to electron microscopy images showing tripartite organization in both conditions. B) Quantification of the average of the nucleolar area in control and stretched for 4 hours (N=3 independent experiments, each point on the graph representing one nucleolus, the total number of analyzed nucleoli n=471 per condition. (one-way ANOVA multiple comparisons, Dunnett's test). C) Stacking graph representing the percentage of nuclei showing the number of nucleoli per nuclei. N=6 independent experiments, the total number of analyzed nuclei n=120 for the control and n=99 for the stretched per each experiment.

  the impact of compressive stress on nucleolar function I.1 Compressive forces do not activate the p53 nucleolar stress pathway.

Figure 27 :

 27 Figure 27: Compressive forces do not activate p53 nucleolar stress pathways. A) i-Representative images of p53 (green), co-localized with nucleolin (Magenta) in MCF10A control and mechanically compressed after 4 hours, scale 8µm, ii-Normalized p53 nuclear intensity quantified based on DAPI staining (N=4 total number of independent experiments, n=259 total number of quantified nuclei per condition), iii-Western blotting of total p53 abundance in control and mechanically compressed MCF10A cells, (N=3 total number of experiments), normalized to GAPDH. B) i-Representative confocal images of immunolabeling against p53 (green), and staining nuclei (DAPI, blue) in MCF10A control and mechanically stretched for 4 hours, and cells treated with Act-D. Scale bar =8µm. ii-Representative graph of nuclear p53 intensity, in mechanically stretched cells for 4 hours and control. Quantified intensity based on DAPI staining. **p<0.0079, (tailed two t-test).

Figure 28 :

 28 Figure 28: Mechanical compression regulates UBF abundance at 2 hours and localization after 4 hours. A) Representative confocal images of MCF10A mechanically compressed overtimes and control stained for UBF (Green) co-stained with nucleolin (Magenta) and nucleus (DAPI, Blue). Scale bar=8um. B) Graphs represent average nucleolar UBF intensity based on nucleolin staining. N=3 total number of independent experiments, n=329 total number of quantified nucleoli per condition **p<0.0019 (ordinary one-way ANOVA multiple comparisons, Dunnett's test). C) Representative zoomed confocal images of MCF10A nucleoli stained for nucleolin (Magenta) co-localized with UBF (Green). The distribution of UBF is indicated by star, arrowhead, and arrow at different time points. Scale bar= 1um. D) i-Western blot analysis of total UBF abundance in the whole cell lysates, GAPDH (Housekeeping gene). Semi-quantification of intensities of total UBF protein to GAPDH by densitometry analysis of Western blots. Error bars represent standard deviation. The total number of experiments analyzed=6, (tailed two t-test) in each condition, ii-Western blot of total UBF abundance in control and at 2 hours post compression, Semi-quantification was done against the housekeeping gene GAPDH, iii-Expression level of UBF gene in control and at different time points of induces compressive stress, analyzed by RT-qPCR.

  , nucleolin inverted images). (a known effect of Act D treatment (Shav-Tal et al. 2005)). Interestingly, this phenotypic change in nucleolin positioning strongly after 4 hours of compression (figure 29D), suggesting potential role of nucleolin in regulating the response of epithelial cells to mechanical compression. ………………………………………………… Figures are presented on the next page.

Figure 29 :

 29 Figure 29 : Compressive forces for 4 hours attenuates rDNA transcription. A) Representative confocal images showing 5'EU intensity in the nucleus and nucleolus of MCF10A in control and mechanically compressed cells for 4 hours, stained for nucleus (DAPI, Blue) and B23 (Red). Scale bar=8μm; zoom=2 and zoom=1 is shown. B) i-Quantification of 5'EU intensity in the nucleus and ii-nucleolus. N= 3 total number of independent experiments, n= 507 total number of analyzed n'=942 total number of analyzed nucleoli. ****p<0.0001, (ordinary one-way ANOVA multiple comparisons, Dunnett's test). C) Total RNA was isolated in control and compressed MCF10A at different time points, and RT-qPCR was performed using primers specific for 45S pre-RNA. (N= 3 total number of independent experiments) ****p< 0.0001, **p=0.0052, *p=0.0156. D) Representative confocal images of MCF10A cells costained for nucleolin and UBF and stained for nucleus (DAPI, Blue) for the three conditions (control, compression 4 hours, Act-D treated cells (10 ug/ml).

Figure 30 :Figure 31 :

 3031 Figure 30 : Mechanical compression for 4 hours induces nucleolin release from the nucleolus. A) Representative confocal images MCF10A mechanically compressed overtimes and control stained for nucleolin (Magenta) and nucleus (DAPI, Blue), the last column represents inverted images of nucleolin staining in each condition. Scale bar=8um. B) Representative graph showing the ratio of quantified Nucleolin intensity divided by the intensity within the nucleoplasm. N= 3 total number of independent experiments, n=276 total number of quantified nucleoli per condition, ****p<0.0001, ***p=0.0010 (ordinary one-way ANOVA multiple comparisons, Dunnett's multiple comparison test). C) Western blot analysis of total Nucleolin abundance in the whole cell lysates, GAPDH (Housekeeping control). Semiquantification of intensities of total Nucleolin protein to GAPDH by densitometry analysis of Western blots. Error bars represent standard deviation. The total number of experiments analyzed=6, (two tail t-test).

  123

Table 1 :

 1 Antibodies used for immunofluorescence labeling

	Antibody	Dilution	Reference	Source
	Anti-Nucleolin	1/200	#14574	Cell Signaling Technology®
	Anti-UBF	1/200	sc-1312	SANTA CRUZ BIOTECHNOLOGY
	Anti-B23	1/200	sc-271737	SANTA CRUZ BIOTECHNOLOGY
	Anti-lamin A/C	1/200	sc-376248	SANTA CRUZ BIOTECHNOLOGY
	Anti-β-catenin	1/200	ab16051	abcam
	Anti-P53	1/200	sc-47698	SANTA CRUZ BIOTECHNOLOGY
	Phalloidin	1/500	A-2228	ThermoFisher Scientific
	Anti-mouse IgG (H+L), Alexa Fluor 488	1/500	A-11059	ThermoFisher Scientific
	Anti-mouse IgG (H+L), Alexa Fluor 568	1/500	A-11004	ThermoFisher Scientific
	Anti-Rabbit IgG (H+L), Alexa Fluor 647	1/500	A-21245	ThermoFisher Scientific

Table 2 :

 2 Antibodies used in to analyze the expression level of protein in western blotting.

	Antibody	Dilution	Reference	Source
	Anti-Fibrillarin	1/1000	ab4566	abcam
	Anti-GAPDH	1/5000	MA5-15738	ThermoFisher
				Scientific
	Anti-Actin	1/5000	A-2066	SIGMA ALDRICH
	Anti-mouse IgG-HRP	1/3000	7076P2	Cell Signaling
				Technology®
	Anti-rabbit IgG-HRP	1/3000	7076P2	Cell Signaling
				Technology®

As I mentioned in Part I, ribosome biogenesis is a highly coordinated process that occurs in the nucleolus, requiring several ribosomal proteins and assembly factors. In addition to the small-nucleolar RNA that acts as a scaffold to guide rRNA modifications, which are crucial for rRNA maturation and hence ribosomal subunits formations.

3.Thesis objectives

4.Materials and Methods

Scientific Reports 6 (1): 36171. https://doi.org/10.1038/srep36171.

Acknowledgment

All work presented in the manuscript was carried out in the team of Dr. Christophe Guilluy under the supervision of Dr. Monika Dolega at the Institute for

Supplementary Data