The extracellular matrix protein Fibronectin (FN) plays an important role in cell contractility, differentiation, growth, adhesion, and migration. The 12th -14th Type III repeats of FN (FNIII 12-14), also referred to as the Heparin-II domain, comprise a highly promiscuous growth factor (GF) binding region. This binding domain aids in cellular signaling initiated from the ECM. Additionally, FN has the ability to assemble into fibrils under certain conditions, mostly observed during cell contractile processes such as those that initiate due to upregulation of Transforming Growth Factor Beta 1 (TGF-β1) [1], [2]. Previous work from our lab has shown that self-assembly of FN into insoluble fibrils is crucial for Epithelial-Mesenchymal Transition (EMT) [3]. The transition from epithelial to mesenchymal cell type has been implicated as an early event in tumor formation and breast cancer. We were previously able to find that upregulation of FN fibrils drive EMT through contractility due to the increase of the GF latent TGF-β complex concentration at the cell membrane [3]. The challenge in the current work is to exploit the role of Heparin-II binding domain and to concentrate growth factors of interest, such as those that are pro-EMT or anti-EMT at the signaling sites of the cell membrane. Initially, we investigated the localization of the fragments FNIII 12-14 delivered to cell membrane using FITC conjugated protein. We then investigated the effects of exogenous FNIII 12-14 on EMT using breast epithelial cells (MCF10A) in the presence or absence of TGF-β1 to determine whether FNIII 12-14 alters EMT signaling. Quantification of mRNA expression, for EMT markers such as Slug, Snail, Twist, and ZEB1 were analyzed. Results showed that dosage increase of FNIII 12-14 appears to inhibit EMT transcription factors. This study will develop a new understanding of disease and gene control using ECM proteins. The exploitation of ECM natural protein interactions could become a new method in turning on/off genes of interest. While we are currently investigating this as a mechanism of blocking EMT, it could also have implications in wound healing, fibrosis, and tissue engineering, where EMT is an important aspect of the physiologic progression.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

Les vaisseaux sanguins sont composés de cellules enchâssées dans la matrice extracellulaire (MEC). Certaines interactions entre les cellules et les composés de la MEC sont impliquées dans plusieurs changements pathologiques et physiologiques au niveau de la paroi artérielle. L'hypertension systolo-diastolique provoque deux formes de remodelage vasculaire : hypertrophique dans les artères de conductance et eutrophique dans les artères de résistance. Les mécanismes régissant le remodelage eutrophique ne sont pas encore bien éclaircis. Afin de les étudier, nous avons utilisé un modèle d'hypertension systolodiastolique induite par l'inhibition de la synthèse de monoxyde d'azote. Nous avons démontré que la contribution des protéines de désadhésion semblait différer d'un type de remodelage à l'autre et que les métalloprotéinases matricielles (MMPs) ne jouaient pas un rôle crucial dans le développement du remodelage eutrophique, contrairement au remodelage hypertrophique. Au cours du vieillissement, on observe des modifications de la paroi vasculaire des artères de conductance, augmentant leur rigidité : une fragmentation des fibres élastiques, leur calcification (élastocalcinose), une augmentation des liens covalents entre les protéines de la MEC et une fibrose. Cette rigidité conduit au développement de l'hypertension systolique isolée. Afin d'évaluer l'implication des MMPs dans la fragmentation de l'élastine et leur rôle dans le développement de l'élastocalcinose, nous avons utilisé un modèle animal d'élastocalcinose basé sur l'inhibition de la maturation de la matrix Gla protein (MGP), une protéine anti-calcifiante, par la warfarine. Nous avons observé une augmentation rapide et transitoire de l'activité de la MMP-9, suivie de l'activation du transforming growth factor-ß (TGF-ß). L'inhibition de l'activité des métalloprotéinases et de TGF-ß a permis de prévenir l'élastocalcinose. Par ailleurs, l'élastocalcinose est accélérée par le diabète. Cette accélération est reliée à la durée et à la sévérité du diabète. Or, ce dernier est associé à une augmentation de la synthèse des produits avancés de glycation (AGEs) pouvant former des liens covalents iv entre les protéines de la MEC. Ne disposant pas de modèle animal pour étudier l'implication des AGEs dans l'élastocalcinose associée au diabète, nous en avons créé un. Dans ce modèle, le diabète est induit par une diète riche en lipides et une injection de streptozotocine (30 mg/kg/jr). L'élastocalcinose est provoquée par l'inhibition de la maturation de la MGP. Nous avons observé une accélération de l'élastocalcinose, liée à la durée du diabète, et une accumulation de AGEs dans la paroi des artères fémorales. L'utilisation de pyridoxamine, un inhibiteur de la formation des AGEs, et de l'ALT711, un briseur des liaisons covalentes formées par les AGEs, a permis de prévenir et de limiter, respectivement, la calcification dans ce modèle. En outre, nous avons montré que la stimulation du récepteur des AGEs (RAGE), pouvait être impliquée dans l'élastocalcinose et élucidé certains éléments de signalisation dans ce processus. Ces travaux ont fait émerger le rôle des MMPs, de TGF-ß et des produits avancés de glycation dans le remodelage hypertrophique et l'élastocalcinose associée ou non au diabète. Ceux-ci sont reliés à l'apparition de l'hypertension systolo-diastolique et systolique isolée, respectivement. L'inhibition de ces acteurs pourrait constituer de nouvelles thérapies anti-hypertensives.

<p>Thyroid growth and function is partly regulated by growth factors binding to receptors on the cell surface. In the present thesis, the transforming growth factor-β (TGF-β) superfamily members have been studied for their role in regulation of growth and differentiation of both normal and neoplastic thyroid epithelial cells.</p><p>TGF-β1 is a negative regulator of thyrocyte growth and function. However, the importance of other TGF-β superfamily members has not been fully investigated. TGF-β1, activin A, bone morphogenetic protein (BMP)-7 and their receptors were found to be expressed in porcine thyrocytes. In addition to TGF-β1, activin A was also found to be a negative regulator of thyroid growth and function, and both stimulated phosphorylation and nuclear translocation of Smad proteins. Furthermore, TGF-β1 and epidermal growth factor (EGF) demonstrated a synergistic negative effect on thyrocyte differentiation. Simultaneous addition of the two factors resulted in a loss of the transepithelial resistance and expression of the epithelial marker E-cadherin. This was followed by a transient expression of N-cadherin.</p><p>Despite the extremely malignant character of anaplastic thyroid carcinoma (ATC) tumor cells, established cell lines are still responsive to TGF-β1. A majority of the cell lines were also found to be growth inhibited by BMP-7. BMP-7 induced cell cycle arrest of the ATC cell line HTh 74 in a dose- and cell density-dependent manner. This was associated with upregulation of p21^CIP1 and p27^KIP1, decreased cyclin-dependent kinase (Cdk) activity and hypophosphorylation of the retinoblastoma protein (pRb). TGF-β1, and to some extent also BMP-7, induced the expression of N-cadherin and matrix metalloproteinase (MMP)-2 and -9. Stimulation of HTh 74 cells with TGF-β1 increased the migration through a reconstituted basement membrane indicating an increased invasive phenotype of the cells.</p><p>Taken together, these data show that TGF-β superfamily members not only affect growth and function of normal thyroid follicle cells but may also, in combination with EGF, play a role in cell dedifferentiation. This study additionally suggests that the TGF-β superfamily members may be important for the invasive properties of ATC cells.</p>

<p>Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. </p><p>In order to study the BMP role for catecholaminergic neurons <i>in vivo</i>, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. </p><p>Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.</p><p>The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.</p><p>Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. </p><p>These results demonstrate a positive role of BMPs for MDN fate<i> in vivo</i>. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.</p>

<p>The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.</p><p>The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, <i>Id2</i> and <i>Id3</i>. Interestingly, we found that TGF-β-induced repression of <i>Ids</i> is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of <i>Ids</i> and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.</p><p>Furthermore, we identified a serine/threonine kinase, <i>SNF1LK</i>, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling. </p>

The goal of this study is to assess the osteogenic potential of two types of dental stem cell lines within a tissue engineering application. More specifically, the goal of this study is to find a readily abundant cell source with capacity to express an osteogenic phenotype. There are two parameters utilized to evaluate tissue engineering potential of cells: proliferation rate and differentiation potential. Briefly, proliferation rate is the speed at which cells divide and differentiation potential determines if cells are capable of committing towards specific lineages (e.g. osteogenic). These components are important, because if cells are not expanding at a specific rate and are not differentiating towards the lineage desired, the tissue engineered will not mirror the characteristics of native tissue. Therefore, both components are necessary for osteogenic tissue engineering applications. Several stem cell lines have been isolated from different sources (e.g. umbilical, bone marrow) and characterized for their proliferative capacity and their potency. Among these progenitor or stem cell lines, are those isolated from human dental tissue. Due to the similarities between teeth and bone, this specific cell line may be useful in osteogenic tissue engineering applications. In this study, stem cells extracted from human exfoliated deciduous teeth (SHEDs) and periodontal ligament stem cells (PERIOs), were evaluated and compared. Briefly, to evaluate the proliferation rate an ex-vivo expansion study was conducted. This experiment found that both SHEDs and PERIOs were proliferative lines with doubling times of 23 hours and 19 hours respectively. Subsequently, osteogenic differentiation of SHEDs and PERIOs was assessed utilizing a 3-D fibrin gel suspension treated with osteogenic media containing either dexamethasone (DEX) or Retinoic Acid (RA) for 28 days. At day 28, osteogenic markers for collagen 1 (Col1), osteocalcin (OCN), and alkaline phosphatase (ALP) were evaluated using qPCR. Results demonstrated both SHEDs and PERIOs exhibited significant (p<0.05) increases in osteogenic gene expression under the influences of DEX and RA. However the most significant increases were expressed by the SHEDs that received the DEX treatment. Additionally, the synergistic ability of TGF-beta 3 on the osteogenic differentiation of the stem cells was evaluated. Cells were cultured in a 3-D fibrin gel suspension and allowed to differentiate in DEX osteogenic media with and without the supplementation of TGF-beta 3 for 21 days. Using qPCR the cells were evaluated for expression of Col1, OCN, and ALP. In both the SHEDs and PERIOs, the samples treated with TGF-beta 3 the osteogenic gene expression increased in reference to the control, but had a hindering effect compared to cells treated in DEX without the TGF-beta 3. These results from this study suggested, SHED cells grown in 3-D fibrin gel suspension, may be better than PERIO cells for osteogenic tissue engineering applications when treated with DEX media without the supplementation of TGF-beta 3.

Thyroid growth and function is partly regulated by growth factors binding to receptors on the cell surface. In the present thesis, the transforming growth factor-β (TGF-β) superfamily members have been studied for their role in regulation of growth and differentiation of both normal and neoplastic thyroid epithelial cells. TGF-β1 is a negative regulator of thyrocyte growth and function. However, the importance of other TGF-β superfamily members has not been fully investigated. TGF-β1, activin A, bone morphogenetic protein (BMP)-7 and their receptors were found to be expressed in porcine thyrocytes. In addition to TGF-β1, activin A was also found to be a negative regulator of thyroid growth and function, and both stimulated phosphorylation and nuclear translocation of Smad proteins. Furthermore, TGF-β1 and epidermal growth factor (EGF) demonstrated a synergistic negative effect on thyrocyte differentiation. Simultaneous addition of the two factors resulted in a loss of the transepithelial resistance and expression of the epithelial marker E-cadherin. This was followed by a transient expression of N-cadherin. Despite the extremely malignant character of anaplastic thyroid carcinoma (ATC) tumor cells, established cell lines are still responsive to TGF-β1. A majority of the cell lines were also found to be growth inhibited by BMP-7. BMP-7 induced cell cycle arrest of the ATC cell line HTh 74 in a dose- and cell density-dependent manner. This was associated with upregulation of p21CIP1 and p27KIP1, decreased cyclin-dependent kinase (Cdk) activity and hypophosphorylation of the retinoblastoma protein (pRb). TGF-β1, and to some extent also BMP-7, induced the expression of N-cadherin and matrix metalloproteinase (MMP)-2 and -9. Stimulation of HTh 74 cells with TGF-β1 increased the migration through a reconstituted basement membrane indicating an increased invasive phenotype of the cells. Taken together, these data show that TGF-β superfamily members not only affect growth and function of normal thyroid follicle cells but may also, in combination with EGF, play a role in cell dedifferentiation. This study additionally suggests that the TGF-β superfamily members may be important for the invasive properties of ATC cells.

Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia. The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor. Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread. The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, Id2 and Id3. Interestingly, we found that TGF-β-induced repression of Ids is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of Ids and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways. Furthermore, we identified a serine/threonine kinase, SNF1LK, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling.