No description available.

Tissues arising late in evolutionary time, such as lung alveoli that are unique to air breathing organisms, have been challenging to generate in vitro from pluripotent stem cells (PSCs), in part because there are limited lower organism model systems available to provide the necessary developmental roadmaps to guide in vitro differentiation. Furthermore, pulmonary alveolar epithelial type II cell (AEC2) dysfunction has been implicated as a primary cause of pathogenesis in many poorly understood lung diseases that lack effective therapies, including interstitial lung disease (ILD) and emphysema. Here we report the successful directed differentiation in vitro of human PSCs into AEC2s, the facultative progenitors of lung alveoli. Using gene editing to engineer multicolored fluorescent reporter PSC lines (NKX2-1GFP;SFTPCtdTomato), we track and purify human SFTPC+ alveolar progenitors as they emerge from NKX2-1+ endodermal developmental precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells are able to form monolayered epithelial spheres (“alveolospheres”) in 3D cultures without the need for mesenchymal co-culture support, exhibit extensive self-renewal capacity, and display additional canonical AEC2 functional capacities, including innate immune responsiveness, the production of lamellar bodies able to package surfactant, and the ability to undergo squamous cell differentiation while upregulating type 1 alveolar cell markers. Guided by time-series global transcriptomic profiling we find that AEC2 maturation involves downregulation of Wnt signaling activity, and the highest differentially expressed transcripts in the resulting SFTPC+ cells encode genes associated with lamellar body and surfactant biogenesis. Finally, we apply this novel model system to generate patient-specific AEC2s from induced PSCs (iPSCs) carrying homozygous surfactant mutations (SFTPB121ins2), and we employ footprint-free CRISPR-based gene editing to observe that correction of this genetic lesion restores surfactant processing in the cells responsible for their disease. Thus we provide an approach for disease modeling and future functional regeneration of a cell type unique to air-breathing organisms.

No description available.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ronette Gehring / Milk composition has a dynamic nature, and the composition varies with stage of lactation, age, breed, nutrition and health status of the udder. The changes in milk composition seem to match the changes in the expression of membrane proteins in secretory mammary epithelial cells that are needed for the movement of molecules from blood to milk and vice versa (Nouws and Ziv, 1982). Thus, an understanding of transporter expression, function and regulation in mammary epithelial cells can provide insight into mammary gland function and regulation. The goal of this project was to elucidate (molecularly and functionally) the role of drug transporters in the barrier function of an epithelial monolayer cultured from an immortalized bovine mammary epithelial cell line (BME-UV). To characterize the regulation (expression and function) of these drug transporters in BME-UV cells after exposure to cytokine TNF-α for selected periods of time. Representative members of drug transporters of the SLC (OCT and OAT) and ABC (P-glycoprotein) superfamilies were chosen for this project. In the first study, the involvement of a carrier-mediated transport system in the passage of organic cation (TEA) and anion (EsS) compounds was elucidated across the BME-UV monolayer. In the second study, molecular and functional expression of bOAT isoforms in BME-UV cells were studied. The final study characterized the effects of cytokine TNF-α on the expression and function of P-glycoprotein, an efflux pump, in BME-UV cells. Cytokine TNF-α exposure induced the expression of ABCB1 mRNA and increased P-glycoprotein production in BME-UV cells, resulting in a greater efflux of digoxin, a known P-glycoprotein substrate, back into the apical fluid. The expression, function, and regulation of these transporters in the mammary gland has important implications for understanding the barrier function of the mammary epithelium and, in more specific, for characterizing the role of these transporters in the accumulation and/or removal of specific substrates from milk and/or plasma. Moreover, this study provides an in vitro cell culture model of mammary epithelium to characterize mammary epithelial cell function during inflammation.

Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell-specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold

The current study investigated the distribution of periodontal epithelial cells and nerve fibres within the furcations of rat maxillary molar teeth subjected to hypothermic injury. The upper right first molars of 30 Sprague-Dawley rats were subjected to a single 20 minute application of dry ice in order to produce aseptic necrosis within the periodontal ligament, while the contralateral first molar served as an untreated control. Five animals were each sacrificed via cardiac perfusion after 7, 10, 14, 18, 21 and 28 days respectively and the maxillae were dissected out. After fixation in paraformaldehyde and processing, the tissues were embedded in paraffin wax and cut into 7µm serial coronal sections through the furcation region. Consecutive sections were then stained with H&E, cytokeratin AE1/AE3 and PGP 9.5 immunostains. Light microscopic examination of the H&E stained sections revealed that ankylosis had not developed in all of the experimental teeth, and in some of the observation groups fewer teeth were ankylosed than unaffected. The morphology of the ankylotic areas appeared to change with time, initially consisting of fine bony trabeculae, then progressing to solid bone occupying the entire furcation before becoming less solid again by the latest observation periods. Root resorption was often seen adjacent to areas of ankylosis, but the cementum of the tooth root at the point of ankylotic union was usually intact and free of resorption. Changes within the pulp chambers of the experimental teeth were also noted, with reduction in cellularity and tissue disorganisation initially, then increasing cellularity and formation of a cementum-like material on the chamber walls later. Cytokeratin AE1/AE3 immunostaining successfully identified epithelial cells within the periodontal ligament and their distribution around control teeth was similar to previous reports. Counting of these cells revealed lower numbers around experimental teeth, with the lowest counts around experimental teeth which had developed ankylosis. No change in the epithelial cell counts was detected over time, and these cells did not appear to regenerate after necrosis regardless of whether or not ankylosis developed. Statistical analysis indicated that the probability of ankylosis decreased as the number of epithelial cells increased. The PGP 9.5 immunostain identified periodontal nerve fibres, but the use of this stain was quite technique sensitive. The furcations of the molar teeth were noted to have relatively sparse innervation, with most of the visible nerve fibres being closely associated with blood vessels and located in the outer two-thirds of the ligament. Counting of the nerve fibres revealed fewer fibres around experimental teeth compared to control teeth, especially in the part of the ligament closest to the tooth root. There was no relationship detected between nerve count and time or between nerve and epithelial cell counts. Resorption was found to be more prevalent in experimental teeth, and the probability of resorption in a given tooth decreased as the epithelial cell count increased. The findings of this study suggest that the epithelial cells within the periodontal ligament have a protective function in the prevention of dentoalveolar ankylosis and resorption. Evidence of an intimate interrelationship between periodontal nerve fibre and epithelial cell numbers could not be confirmed. The null hypothesis that epithelial cell rests of Malassez do not provide a protective function against ankylosis and external root resorption was rejected, and the null hypothesis that nerve fibres and epithelial cells are not inter-dependent was retained. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297409 / Thesis (D.Clin.Dent.) -- School of Dentistry, 2007

<p>RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. </p><p>We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The <i>Caenorhabditis elegans</i> homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the <i>par</i> (for partitioning defective) gene family, first identified in <i>C.elegans</i>. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.</p><p>Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. </p><p>To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. </p><p>In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.</p>

RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex. Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.

The current study investigated the distribution of periodontal epithelial cells and nerve fibres within the furcations of rat maxillary molar teeth subjected to hypothermic injury. The upper right first molars of 30 Sprague-Dawley rats were subjected to a single 20 minute application of dry ice in order to produce aseptic necrosis within the periodontal ligament, while the contralateral first molar served as an untreated control. Five animals were each sacrificed via cardiac perfusion after 7, 10, 14, 18, 21 and 28 days respectively and the maxillae were dissected out. After fixation in paraformaldehyde and processing, the tissues were embedded in paraffin wax and cut into 7µm serial coronal sections through the furcation region. Consecutive sections were then stained with H&E, cytokeratin AE1/AE3 and PGP 9.5 immunostains. Light microscopic examination of the H&E stained sections revealed that ankylosis had not developed in all of the experimental teeth, and in some of the observation groups fewer teeth were ankylosed than unaffected. The morphology of the ankylotic areas appeared to change with time, initially consisting of fine bony trabeculae, then progressing to solid bone occupying the entire furcation before becoming less solid again by the latest observation periods. Root resorption was often seen adjacent to areas of ankylosis, but the cementum of the tooth root at the point of ankylotic union was usually intact and free of resorption. Changes within the pulp chambers of the experimental teeth were also noted, with reduction in cellularity and tissue disorganisation initially, then increasing cellularity and formation of a cementum-like material on the chamber walls later. Cytokeratin AE1/AE3 immunostaining successfully identified epithelial cells within the periodontal ligament and their distribution around control teeth was similar to previous reports. Counting of these cells revealed lower numbers around experimental teeth, with the lowest counts around experimental teeth which had developed ankylosis. No change in the epithelial cell counts was detected over time, and these cells did not appear to regenerate after necrosis regardless of whether or not ankylosis developed. Statistical analysis indicated that the probability of ankylosis decreased as the number of epithelial cells increased. The PGP 9.5 immunostain identified periodontal nerve fibres, but the use of this stain was quite technique sensitive. The furcations of the molar teeth were noted to have relatively sparse innervation, with most of the visible nerve fibres being closely associated with blood vessels and located in the outer two-thirds of the ligament. Counting of the nerve fibres revealed fewer fibres around experimental teeth compared to control teeth, especially in the part of the ligament closest to the tooth root. There was no relationship detected between nerve count and time or between nerve and epithelial cell counts. Resorption was found to be more prevalent in experimental teeth, and the probability of resorption in a given tooth decreased as the epithelial cell count increased. The findings of this study suggest that the epithelial cells within the periodontal ligament have a protective function in the prevention of dentoalveolar ankylosis and resorption. Evidence of an intimate interrelationship between periodontal nerve fibre and epithelial cell numbers could not be confirmed. The null hypothesis that epithelial cell rests of Malassez do not provide a protective function against ankylosis and external root resorption was rejected, and the null hypothesis that nerve fibres and epithelial cells are not inter-dependent was retained. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297409 / Thesis (D.Clin.Dent.) -- School of Dentistry, 2007

Dans les tumeurs, les cellules cancéreuses invasives induisent l’expression de la métalloprotéase matricielle 11 (MMP-11) dans les adipocytes adjacents (cancer-associated adipocytes) entrainant leur « dédifférenciation » en fibroblastes, la MMP-11 régulant négativement l’adipogénèse. Au cours de ma thèse, j’ai étudié la fonction de la MMP-11 sur le développement mammaire postnatal. La structure de la glande ainsi que la production de lait sont altérées dans les souris déficientes pour la MMP-11. De plus, la MMP-11 régule l’homéostase du collagène. In vivo, des transplantations montrent une fonction locale paracrine de la MMP-11 essentielle à la morphogenèse mammaire. In vitro, la MMP-11 adipocytaire favorise le branchement d’organoïdes primaires. Ainsi, la MMP-11 est un facteur paracrine majeur pour le développement de la glande mammaire. Enfin, pour poursuivre ces travaux, j’ai établi des souris transgéniques conditionnelles ciblant l’expression de la MMP-11 dans les adipocytes. / In tumors, invasive cancer cells induce matrix metalloproteinase 11 (MMP-11) expression in adjacent adipocytes (cancer-associated adipocytes), leading to their « dedifferentiation » into fibroblast-like cells. Indeed, MMP-11 is a negative regulator of adipogenesis. During my PhD, I have investigated MMP-11 function during postnatal mammary gland development. The ductal tree and alveolar structures, and milk production are reduced in MMP-11-deficient mice. Moreover, MMP-11 plays a role in collagen homeostasis. Transplantation experiments show that MMP-11 exerts an essential local paracrine function for ductal morphogenesis. Using primary mammary organoids, I show that adipocyte-related MMP-11 is a pro-branching factor in vitro. Thus, MMP-11 is a master paracrine factor during mammary gland development. Finally, to further investigate the adipocyte-related MMP-11 function, I have developed conditional transgenic mice targeting MMP-11 expression specifically into adipocytes.