Browsing by Author "Childs, Sarah J."

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    Absence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair
    (2022-11-01) Larijani, Leila; Rancourt, Derrick; Krawetz, Roman; Childs, Sarah J.; Bob, Argiropoulos
    p21 is a multifunctional protein that is critical to the control cellular proliferation and plays a number of other functions in the cell. In the nucleus, p21 functions as a tumour suppressor, while in the cytoplasm, phosphorylated p21 functions as a proto-oncogene and can also suppress apoptosis. Because p21 deficiency has been linked to increased tissue regeneration, in this thesis, I sought to investigate if p21 mutations can improve the regenerative capacity of induced pluripotent stem cells in a cellular therapy approach for cartilage injuries in mice. However, because p21 is a tumor suppressor and its loss can result in tumorigenesis, I employed the inducible Caspase9 cell suicide system to purge iPSCs through forced apoptosis. In vitro, iCaspase9-mediated cell death resulted in a statistically significant increase in the apoptosis rate in p21-/- iPSCs compared to p21+/+ iPSCs (wild-type). RNA sequencing was undertaken to determine which pathways are involved in p21 mediated apoptosis. Increased expression of 41 apoptotic and 6 healing-related genes were observed in p21-/- iPSCs compared to wildtype. In vitro chondrogenesis of p21-/- iPSCs showed an increase in chondrogenic genes relative to the wild-type cells. When these iPSCs were transplanted into a focal cartilage injury in mice, ectopic cartilage formation was observed. Neither tumors, nor ectopic cartilage formation was observed in mice that were treated with CID drug to induce iCasp9 mediated apoptosis. Finally, I performed a drug screen to discover compounds that promote apoptosis with iCasp9. In addition to identifying several epigenetic drugs that promoted iCasp9 function in vitro, I also demonstrated that p21 inhibitors could phenocopy the enhanced iCasp9 mediate cell killing seen previously in the p21 mutant iPSCs. Overall, my findings indicate that p21 does play a role in protecting iPSCs from apoptosis and its downregulation can be exploited to increase cell-suicide approach effectiveness. I was also able to demonstrate that iPSCs can induce robust cartilage repair in mice regardless of the expression of p21. Therefore, modulation of p21 warrants further research to determine if exploiting this protein can be used in iPSC clinical trials safely in the future.
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    Characterization of Muscle-Associated Cells in Adult Zebrafish
    (2019-12-20) Ruel, Tyler David; Huang, Peng; Childs, Sarah J.; Biernaskie, Jeff A.
    Skeletal muscles make up 40% of body weight in humans. Any compromises in muscle function will cause major consequences to the quality of a person’s life. It is therefore extremely important that this tissue is maintained in a state of homeostasis. To do this, muscle fibres that become damaged must be repaired by tissue-resident muscle-stem cells throughout the life of an animal. Several different kinds of muscle-associated cells have been described, including the two main populations: satellite cells (a population of muscle stem cells) and fibro/adipogenic progenitors (FAPs) (a population of mesenchymal stem cells). Using zebrafish as a model, the importance of muscle-associated cells in maintaining muscle homeostasis is demonstrated. Our lab has previously generated a col1a2¬-based transgenic line that labels collagen-expressing cells in zebrafish. Using a combination of immunohistochemistry and confocal microscopy, I characterize the dynamics and function of col1a2+ muscle-associated cells. A developmental time course shows that col1a2+ intramuscular cells increase in numbers during juvenile stages. In response to muscle injury, col1a2+ muscle-associated cells are expanded and contribute to muscle regeneration. Genetic ablation of col1a2+ cells, results in a compromised regenerative response. Using Cre-mediated lineage tracing, the developmental origin of intramuscular cells is traced to the dermomyotome and sclerotome, two sub-compartments of the embryonic somite. Finally, characterization of a col1a2 mutant line of zebrafish suggests that Type-I collagen is important for maintaining muscle integrity. These observations suggest the importance of col1a2+ muscle-associated cells in maintaining muscle homeostasis and for producing the extracellular matrix (ECM) within the skeletal muscle tissue to prevent degeneration.
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    Characterization of the Derlin protein, CUP-2, in promoting stem cell proliferation in the C. elegans germ line
    (2018-03-27) Singh, Ramya; Hansen, David; Cobb, John A.; Muench, Douglas G.; Childs, Sarah J.; Labbé, Jean Claude
    Understanding of how tissue homeostasis is maintained is not only essential for furthering our understanding of a fundamental area in Biology, but also has important implications for regenerative medicine and the design of therapeutics for human health. The germline of the C. elegans nematode serves as an excellent model to study adult stem cells and how the balance between proliferation and differentiation is maintained in this vital tissue. GLP-1/Notch signalling is the principal pathway that promotes proliferation of Germline Stem Cells (GSCs) in this system. In this thesis, the role of a Derlin (Degradation in the ER) protein, CUP-2, in promoting GLP-1/Notch signalling-mediated proliferation of GSCs is presented. Reducing cup-2 activity strongly suppresses germline tumours caused by increased Notch signalling. However, cup-2(0) is unable to suppress Notch-independent germline tumours, which suggests that cup-2 promotes Notch signalling specifically and not cell proliferation in general. This is further supported by cup-2’s ability to influence the expression of a read-out for Notch signalling, SYGL-1. The cup-2 gene was tagged using CRISPR/Cas9 editing with an epitope tag and CUP-2 was found to be expressed in the germ line, which is consistent with its role in promoting Notch signalling. The ability of cup-2 to promote GLP-1/Notch signalling likely represents a conserved function, since der-2, a Derlin homolog, was also found to contribute to the development of Notch-dependent germ line tumours. In fact, cup-2 and der-2 appear to additively promote GLP-1/Notch signalling. Derlin proteins are known to function in Endoplasmic Reticulum-Associated Degradation (ERAD) of proteins. Therefore, this work sheds light on the importance of ERAD in maintaining the balance between proliferation and differentiation in stem cells. Investigation into the mechanism by which cup-2 promotes GLP-1/Notch signalling led to the discovery that cup-2 is involved in anchoring the niche cell, the Distal Tip Cell, in place in the aging gonad. This is a novel phenotype not reported before in the literature. The CRISPR/Cas9 tagged cup-2 alleles developed as part of this thesis could serve as a useful reagent for further studies in determining the mechanism by which cup-2 promotes GLP-1/Notch signalling.
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    Defining the Role of Proneural Genes in Neurogenesis, Specification, and Migration of Mouse VMH Neurons
    (2020-01) Aslan Pour Kal Bolandi, Shaghayegh; Kurrasch-Orbaugh, Deborah M.; Childs, Sarah J.; Yang, Guang
    The ventromedial hypothalamus (VMH) is a hypothalamic nucleus important for controlling satiety and reproductive behaviors, among other physiologies. Despite these important roles, the genetic programs driving VMH development are just starting to be explored. Since proneural genes are one of the key drivers of neurodevelopment throughout the brain, we asked whether the proneural genes Achaete-scute homolog 1 (Ascl1) and Neurogenin 2 (Neurog2) play key roles in VMH development, given their expression in VMH progenitors. My hypothesis is that proneural genes play a role in neurogenesis, cell fate decisions and/or in the migration of VMH neurons. To start, I investigated the role of Ascl1 in the specification and fate of VMH neurons. I showed that Ascl1+ lineages give rise to a large population of VMH neurons and that Ascl1 is necessary for promoting VMHDM and VMHC neuronal specification. In addition, my results revealed that Ascl1 played an important role in the timing of neurogenesis within the tuberal hypothalamus. Next, I examined the role of Neurog2 in the neurogenesis of VMH specific neurons and showed that Neurog2 was necessary for timely cell cycle exit and birth of VMH neurons. Moreover, I showed Neurog2 was required for proper development of the VMH at both early and late embryonic developmental stages. Neurog2 is particularly important for proper differentiation of VMHVL neurons. Finally, I studied the role of Ascl1 and Neurog2 in cell sorting within the maturing VMH. My results revealed that between E15.5 to E17.5 and in the absence of either Ascl1 or Neurog2, there is a change in the final positioning of cells. Since Ascl1 and Neurog2 can act through Rnd3 and Rnd2 proteins to regulate cortical neuronal migration, I asked whether Ascl1 and Neurog2 potentially employ a similar mechanism to regulate VMH cell sorting. My results showed both Rnd2 and Rnd3 were expressed within the VMH nucleus across VMH embryonic developmental stages. In addition, at later stages such as E15.5 and E19.5, the expression of both Rnd2 and Rnd3 was significantly decreased in the absence of Neurog2 and Ascl1 respectively. Finally, using a live cell imaging technique, I assayed neuronal movement during cell sorting events within the VMH. Together, these studies provide insight into the varying roles proneural genes play in the developing tuberal hypothalamus.
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    Development of vascular regulation in the zebrafish embryo.
    (2019-09-19) Bahrami, Nabila; Childs, Sarah J.; Cole, William C.; Gordon, Grant Robert J.
    The vascular system is placed under enormous stress at the onset of cardiac contractility and blood flow. Nascent blood vessel tubes initially consist of a thin endothelial wall and rapidly acquire support from mural cells (pericytes and vascular smooth muscle cells; vSMCs). Following their association with vessels, mural cells acquire vasoactive ability (contraction and relaxation). However, we have little information as to when this vasoactivity first develops, and the extent to which each mural cell type contributes to vascular tone regulation during development. For the first time in an in vivo system, we highlight the dynamic changes in mural cell vasoactivity during development. We assess mural cell vasoactivity in the early zebrafish (Danio rerio) cerebral vasculature in response to pharmacological agents. We determine that pericyte-covered vessels constrict and dilate at 4 days post fertilization (dpf) but not at 6 dpf. The prostaglandin EP4 receptor contributes to pericyte-covered vessel dilation at 4 dpf. In contrast, vSMC-covered vessels constrict but do not dilate at 4 dpf. At 6 dpf, vSMC-covered vessels continue to constrict but only dilate from a pre-constricted state. Using genetic ablation, we demonstrate that mural cell contraction and relaxation is an active response by pericytes and vSMCs. Thus, we show that both pericytes and vSMCs have the ability to regulate cerebral vascular tone but at different stages of development. Pericytes are involved in regulating vessel diameters prior to the maturation of the vSMCs. Once vSMCs mature, pericytes are no longer active, and only vSMCs mediate vasomotor activity in the developing embryonic brain of zebrafish. The onset of vSMC vasoactivity corresponds to the development of increased neuronal activity and neurovascular coupling.
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    Elucidating the role of tcf15 in somite development
    (2024-06-25) Lim, Nicholas Farn Wei; Huang, Peng; Childs, Sarah J.; Cobb, John; Chu, Li-Fang
    Somites are transient embryonic structures that give rise to the axial musculoskeletal system. Eventually, the somite gives rise to three distinct compartments: the dermatome, myotome, and sclerotome. These compartments give rise to the skin, skeletal muscles, and the axial skeleton, respectively. Thus, proper formation of somitic compartment is necessary for a functional body. Despite many early studies elucidating the development of the somite, little is known about the regulator of somite compartment proportion. Here I characterized the basic helix-loop-helix transcription factor, tcf15, as a key regulator of somitic compartments in zebrafish. I showed that tcf15 expression is highly dynamic during somite development, with initial robust expression throughout the presomitic mesoderm, followed by restricted expression in the dermomyotome and ultimately in dermomyotome-derived muscle progenitors and in sclerotome-derived tenocytes along the somite boundary. Through mutant analysis, I found that loss of tcf15 results in a reduction in the pax7a+ dermomyotome and the myoD+ myotome compartment accompanied by an expansion of the nkx3.1+ sclerotome. Interestingly, migration of sclerotome cells and the development of sclerotome-derived cells such as tenocytes and fin mesenchymal cells were unaffected in tcf15 mutants. To manipulate Tcf15 activity, I developed various gain-of-function and loss-of function tools. However, the broad application of these tools was limited by technical challenges. Nonetheless, by combining the expression and mutant analysis, my results suggest that Tcf15 functions as an important regulator of somite compartmentalization in zebrafish, promoting the dermomyotome/myotome fate while repressing the sclerotome fate.
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    Fluid flow effects on nanoparticle localization in zebrafish vessels and cultured human endothelial cells
    (2017) Gomez, Maria Juliana; Rinker, Kristina D.; Childs, Sarah J.; Cramb, David T.; Di Martino, Elena
    Assessment of nanoparticle distribution in the vasculature is important for determining drug delivery, molecular imaging efficacy, and risk profiles. Even though most medical nanoparticle applications require a vascular administration, factors affecting nanoparticle association with vessel walls in the presence of fluid forces are poorly understood. We evaluated the effect of fluid flow on the distribution of 200 nm carboxylate-coated polystyrene nanoparticles in flow-exposed endothelial cell cultures and zebrafish embryos. We combined confocal imaging of nanoparticle injected transgenic zebrafish, 3D modeling, and computational fluid dynamics to assess nanoparticle distribution under flow. Highest nanoparticle localization occurred in regions of disturbed flow and low shear stress found at branch points and downstream of bumps and curves in the zebrafish vasculature. Similar findings were obtained in human endothelial cells in vitro. Overall, fluid shear stress magnitude, flow disturbances, and flow-induced changes in endothelial physiology contribute to the vascular localization of nanoparticles.
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    FOXO as a Regulator of Hypoxia Tolerance in Drosophila
    (2020-12) Barretto, Elizabeth; Grewal, Savraj S.; Childs, Sarah J.; Brook, William J.
    Animals often live in conditions where environmental oxygen levels fluctuate. When oxygen is abundant, growth is promoted, but when oxygen is scarce, metabolic processes are altered to limit growth and promote survival. One important mechanism controlling responses in hypoxia is the regulation of metabolic gene expression. The conserved hypoxia-inducible factor (HIF) is a transcriptional regulator of genes important for hypoxia-responses. However, less is known about other transcription factors important in hypoxia adaptation. We have explored this question in Drosophila melanogaster. Drosophila can tolerate low oxygen conditions. At 5% oxygen, larvae slow their growth and development, but viability remains unaffected. At 1% oxygen, larvae and adults exhibit a state of ‘suspended animation’ but can survive for up to 12 or 30 hours respectively. We found that upon switching larvae from normoxia to hypoxia, the transcription factor Forkhead Box O (FOXO) was rapidly relocalized from the cytoplasm to the nucleus in larval tissues even though animals maintain normal feeding. Moreover, we saw that many known FOXO target genes were induced in animals exposed to hypoxia. FOXO is important for regulating starvation and stress responses and can regulate aging in Drosophila and Caenorhabditis elegans. We found that foxo null mutant larvae and adults show reduced survival in hypoxia. We also identified two downstream FOXO target genes involved in hypoxia tolerance. The first is the translational repressor 4e-bp. Upon exposure to hypoxia, 4e-bp expression is increased in a FOXO-dependent manner, while 4e-bp mutants showed a modest decrease in hypoxia survival. The second factor is the NF-kB transcription factor relish, a mediator of innate immune responses. We found that expression of relish and antimicrobial peptides was increased in a FOXO-dependent manner in hypoxia, and that relish null mutant animals have reduced survival in hypoxia. To better understand the breadth of FOXO responses, we examined metabolites and found that FOXO is necessary for maintenance of glycogen and lipid stores during hypoxia. foxo null mutants also have deregulated expression of lipases and glycolytic genes. Transcriptomic analyses further revealed that FOXO may modulate expression of genes involved in Notch and Hippo signaling, oocyte development, and translational effectors such as Ribosomal Proteins. Together, these data indicate that FOXO is a hypoxia-inducible factor that mediates tolerance to hypoxia by controlling protein synthesis, immune signaling, and metabolism.
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    Interaction of the SHOX2 transcriptional regulator with the Sonic hedgehog signalling pathway in the developing mouse limb
    (2019-12) Ljubojevic, Anja; Cobb, John A.; Chua, Gordon; Childs, Sarah; Hansen, David V.; Childs, Sarah J.
    The mouse Shox2 gene is a critical transcription factor for embryonic limb patterning. This work explored how Shox2 is regulated by employing the 4C technique, which allows one to identify DNA sites that frequently interact with a genomic region of interest. Our findings showed that many sites contact Shox2, indicating that its expression is controlled by a collection of enhancers. By using a transgenic reporter line, we found that the SHOX2 protein negatively regulates an enhancer of Shox2. To investigate the genetic relationship between Shox2 and the Sonic hedgehog pathway, we generated compound mutant mouse embryos and found that the characteristic proximal limb truncation in conditional Shox2 mutants was significantly rescued in the absence of functional Shh. Finally, we focused on identifying shared downstream targets of SHOX2 and Sonic hedgehog. Through whole mount in situ hybridization, we tracked changes in gene expression of candidate common target genes in mutant backgrounds.
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    Investigating the role of splicing in disorders of craniofacial development
    (2017) Lynch, Danielle C; Parboosingh, Jillian S.; Innes, A Micheil; Bernier, Francois P; Childs, Sarah J.; Rancourt, Derrick E; Zimmerly, Steven; Trainor, Paul A
    Following clinical delineation of a rare disease, identification of the causative gene(s) is a crucial first step towards providing enhanced patient care and understanding disease aetiology. Disease gene discovery, especially when considered in the context of related disorders, can also provide new insight regarding normal development and physiology. Herein, I present two novel disease-causing genes: the ribosomal gene RPLP2 in Nager syndrome (NS) and the spliceosomal gene SNRPB in cerebro-costo-mandibular syndrome (CCMS). The mutations identified in SNRPB are the first example of de-regulated alternative splicing-coupled nonsense-mediated decay as a mechanism for human disease. NS and CCMS are both disorders of first and second pharyngeal arch development, with the jaw and ears being affected. Over the past five years, the association between disorders of craniofacial development and mutations in spliceosomal genes has become apparent with the discovery of SF3B4 in NS, EFTUD2 in mandibulofacial dysostosis type Guion-Almeida, and TXNL4A in Burn McKeown syndrome. The specificity of the phenotypes resulting from mutations in such ubiquitous genes has perplexed the scientific community, but it is at least clear that spliceosomal genes play a more prominent yet subtle role in development than previously thought. The association between Treacher Collins syndrome, which overlaps phenotypically with CCMS and NS, and the ribosomal genes TCOF1, POLR1C, and POLR1D has been known for longer. This work establishes NS as having both spliceosomal and ribosomal defects as a cause. In this thesis I discuss potential links between the mechanisms underlying ribosomal and spliceosomal defects in disorders of craniofacial development, particularly increased sensitivity to reactive oxygen species (ROS).
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    Polo-like kinase-1 (PLK-1) inhibitors as novel therapeutics in oral squamous cell carcinoma
    (2019-07-22) Sarkar, Subhanwita; Bose, Pinaki; Narendran, Aru; Bonni, Dr. Shirin; Childs, Sarah J.
    Polo-like kinase-1 (PLK-1) belongs to a family of conserved serine/threonine kinases and is an oncogenic protein in many cancers. Therefore, PLK-1 is an attractive therapeutic target and clinical trials are ongoing to test the efficacy of PLK-1 inhibitors in several cancer-types. Oral squamous cell carcinoma (OSCC) is a common head and neck cancer. OSCC is associated with frequent recurrences after initial curative therapy and overall poor prognosis. We analysed RNA sequencing data from The Cancer Genome Atlas (TCGA) and found that PLK-1 mRNA levels are elevated in OSCC compared to normal oral cavity squamous epithelium and high PLK-1 expression in OSCC is associated with worse survival. Based on these results, we tested the efficacy of PLK-1 inhibitors in a panel of ten OSCC cell lines. The PLK-1 inhibitor, volasertib effected cell death at low nanomolar concentrations in most tested OSCC cell lines but not in normal oral keratinocytes. Flowcytometry analysis showed that volasertib induces G2/M arrest in sensitive cell lines. Western blot analysis showed that levels of total PLK-1 and phospho PLK-1 were reduced after volasertib treatment in sensitive cell lines. Volasertib also triggered apoptosis confirmed by the cleavage of PARP and Caspase 3. Cell lines resistant to volasertib did not show any alteration in total PLK-1 and pPLK-1 after volasertib treatment. Post-operative radiotherapy is a common treatment modality in OSCC patients. In two OSCC cell lines that were refractory to volasertib treatment, a combination of volasertib and γ-radiation significantly lowered cell survival compared to volasertib or γ-radiation alone. Combination therapy with γ-radiation and volasertib in resistant cell lines resulted in S-phase arrest. Western blot analysis showed a significant reduction of total PLK-1 and pPLK-1 after combinatorial therapy. Apoptosis was induced in volasertib resistant cells as a result of combination therapy. Taken together, these in vitro studies establish the rationale for further investigation of volasertib efficacy in orthotopic OSCC xenograft models and clinical trials.
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    Semaphorin3f as a Spatial Regulator of Embryogenesis
    (2019-01-22) Halabi, Rami; McFarlane, Sarah; Childs, Sarah J.; Huang, Peng
    During embryogenesis, cells integrate both spatial and temporal information from their surroundings to influence proliferation, migration, differentiation and physiological functions. Understanding the molecular mechanisms which confer spatial identity is essential to our understanding of tissue development and human disease. In this thesis I explore multiple roles for the secreted chemotactic ligand Semaphorin3f (Sema3f) in different biological contexts. Using zebrafish (Danio rerio) as a model I take advantage of the duplicated genome to study loss of function of both orthologs, Sema3fa and Sema3fb, in discrete contexts due to their differential expression. First, I show that in the eye Sema3fa produced by progenitors is necessary for the generation of amacrine cells within the temporal retina and the spatially-organized transcriptome of stem cells in the ciliary marginal zone (CMZ). Second, I define an endogenous role of Sema3fa to maintain the avascularity of the neural retina and refine the branch pattern of intraocular vessels. Loss of Sema3fa results in the pathologic angiogenesis of leaky blood vessels into the neural retina. Last, I unveil a role for Sema3fb produced by cardiomyocyte progenitors in the differentiation of the ventricle of the developing heart. Overall, my work provides the first evidence of a Sema3 involved in retinal progenitor cell and cardiomyocyte differentiation, and elucidates the endogenous role of Sema3fa as a negative regulator of retinal blood vessels in the embryo and adult. My data exemplifies the necessity of spatial information conferred by a single chemotactic molecule, Sema3f, to impact differentiation and cellular biology.
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    Semaphorin3f in the maturation of the outer retina
    (2020-09-25) Mori-Kreiner, Risa; McFarlane, Sarah; Childs, Sarah J.; Guo, Jiami; Ungrin, Mark D.
    Cells of multicellular organisms have the remarkable ability to coordinate and control dynamic cellular activities in response to changes in their environment. From development into tissue homeostasis, cells communicate with each other through a myriad of intercellular signalling mechanisms. The large family of Semaphorins is a group of well-known extracellular signalling molecules implicated in a wide range of diverse physiological functions. In particular, the expression of secreted Class III Semaphorins (Sema3s) in the retina, not only during development but also in adult tissue, raises interesting questions about their tissue-specific spatiotemporal roles. The vertebrate retina is a highly complex, light-sensitive tissue that lines the back of our eyes. Within the retina, there are two key players that enables our ability to see: the retinal pigment epithelium (RPE) and the photoreceptors. Located in the outermost layer of the retina, the RPE and photoreceptors develop and mature together throughout the lifetime of the organism, forming an interdependent relationship that is highly critical for visual function. This thesis explores, using CRISPR/Cas9-generated loss-of-function mutants (sema3faca304), cell autonomous and non-cell autonomous roles of an RPE-secreted protein, Semaphorin3f (Sema3f), in the zebrafish retina. First, I demonstrate that both cell types, the RPE and photoreceptors, express multiple members of well-known Sema3 receptors, Nrp and PlxnA families. Second, I define a cell autonomous role of Sema3fa in maturing RPE. The loss of Sema3fa does not affect the maturation of the RPE at the transcriptional and morphological level, but does result in the perturbation of appropriate physiological responses to light conditions. Last, I demonstrate the non-cell autonomous role of Sema3fa in the development and specification of maturing photoreceptors. My work elucidates one of the many endogenous roles of Sema3fa as a regulator of the maturing retina to add to the growing literature of Sema signalling in events other than development.