Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.

Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.

The signal transducer and activator of transcription-3 (Stat3) is a latent cytoplasmic protein that is activated through phosphorylation of tyrosine-705 by a number of receptor and non-receptor tyrosine kinases. This leads to Stat3 dimerization by reciprocal SH2-ptyr interactions, followed by translocation to the nucleus to initiate transcription of genes involved in cell growth, survival, and differentiation. Many of these signaling molecules known to activate Stat3 concentrate in specialized plasma membrane microdomains called caveolae, and are sequestered in an inactive state to the caveolin scaffolding domain (CSD) of the main caveolae resident protein, caveolin-1 (cav1). Since many of these signaling molecules are known, potent Stat3 stimulators, we set out to examine the effect of cav1 upon Stat3 activity. To this effect, cav1 was downregulated using a cholesterol chelator (methylcyclodextrin), or an antisense approach. Since we previously found that cell density can dramatically activate Stat3, all experiments were conducted at several densities. The results show that cav1 downregulation causes an increase in Stat3-tyr705 phosphorylation at all densities examined. We next examined the effect of cav1 upregulation upon Stat3 activity by transfecting an EGFP-cav1 construct. The results revealed that cav1 overexpression using this construct reduces Stat3 activity and induces apoptosis, which can be overcome by expression of a constitutively active form of Stat3. Finally, by expressing a Stat3 shRNA with an adenovirus vector, we demonstrated that Stat3 downregulation leads to an increase in cav1 levels. These results reveal the presence of a potent, negative regulatory relationship between cav1 and Stat3 phosphorylation. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2008-11-09 20:53:54.787

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 160 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

abstract: In the U.S., breast cancer (BC) incidences among African American (AA) and CA (CA) women are similar, yet AA women have a significantly higher mortality rate. In addition, AA women often present with tumors at a younger age, with a higher tumor grade/stage and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Even within the TNBC subtype, AA women have a worse clinical outcome compared to CA. Although multiple socio-economic and lifestyle factors may contribute to these observed health disparities, it is essential that the underlying biological differences between CA and AA TNBC are identified. In this study, gene expression profiling was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage TNBC. Initial analysis revealed a pattern of differential expression in the AA cohort compared to CA. Further molecular characterization results showed that the AA cohort segregated into 3-TNBC molecular subtypes; Basal-like (BL2), Immunomodulatory (IM) and Mesenchymal (M). Gene expression analyses resulted in 190 differentially expressed genes between the AA and CA cohorts. Pathway enrichment analysis demonstrated that differentially expressed genes were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC -cohort. Furthermore, genes in the Wnt/β-catenin pathway were over-expressed. These results were validated using RT-qPCR on an independent cohort of FFPE samples from AA and CA women with early stage TNBC, and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort. Furthermore, CAV1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, silencing of CAV1 expression by siRNA resulted in a significant decrease in proliferation in each of the TNBC cell lines. These observations suggest that CAV1 expression may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Abstract In the kidney, Wnt is involved in ureteric bud branching and nephrogenesis. Wnt mutation may lead to specific developmental dysfunctions and diseases. As part of this thesis, I show that Wnt11 is expressed in the renal tubules, except for the ureteric epitheliums, and I examine the function of Wnt11 in renal tubule organization using the new C57Bl6 Wnt11-/- mouse model. Convoluted and dilated tubules were observed in the Wnt11 mutated kidneys that may cause glomerular cysts and kidney dysfunction. More specifically, a lack of Wnt11 in the kidney reduced Six2, Hoxd1, and Hox10 expression, which may have contributed to the anomalies in the kidney tubular system. Embryogenesis and carcinogenesis share molecular characteristics. Gene expression changes take place during development to meet the demands of the tissue formation, but ectopic expression of embryonic genes by deletion, SNPs, or epigenetic modification in adult may lead to cancer. The research carried out as part of this thesis identified genes that were differentially expressed in both induced metanephric mesenchymes (MM) and human ccRCC. Gene silencing of Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr, and Itgb2 mediated by siRNA inhibited the migration, viability and invasion capacity of Renca cells (RCC). Furthermore, by using the novel 3D in vitro MM-Renca co-culture setup, the result revealed that downregulation of Bnip3, Cav1 or Gsn in Renca cells partly rescued the RCC-mediated inhibition epithelial tubules formation during nephrogenesis. Altogether, the data demonstrates that renal ontogenesis control genes play a role both in normal kidney development and in kidney cancer. The 3D RCC-MM chimera organoids developed as part of the research may also serve as a novel model for kidney cancer study. / Tiivistelmä Wnt-viestit ohjaavat munuaisen kehityksen yhteydessä sekä virtsanjohtimen että munuaiskeräsen kasvua. Virhe Wnt-proteiinia tuottavassa geenissä johtaa puolestaan vakavaan kehityshäiriöön ja syöpään, jos geeni aktivoituu aikuisvaiheessa. Tässä väitöskirjassa osoitetaan, että Wnt11-geeni osallistuu alkion munuaisen epiteeliputkiston kehityksen säätelyyn. Wnt11-signaalin tehtävää tutkittiin munuaisen putkiston rakenteen synnyssä poistogeenisen C57Bl6-hiirimallin avulla. Wnt11-puutos häiritsi virtsan kokoojatiehyiden rakenteiden kehitystä. Tämä on mahdollinen syy siihen, että Wnt11-poistogeenisen hiiren munuaiseen kehittyy hiusverisuonikerästen laajentumia. Munuaisen toiminta on myös heikentynyt verrokkiin verrattuna. Munuaisen epiteeliputkien kehitykseen osallistuvien geenien Six1, Hoxd1 ja Hox10 aktiivisuus oli niin ikään heikentynyt Wnt11-signaloinnin puutoksen vuoksi. Nämä seikat voivat olla puutoksen aiheuttamien kudosrakenteellisten muutosten taustalla. Solun kasvusäätely on keskeinen tekijä sekä alkion että syövän kehityksessä. Prosessien taustalla ovat myös samankaltaiset molekyylit. Wnt-solusignaalit esimerkiksi säätelevät normaalin kehityksen aikana solujen itsesäätelyä, solujen jakautumista, solujen vaeltamista ja solujen invaasiota kudoksiin ja osallistuvat syövän syntyyn niiden viestinnän häiriintyessä. Kehitystä säätelevien geenien tuotteet ohjaavat solujen jakautumista, mikä on edellytys kudosrakenteen synnylle. Jos nämä geenit syystä tai toisesta aktivoituvat uudelleen aikuisvaiheen aikana, se voi johtaa syöpäkasvaimen syntyyn. Tässä työssä verrattiin keskenään munuaisen elinkehitykseen ja ihmisen syövän kasvuun osallistuvia geenejä. siRNA-välitteinen hiljennys geeneille Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr ja Itgb2 inhiboi Renca-solujen migraatiota, elinkykyä ja invaasiota. Lisäksi tulokset paljastivat, että käytettäessä uutta 3D in vitro MM Renca -kasvatusmenetelmää Bnip3-, Cav1- tai Gsn-geenien hiljennys Renca-soluissa osittain pelastaa RCC-välitteisen inhibition epiteeliputkissa nefrogeneesin aikana. Kaiken kaikkiaan työssä seulottiin uusin tavoin sekä ihmisen munuaissyöpään liittyviä geenejä että kehitettiin lisäksi uusia niin kutsuttuja kokeellisia funktionaalisia organoidimenetelmiä. Työn tulokset tarjoavat uuden strategian, jolla geenien osuutta munuaissyöpään voidaan tutkia seikkaperäisesti yhdessä 3D-kasvatusmenetelmien kanssa

Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases. / Medicine, Faculty of / Graduate

No description available.

Die strahleninduzierte orale Mukositis (OM) gilt als limitierender Faktor in der Therapie von Kopf-Hals-Tumoren (KHT). Jährlich erkranken an KHT weltweit zwischen 400.000 und 600.000 Menschen sowie eine Vielzahl an Hunden und Katzen. Die fraktionierte Radiotherapie wird allein oder in Kombination mit anderen Therapieformen zur Behandlung von KHT eingesetzt. Neben dem Tumorgewebe werden immer auch Normalgewebsanteile der Strahlung ausgesetzt. Dabei besteht ein gewisses, akzeptiertes Risiko für unerwünschte Wirkungen. Die fast bei allen Bestrahlungspatienten auftretende OM stellt die wichtigste frühe Nebenwirkung dar. Zahlreiche Untersuchungen zu Therapie- und Prophylaxemöglichkeiten blieben bisher ohne eindeutigen Konsens bezüglich Behandlung beziehungsweise Prävention der OM. Pentoxifyllin (Ptx), Dermatansulfat (Ds) und Thalidomid (Th) haben in verschiedenen präklinischen Studien eine verbessernde Wirkung auf die strahleninduzierte OM gezeigt. In der vorliegenden Arbeit soll die Caveolin-1 (Cav-1)-Expression und deren Modifizierung durch die drei oben genannten Wirkstoffe untersucht werden, um einen möglichen Zusammenhang zwischen Cav-1, OM und Mukoprotektivität herstellen zu können.

No description available.