Dissertations / Theses on the topic 'CCNE2'

Biomedical Sciences
Ph.D.
The mammalian palate develops early in embryogenesis by way of a carefully orchestrated series of temporally and spatially regulated signaling events. Molecular signaling pathways that have been proven to be vital to the process of palatogenesis include TGF-βs, BMPs, FGFs, EGF, and Wnts. The absence of connective tissue growth factor (CTGF/CCN2) has been shown previously to cause failure of proper palatogenesis, i.e. cleft palate. However, the details about the phenotype of this model of cleft palate were scarce. Additionally, CCN2 is known to interact with TGF-βs, BMPs, FGFs, EGF, and Wnts, though information on how these pathways were impacted in the developing palate lacking CCN2 were also not available. In Chapters 2 and 3, through our use of gross specimen and histological examination combined with cell and organ culture, we produced the most detailed characterization of the CCN2 knockout (KO) model of cleft palate with identification of negatively affected signaling pathways that lead to the clefting phenotype. Collection and examination of gross and histological sections revealed at 100% penetrance of cleft palate in which development is impaired around the phase of palatal shelf elevation. Organ culture also revealed that when artificially apposed, the CCN2 KO model system also suffers from a fusion deficit. Finally, utilizing cells isolated from the developing palates, we found a reduction in proliferation, adhesion, and spreading with an enhanced migratory ability. Addition of recombinant CCN2 was able to rescue cell spreading but not proliferation. CCN2 as an immobilized substrate did not rescue adhesive ability. Decreased adhesion and spreading in the KO cells are attributed to the inability of the KO cells to activate Rac1 and RhoA. Examination of gene expression differences by mRNA-sequencing and qRT-PCR revealed numerous gene expression alterations between the wild type (WT) and the KO palates, most notably FGF4 and EGFR. Addition of FGF4 or EGF to cell culture was unable to promote increased proliferation in the KO cells while producing a response in the WT cells. Examination of downstream signaling revealed highly amplified and prolonged ERK1/2 signaling in the FGF4 treated palate cells indicating that FGF signaling is significantly altered in the absence of CCN2. Treatment of the cells with EGF produced a response proportional to EGFR expression differences indicating that EGFR signaling is not impacted beyond the receptor protein levels. The link between EGFR protein levels and FGF mediated ERK1/2 activation is a protein called Spry2. We found greatly reduced Spry2 mRNA levels in the KO palates and upon FGF4 stimulation at 24 hours of exposure indicating that in the absence of CCN2, proper inhibition of FGF signaling and EGFR degradation is negatively altered. Collectively, the data demonstrate that CCN2 is vital to palatogenesis by impacting proliferation, shelf elevation, and shelf fusion through increased FGF signaling and reduced EGFR signaling resulting partially from reduced Spry2 activity.
Temple University--Theses

Cutaneous wound healing is a complex process involving the migration of inflammatory cells to the wound site, deposition of extracellular matrix, and the reestablishment of an intact epithelial barrier. Re-epithelialization depends on the proliferation and directional migration of keratinocytes from the wound edges. Initially, keratinocytes migrate over a provisional wound matrix that is rich in fibronectin, and as the wound heals the provisional matrix becomes replaced by one consisting of collagen and proteoglycans. Re-epithelialization is tightly regulated by a variety of peptides such as growth factors, cytokines and proteases, and abnormalities may result in chronic non-healing wounds or hypertrophic scars. CCN2 (Connective Tissue Growth Factor) is a multifunctional protein with effects on cells and their interactions with the connective tissue. CCN2 is expressed in a variety of cell types and regulates numerous cell functions including proliferation, differentiation, adhesion, migration and stimulation of collagen production. While the importance of CCN2 for the fibrotic response has been well studied, its involvement in keratinocyte function has not yet been fully explored. Using an in vivo wound model, the expression of CCN2 was captured at the leading keratinocyte edge during re-epithelialization. In vitro, exogenous addition of CCN2 to human keratinocyte cultures promoted keratinocyte migration. Subsequently, integrin a5b1 was identified as an important mediator of CCN2 enhancement of keratinocyte adhesion to fibronectin. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1 specific inhibitor PD98059 markedly reduced CCN2-promoted keratinocyte migration. In vitro, CCN2 expression was induced by TGF-β1. Compared with inhibiting the SMAD pathway, blocking MAPK was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. In addition, CCN2-induced keratinocyte spreading required FAK. Treatment with CCN2 led to actin disassembly and altered the activity of the Rho proteins and p190RhoGAP in keratinocytes. Furthermore, Cdc42 mediated CCN2-induced cell polarity. In conclusion, using in vivo and in vitro models, CCN2 was shown to regulate keratinocyte function by promoting keratinocyte adhesion, spreading and migration. A complete understanding of CCN2 expression in keratinocytes is crucial in order to develop novel therapies for wound healing.

The pericellular matrix (PCM) is a distinct zone of matrix that surrounds individual chondrocytes throughout articular cartilage. Although not much is known about its function, our group has shown that it has a role in mechanotransduction by controlling the bioavailability of perlecan-bound FGF-2. The aim of this project was to determine the other structural components of the PCM, and to search for other heparin binding growth factors which might be sequestered in the matrix. By confocal microscopy I confirmed that the PCM was rich in type VI collagen and perlecan, although devoid of type II collagen and aggrecan. Isolation of individual chondrons (the chondrocyte together with its PCM) was performed by partial digestion of the matrix using dispase and collagenase, and proteomic analysis using mass spectrometry was performed to identify new proteins. As this method was only partially successful I looked for the presence of a known heparin binding growth factor, connective tissue growth factor (CCN2), in chondron preparations. CCN2, which is an abundant secreted protein of articular cartilage, was present in both the chondron preparation by western blot, and was visualised in the PCM of porcine and human articular cartilage by confocal microscopy. CCN2 is not commercially available so His-tagged recombinant protein was stably expressed and purified using nickel affinity chromatography. Biological activity of the purified protein was investigated in a number of established assays. No biological activity was demonstrated when purified CCN2 was used alone on murine mesenchymal stem cells, but was evident when assayed in combination with low dose TGF-β. The effect of exogenous CCN2 on fibroblasts was limited by the significant release of endogenous CCN2. High constitutive expression of CCN2 in articular cartilage may limit the effects seen by exogenous CCN2. However, the results presented in this thesis support the role of CCN2 as a modulator of TGF beta signalling, and suggest that, through potentiating TGF beta it may regulate matrix turnover in articular cartilage.

CCN protein family members (CYR61, CTGF, Nov, Wisp-1, Wisp-2 and Wisp-3) have important roles in many different processes including angiogenesis, inflammation, remodelling of extracellular matrix and tumorigenesis. In bone, CCN1 increases osteoblastogenesis via Wnt3A signalling and activation of -catenin which, in turn, upregulates CCN1 expression. The exact role of CCN1, CCN2, and CCN6 in osteoclast physiology are not known but we have recently shown that recombinant human (rh)CCN1 inhibits osteoclastogenesis in vitro. The aim of this study was to determine: 1) the expressions of all six members of the CCN protein family in osteoblasts and osteoclasts; 2) the functions of recombinant human CCN2, CCN6 in osteoclastogenesis; 3) whether osteoblast-derived CCN1 may mediate the effect of CCN1 on osteoclast formation and the roles of osteoblast-derived CCN1 and/or osteoclast-derived CCN1 in osteoblast and/or osteoclast differentiation; 4) which signalling pathways are involved in the function of CCN1 in osteoclasts and osteoblasts. We found CCN1-5 but not CCN6 expressed in murine osteoclasts and osteoblasts. All six members were expressed in human OA osteoblasts but only CCN1-3 were detected in human osteoclasts using quantitative RT-PCR. rhCCN1 (in agreement with our previous observations), and also 2 and 6 inhibited human and mouse osteoclast formation in a concentration-dependent manner. We generated and validated an expression construct to specifically overexpress CCN1 in osteoblasts. Incorporation of CCN1-specific siRNA reduced CCN1 expression to between 12.5% and 50% of control osteoblast cultures. In both co-cultures with direct contact between osteoblasts and osteoclast co-cultures as well as Transwell cultures, overexpression of CCN1 in osteoblasts decreased the formation of TRAP positive multinucleated osteoclast-like cells, while siRNA mediated knockdown of CCN1 in the osteoblasts resulted in increased osteoclast-like cell formation. These data suggest that osteoblast-derived CCN1 is a secreted negative regulator of osteoclastogenesis. Moreover, overexpression or knockdown of CCN1 in osteoclast precursors inhibited or increased osteoclast differentiation whilst overexpression or knockdown CCN1 in osteoblasts increased or inhibited osteoblast mineralization respectively. Further investigation found that CCN1 increased Wnt and MAPK signalling in osteoblasts cultured in mineralization medium and inhibited Wnt and IGF-1 signalling during osteoclast differentiation. In conclusion, paracrine and autocrine effects of CCN1 have been demonstrated in osteoclasts and osteoblasts in this study and Wnt, MAPK, amd IGF-1 signalling pathways, may be involved in these effects.

Systemic sclerosis (Scleroderma, SSc) is a chronic, connective tissue disease of unknown etiology, characterised by vascular dysfunction, iriflarnmation and organ fibrosis. Involving both genetic and environmental components, the specific mechanisms which result in fibrosis remain largely unknown. A cardinal feature of SSc is increased synthesis of extracellular matrix (ECM). Dermal fibroblasts cultured from SSc patients maintain many of the abnormal properties seen in vivo, including excess production of collagen type I, and growth factors such as connective tissue growth factor (CTGF/CCN2). CTGF, like many genes dysregulated in SSc, is induced by TGF- p in normal fibroblasts. The overall aim of my studies was to determine the mechanism(s) controlling CTGF over-expression in SSc dermal fibroblasts (SDF). Induction of CTGF by TGF-p was found to be dependent upon elements in the proximal portion of the CTGF promoter, distinct from those of the previously characterised TGF- P response element (TRE). The TRE acts, in NIH/3T3 and HFF cells, as a regulator of basal expression, and is not essential for TGF-P induction of CTGF. Instead TGF-p induces CTGF expression via a Smad3 complex, binding to a bona fide SMAD transcription factor binding site. Over-expression CTGF in SDF is independent of autocrine expression of TGF-P and the SMAD binding element and rather dependent on a functional Sp-binding site. Inhibition of Spl-like DNA binding reduces excessive CTGF expression in SDF. Consistent with this Spl-DNA binding activity is elevated in SDF nuclear extracts. Investigation of the mechanism of elevated Spl-like binding found that SDF exhibited constitunVely active ERK1/2 and JNK1. Inhibition of ERK1/2 repressed elevated Sp-binding and CTGF over-expression observed in SDF. In summary, the data presented in this thesis provide evidence that dysregulation of ERK1/2 in SDF is involved in CTGF over-expression via a Spl-like DNA binding. Thus repression of ERK may represent a candidate in targeting fibrosis in SSc.

Cell Biology
Ph.D.
Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF to study the effect of different levels of endogenous CTGF on osteoblast cytoskeleton reorganization and motility. Our assays showed enhanced cell adhesion, spreading and Rac expression in CTGF OE osteoblasts, while in CTGF KO osteoblasts, cell adhesion, spreading and Rac expression were significantly decreased. In contrast, CTGF OE osteoblasts that showed high adhesion and spreading, exhibited diminished cell motility and low levels of RhoA expression, while KO cells migrated quickly and expressed high levels of RhoA. Together, these experiments establish CTGF as an adhesion protein for osteoblasts; they demonstrate that the alpha-v beta1 integrin is a functional signaling receptor for CTGF; they confirm that osteoblast differentiation is enhanced when cultured on CTGF matrix through activation of regulatory signaling molecules; and finally, these experiments establish a role for CTGF in the regulation of small RhoGTPases expression, which in turn implies a significant role for CTGF in cell cytoskeleton reorganization and motility.
Temple University--Theses

Les cellules souches embryonnaires de souris (mESC) présentent un cycle cellulaire atypique caractérisé par l'absence d'une voie Rb fonctionnelle et la forte expression de la cycline E pendant toutes les phases du cycle cellulaire. En conséquence, les mESC sont constitutivement amorcées pour la réplication de l'ADN. Pour comprendre comment la cycline E, un régulateur clé de la transition de la phase G1 à S, est régulée dans les mESC, nous avons analysé la régulation transcriptionnelle de son gène Ccne1 par des facteurs de transcription du réseau de pluripotence naïve. Nous avons observé que les facteurs Esrrb, Klf4 et Tfcp2l1 se lient à la région du promoteur de Ccne1 sur plusieurs sites situés entre 0 et 1kb en amont du site d'initiation de la transcription. Un test luciférase nous a permis de monter qu'une mutation de ces sites de liaison diminue ou abolie l'activité transcriptionnelle du promoteur. De plus, la surexpression inductible à la doxycycline des facteurs Esrrb, Klf4 et Tfcp2l1 augment le niveau d'expression d'ARNm de Ccne1. Ces résultats suggèrent que Esrrb, Klf4 et Tfcp2l1 contrôlent l'expression de la cycline E. Ils mettent en évidence un lien direct entre le réseau de pluripotence naïve et la régulation du cycle mitotique dans les mESC. Nous avons utilisé le système rapporteur FUCCI pour étudier en fonction du cycle cellulaire l'expression des facteurs de transcription qui forment le réseau de pluripotence naïve. Nous avons observé que l'expression de Esrrb, Klf4, Tfcp2l1 et Nanog oscille au cours du cycle cellulaire avec une diminution de l'expression entre la phase G1 précoce et le début de S, puis une ré-augmentation entre le début de S et la phase G2/M. Ces résultats suggèrent que le réseau de pluripotence naïve est déstabilisé transitoirement lors du passage de la phase G1 à la phase S du cycle cellulaire
Mouse embryonic stem cells (mESCs) display an unorthodox cell cycle characterised by the lack of a functional Rb pathway and robust expression of cyclin E during all cell cycle phases. Therefore, mESCs are constitutively primed for DNA replication. To understand how cyclin E, a key regulator of the G1-to-S phase transition, is regulated in mESCs, we analysed the transcriptional regulation of Ccne1 by transcription factors of the naive pluripotency network. We observed that Esrrb, Klf4 and Tfcp2l1 bound the Ccne1 promoter region on multiple sites between 0 and 1kb upstream transcription start site. Disrupting the binding sites reduced or abolished transcriptional activity in a luciferase assay. Moreover, the doxycyclin-inducible expression of Essrb, Klf4 and Tfcp2l1 up-regulated the Ccne1 mRNA level. Taken together, these results strongly suggest that Essrb, Klf4 and Tfcp2l1 control Cyclin E expression and highlight a direct connection between the naïve pluripotency network and regulation of the mitotic cycle in mESCs. We used the FUCCI reporter system to study cell-cycle dependent expression of the transcription factors that form the naïve pluripotency network. Esrrb, Klf4, Tfcp2l1 and Nanog expression oscillated during the cell cycle with a down-regulated expression between the early G1-phase and the beginning of S-phase, and then up-regulated expression between the beginning of S-phase and the G2/M-phase. These results suggest that the naive pluripotency network is destabilized transiently during the transition from the G1-phase to the S-phase of the cell cycle

Cell Biology
Ph.D.
Connective tissue growth factor (CTGF/CCN2) is axiomatically necessary for proper skeletal development and function. We need not look further than the studies that have been done to date utilizing mice genetically engineered to lack CTGF production. These CTGF null or knockout (KO) mice fail to form a normal murine skeleton and instead yield one littered with bony dysmorphisms, including incompetent craniofacial development, kinked limb bones, and misshapen ribs that are not conducive to proper respiratory function. As a result, the global lack of CTGF is incompatible with postnatal life. A closer look at several sites demonstrated defects in physiologic processes necessary for bone formation - angiogenesis, chondrogenesis, and osteogenesis. Therefore, the dogma in the CCN protein field to date has been that systemic ablation of CTGF production in vivo results in global defects in bone development. We believe this dogma is an oversimplification of the role of CTGF on skeletal development. Our initial impetus leading us to this belief was the gross identification of the specific skeletal sites malformed in CTGF KO mice, in particular the bones of the limbs. While in the lower limb of CTGF KO mice the tibiae and fibulae are misshapen, the adjacent femora and digits are phenotypically normal. The same is true for the upper limb, in which the radii and ulnae are phenotypically abnormal while the humeri and digits are normal. Therefore, we believe that the role of CTGF in skeletogenesis is site-specific such that its loss affects local skeletal patterning and/or mechanobiological cues resulting in the unique phenotype seen in CTGF KO mice. The research of this dissertation constitutes a comprehensive skeletal analysis of CTGF KO mice and in so doing we determined the extent and location of skeletal abnormalities. We found skeletal site-specific changes in growth plate organization, bone microarchitecture and shape and gene expression levels in CTGF KO compared to wild-type (WT) mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and non-allometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. We further continued the skeletal characterization of CTGF KO bones with an analysis of bone cell ultrastructure and matrix composition. These studies demonstrated that, while CTGF is not necessary for complete morphologic maturation of bone cells, global ablation results in ultrastructural features not commonly seen in WT bones. Our findings include drastically dilated rough endoplasmic reticulum (RER) in osteoblasts of the tibial diaphyseal region, comprising the phenotypic kink in CTGF KO mice and ultrastructural dysmorphologies of CTGF KO osteoclasts including multi-layered, membranous inclusions, decreased vacuolization and ruffled border extents, and disproportionately large clear zones. Lastly, FT-IR analysis demonstrated heterogeneity in CTGF KO bone composition. The results of this dissertation have revealed a more complex role for CTGF in osteogenesis and have identified potential mechanisms and future research directions to fully understand this intricate story.
Temple University--Theses

BMI1 est une protéine appartenant à la famille des polycombs impliquée dans la régulation épigénétique de la transcription. Il a été montré que cette protéine est essentielle à la régulation de la prolifération, de la sénescence et du métabolisme ainsi qu’à l’auto-Renouvellement des cellules souches hématopoïétiques et cancéreuses. Ce répresseur transcriptionnel au fort potentiel oncogénique est retrouvé surexprimé dans de nombreux types de cancer ; dans le cas de la Leucémie Myéloïde Chronique (LMC) le niveau d’expression de BMI1 augmente avec l’aggravation de la pathologie. Cependant, les voies de signalisation impliquées dans sa surexpression et le rôle qu’il joue au sein de cette maladie demeurent méconnus. En réprimant l’expression de BMI1 par ARN interférence nous avons pu mettre en évidence que ce polycomb était essentiel à la prolifération cellulaire ainsi qu’au potentiel clonogénique des cellules de LMC. Nous avons également démontré pour la première fois que BMI1 soutenait la croissance tumorale à travers la répression d’un processus autophagique délétère pour la cellule cancéreuse. Une approche transcriptomique nous a permis d’identifier la cible transcriptionnelle impliquée dans ce processus, la Cycline G2. Nous avons, pour finir, trouvé une molécule, via une approche bioinformatique, capable de réinduire l’expression de la Cycline G2 dans les cellules de LMC, l’alexidine dihydrochloride. Cette molécule induit une forte autophagie dans les cellules cancéreuses ainsi que de l’apoptose. Elle s’est également montrée capable de resensibiliser à l’imatinib (un inhibiteur de BCR-ABL) une lignée pourtant résistante
The polycomb protein Bmi1 is a major epigenetic regulator. It has been shown that this protein is essential for the regulation of cell proliferation, senescence and metabolism but also self-Renewal of hematopoïetic and cancer stem cells. This transcriptional repressor, with a strong oncogenic potential, is overexpressed in many types of cancer. In case of Chronic Myeloid Leukemia (CML) the expression level of BMI1 is associated with worsening prognosis. However, the signaling pathways involved in its overexpression and its role in this disease remains unclear. By using RNAi to repress BMI1 expression we highlighted that this polycomb was essential for proliferation and clonogenicity of CML cells. We also demonstrated, for the first time, that BMI1 supported tumor growth through repression of deleterious cancer cell autophagy. A transcriptomic approach allowed us to identify a transcriptional target involved in this process: the Cyclin G2. Through a bioinformatic approach, we finally found a molecule capable of expression re-Induction of Cyclin G2 in CML cells : alexidine dihydrochloride. This molecule induced a high level of autophagy as well as apopotosis in cancer cells. It had also been able to re-Sensitize to imatinib a resistant cell line. In conclusion, our results revealed a new role for the polycomb BMI1 in supporting the CML pathology. Moreover, our work allowed the identification of two new approaches for therapeutically targeting this oncogene functions

Anatomy
Ph.D.
Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by transforming growth factor beta 1 (TGF-β1) where it acts as a downstream mediator of TGF-β1 induced extracellular matrix production. The molecular mechanisms that control CTGF induction by TGF-β1 in osteoblasts are not understood. We have previously demonstrated the requirement of Src, Erk and Smad signaling for TGF-β1 induced CTGF promoter activity in primary osteoblasts, however the potential interaction among these signaling pathways in osteoblasts remains unknown. In this study, we demonstrate that CTGF is induced by TGF-β1 in rat osteosarcoma osteoblast like cells (ROS17/2.8). TGF-β1 activates Src and blocking of Src family kinases by PP2 abrogates TGF-β1 induced CTGF up-regulation. Western blot analysis revealed that primary osteoblasts and ROS 17/2.8 cells express not only Src, but also other Src family members, such as Fyn, Yes and Hck. In order to determine whether CTGF up-regulation is controlled by Src or other members, we used either kinase-dead dominant negative Src constructs in primary osteoblasts or Src siRNA in ROS17/2.8 cells to block Src function. Inactivation of Src by both kinase-dead and siRNA prevented TGF-β1 induced CTGF induction, demonstrating that TGF-β1 induced CTGF up-regulation is mediated only by Src not by other members. In addition, we also demonstrated that Erk is activated by TGF-β1 and that blocking of Erk activation using pharmacological inhibitors, PD98059 and U0126, prevents TGF-β1 induced CTGF induction, demonstrating the requirement of Erk for CTGF induction. These results prompted us to further explore the cross-talk between Src, Erk and Smads in ROS17/2.8 cells. Inhibition of Src using PP2 prevented Erk activation, demonstrating that Src is upstream of Erk. To investigate how Src and Erk regulate the canonical TGF-β1 signaling pathway, including Smad2/3 phosphorylation and nuclear translocation of activated Smads, we treated cells with TGF-β1 in the presence or absence of the Src inhibitor, PP2, or the Erk inhibitors, PD98059 or U0126. PP2 pre-treatment prevented the phosphorylation of Smad2/3 at both the SSXS motif and the linker region and consequently blocked their nuclear translocation, demonstrating that Src can regulate Smad signaling. In contrast, the Erk inhibitors did not have any effects on Smad phosphorylation and/or nuclear translocation. To examine whether Erk can modulate Smad signaling indirectly through the activation/ inactivation of required nuclear coactivators/ co-repressors that mediate Smad DNA binding, we used electro-mobility shift assays. These experiments showed that inhibition of Erk activation impaired transcriptional complex formation on the Smad binding element (SBE) and TGF- β responsive element (TRE) of the CTGF promoter, demonstrating that Erk activation is required for SBE and TRE transactivation. Taking together, these data demonstrate that Src is an essential upstream signaling transducer for Erk and Smad signaling in osteoblasts, and that while the Smad and Erk signaling cascades appear to function independent of each other, they are both essential for the formation of a transcriptionally active complex on the CTGF promoter.
Temple University--Theses

Cell Biology
Ph.D.
Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance.
Temple University--Theses

The AACN has asked academic leaders to align the performance of their organizations to the prescribed standards within the Essentials of Baccalaureate Education for Professional Nursing Practice document and has provided indicators of quality suggestions for program enhancement as a means of promoting continuous performance improvement. However, the AACN has not prescribed a strategy that specifies the manner in which colleges should achieve these benchmarked standards, which has created uncertainty among administrators about whether the indicators of quality lead to improvements that are actually indicative of improved performance.

This dissertation used multiple linear regression research design to determine whether predictive relationships exist between the American Association of Colleges of Nursing (AACN) indicators of quality and the Baldrige core values and concepts of performance improvement within Commission on Collegiate Nursing Education accredited baccalaureate colleges of nursing.

The purpose of this study was to determine whether the behaviors associated with specific AACN indicators of quality reflect behaviors that the Baldrige core values and concepts have already proven to be successful in achieving continuous performance improvement. The results revealed nine AACN indicators of quality behaviors most likely to enhance performance improvement outcomes within baccalaureate colleges of nursing. They include; (1) Resources are budgeted for research, development, business operations, public relations, marketing, and human resources; (2) Establishing and upholding policies that reflect faculty and leadership development resources; (3) Student experiences include service learning opportunities; (4) Practice partnerships include collaborative practice initiatives; (5) Collecting data and making program changes that focus on the level of graduate satisfaction with their preparation for the profession; (6) Faculty have input into the governance of the college/school; (7) The majority of faculty have a presence in state, regional, national, and international professional activities; (8) Opportunities for baccalaureate graduate's employment with practice partnerships; and (9) Formal mentoring program for clinical preceptors.

The results underline the fact that continuous performance improvement within baccalaureate colleges of nursing is a deliberate and dynamic analysis-driven endeavor dependent on an organization's ability, willingness, and initiative to continually strive to narrow the chasm between actual and potential performance results.

Introdução: O linfoma difuso de grandes células B é o mais freqüente grupo de linfoma não- Hodgkin, perfazendo quase 50% dos casos no serviço de hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e Instituto do Câncer do Estado de São Paulo. Possui heterogeneidade clínica e biológica traduzida em mais de vinte subtipos na Organização Mundial da Saúde. Sua terapêutica se baseia na associação do anticorpo monoclonal anti-CD20 e quimioterapia com antracíclico, esquema que resulta em 43,5% de sobrevida global em 10 anos. Determinantes de prognóstico clínico como o Índice Internacional de Prognóstico e o Índice Internacional de Prognóstico Revisado carecem de acurácia, pois até 20% dos pacientes de baixo risco falecerão da doença e 60% dos pacientes de alto risco estarão vivos em quatro anos. Essas discrepâncias podem, em parte, ser atribuídas a fatores genéticos. A assinatura gênica do linfoma difuso de grandes células B tipo centro germinativo apresenta sobrevida global superior ao tipo células B ativadas (76% versus 16%, p=0,01), contudo o perfil de expressão gênica por microarray ainda não está disponível na prática clínica. Entretanto, o escore preditivo de mortalidade para linfoma difuso de grandes células B baseado no valor prognóstico da expressão dos genes BCL2, BCL6, CCND2, FN1, LMO2 e SCYA3 por PCR em tempo real quantitativa mostrou-se independente do Índice Internacional de Prognóstico na era pré-rituximabe. Mas não foi significante em pacientes de alto risco clínico tratados com R-CHOP. Os genes BCL2, CCND2 e SCYA3 integram a assinatura de células B ativadas, BCL6 e LMO2 a do centro germinativo e FN1 a linfonodal. Objetivo: Avaliar o impacto da expressão absoluta dos genes BCL2, BCL6, CCND2, FN1, LMO2 e SCYA3 em população brasileira com linfoma difuso de grandes células B tratada com R-CHOP em relação à resposta global, sobrevida livre de doença, sobrevida livre de progressão e sobrevida global. Métodos: A expressão gênica foi analisada por PCR em tempo real quantitativa de RNA extraído de amostras parafinadas de 63 pacientes, porém foi avaliável em 42. Seus valores foram normatizados pelo gene endógeno ABL e transformados em escala logarítmica na base 2 para posterior correlação com variáveis clínicas e de desfecho. Resultados: Com mediana de seguimento de 29 meses, as sobrevidas global, livre de doença e livre de progressão foram, respectivamente, 82,8%, 97,14% e 87,53%, enquanto a resposta completa foi 82,5%. A expressão de LMO2>3logs e BCL6>3,5logs definiu um grupo de maior sobrevida global (91% versus 64,3%, p=0,040) e sobrevida livre de doença (95,5% versus 70,7%, p=0,03), independentemente do Índice Internacional de Prognóstico (p=0,010 e p=0,042) e com significativa hiperexpressão do SCYA3 (p=0,046). Não se observou associação entre escore preditivo de mortalidade baseado nos seis genes e prognóstico. Assim, foi criado novo escore genético prognóstico baseado no poder da expressão concomitante de LMO2 e CCND2, definindo-se grupos de baixo risco (<2,5) e alto risco (>=2,5) com distintas sobrevidas global (92,4% versus 57,1%, p=0,011) e livre de progressão (96,2% versus 66,7%, p=0,013), independentes do IPI. Conclusão: Em pacientes com linfoma difuso de grandes células B tratados com R-CHOP, a hiperexpressão de BCL6, LMO2 e SCYA3 correlacionou-se com melhor prognóstico. O novo escore genético prognóstico definido por LMO2 e CCND2 estratificou grupos de risco de prognósticos distintos independentes do Índice Internacional de Prognóstico
Introduction: Diffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma; which accounts for almost 50% of the cases at the Hematology Department of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and Instituto do Câncer do Estado de São Paulo. Its clinical and biological heterogeneity results in more than twenty subtypes according to the World Health Organization classification. Its treatment is based on a combination of anti-CD20 monoclonal antibody and antracycline-based chemotherapy, with a 10-year overall survival of 43.5%. Clinical prognostic determinants such as the International Prognostic Index and the Revised International Prognostic Index lack accuracy, since up to 20% of low-risk patients will die from the disease and up to 60% of high-risk patients will be alive within four years. Such discrepancies can partially be attributed to genetic factors. Diffuse large B-cell lymphoma germinal center gene signature shows superior overall survival compared to activated B-cell signature (76% versus 16%, p=0.01), however microarray gene expression profile is not yet available in clinical practice. Nonetheless, the Mortality Predictor Score for diffuse large B-cell lymphoma based on the prognostic value of BCL2, BCL6, CCND2, FN1, LMO2 and SCYA3 gene expression by quantitative real-time PCR has proved to be independent from the International Prognostic Index in the pre-rituximab era. But it was not significant in high clinical risk patients treated with R-CHOP. The genes BCL2, CCND2 and SCYA3 compose activated B-cell signature, whereas BCL6 and LMO2 compose the germinal center signature and FN1 the lymph-node signature. Objective: Evaluate the impact of BCL2, BCL6, CCND2, FN1, LMO2 and SCYA3 absolute gene expression in Brazilian population diagnosed with diffuse large B-cell lymphoma and treated with R-CHOP, with respect to overall response, disease free survival, progression free survival and overall survival. Methods: Gene expression was analyzed by quantitative real-time PCR of RNA extracted from paraffin-embedded samples of 63 patients, although evaluable in 42. Their values were normalized by endogenous gene ABL and log- transformed on a base 2 scale for subsequent correlation with clinical and outcome variables. Results: With a median follow-up of 29 months, overall survival, disease free survival and progression free survival accounted for 82.8%, 97.14% and 87.53% respectively, while complete response was 82.5%. The expression of LMO2>3logs and BCL6>3.5logs defined a group with higher overall survival (91% versus 64.3%, p=0.040) and progression free survival (95.5% versus 70.7%, p=0.03), independent of International Prognostic Index (p=0.010 and p=0.042) and with significant overexpression of SCYA3 (p=0.046). It was not identified any association between six gene Mortality Predictor Score and prognosis. As a result, we developed the New Genetic Prognostic Score based on the power of concomitant expression of LMO2 and CCND2, defining low-risk (<2.5) and high-risk (>=2.5) groups with distinct overall survival (92.4% versus 57.1%, p=0.011) and progression free survival (96.2% versus 66.7%, p=0.013), independent of International Prognostic Index. Conclusion: In patients with diffuse large B-cell lymphoma treated with R-CHOP, hyperexpression of BCL6, LMO2 and SCYA3 was correlated with a better prognosis. The New Genetic Prognostic Score, defined by LMO2 and CCND2, stratified risk groups with different prognosis, independent of International Prognostic Index

Il est quasiment devenu un lieu commun aujourd’hui de mourir à l’hôpital. Et s’il fallait traiter ce sujet, le plus souvent c’est la perspective médicale qui tenterait d’en percevoir les enjeux. C’est au tournant du milieu du XXème siècle, au lendemain d’un énième génocide, marquant l’esprit de l’humanité certainement pour l’éternité, qu’un nouveau langage bioéthique va se faire le vecteur d’une considération renouvelée de la personne. L’homme devient personne humaine dans les textes internationaux proclamant dès lors d’inédits droits de l’homme. C’est en prenant la bioéthique comme objet de réflexion qu’un certain droit de la santé, largement inspiré par la production de comités d’éthiques, a émergé d’une doctrine privatiste en premier, afin de cerner de premiers droits définissant celui qu’il serait convenu d’appeler alors le mourant. Toutefois, l’établissement public de santé confronté à la technologisation médicale, permettant alors un allongement de la fin de vie, s’est rapidement trouvé être un lieu de conflit entre ceux qui furent considérés comme des usagers de service public, et les commettants médecins de l’Etat. La primauté du droit privé de la personne semblait alors menacée, le droit administratif prenant le pouvoir afin d’indemniser les victimes de l’hôpital. Cependant, à force de réductions systématiques ne regardant la personne mourante que sous un angle génériciste, relevant d’un droit public, celle-ci s’est progressivement retrouvée parfaitement ignorée en son essence première que le droit privé parvenait à percevoir. Les formes les plus récentes de déresponsabilisation attesteraient d’une consécration de l’indemnisation finissant d’objectiviser l’être sous-jacent à la personne mourante, allant jusqu’à justifier l’acte euthanasique. L’approche réitérée de ce difficile objet d’étude trouverait à générer une réflexion que le philosophe du droit engagerait, réintroduisant la personne irréductible se manifestant comme point de départ et d’arrivée, de telle sorte que se pourrait être harmonisée la rencontre inévitable des domaines public et privé, juridique et politique, afin de rendre toute sa vérité onto-axiologique aux droits premiers du sujet mourant
Today, dying at hospital is the most curently idea agreed in order to protect people. That’s maybe the reason why medical studies took this as an issue more than lawyer studies. It’s around the middle of the twentieth century, after one more genocide, marking the spirit of the humanity certainly for all eternity, that a new bioethical language is going to be made the vector of a consideration renewed by the person. The man becomes a human-person in the international texts, proclaiming from then on of unpublished works human rights. While taking the bioethics as object of reflection a certain health law, widely inspired by the production of committees of ethics, emerged from a privatiste doctrine in the first one, to encircle first rights of the one that it would have been advisable to call then the dying. However, the public institution of health confronted with the medical technologisation, allowing then an extension of the end of life, quickly was to be a place of conflict between those who were then considered as users of public service, and principals doctors of the State. The superiority of the private law of the person seemed then threatened, the administrative law taking the power to indemnify the victims of the hospital. However, by means of systematic reductions looking at the dying person only under an angle génériciste, recovering from a public law, this one gradually found itself perfectly ignored in the first essence which the private law succeeded in perceiving. The most recent forms of deresponsabilisation would give evidence of a consecration of the compensation stopping an objectivisation the underlying being to the dying, going person to justify the euthanasic act. The approach repeated by this difficult object of study would find to generate a reflection which the philosopher of the right(law) would engage, reintroducing the person inflexible as point of departure and arrival, so that could be harmonized the inevitable meeting of the public and private, legal and political domains, to return all its onto-axiological truth to the first rights of the dying subject

碩士
臺北醫學大學
醫學科學研究所
98
According to the Department of Health, Executive Yuan,R.O.C. vital statistics data show that people over the past decade the incidence of female breast cancer and mortality cases are increasing year by year, and there is getting younger and younger, therefore, studies of breast cancer also imperative. This study was to Cyclin E2 and breast cancer cells, clinical disease associated; through cell experiments and clinical samples, observe Cyclin E2 in the role of breast cancer. In cell experiments, by reverse transcription - polymerase chain reaction method, preliminary observations Cyclin E2 expression in cell lines and select the laboratory used MDA-MB-231, MCF-7 breast cancer cells to natural compounds luteolin and artificial compounds treatment 24 Hrs, found that breast cancer cell line MDA-MB-231 significantly inhibited the growth of the phenomenon, and Cyclin E2 mRNA / Protein expression levels decreased as the dose increases. In clinical terms, the use of real-time quantitative polymerase chain reaction method to detect breast cancer patients of 257 normal tissue and cancerous tissue samples of Cyclin E2 expression, and to collect patient diagnostic information, application of SPSS statistical software for analysis , Cyclin E2 was found in cancerous tissue had higher expression; and normal tissue and cancerous tissue of more than 10 times the performance compared to those in the T＞N group is a higher incidence of cases. Immunohistochemical stain of CCNE2 expression in clinical patient tissues, we observed that Cyclin E2 in tumor region of breast cancer tissue showed positive reaction(brown).

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
100
Sorafenib is a drug for standard systemic therapy in patients with advanced HCC (hepatocellular carcinoma), and it is also the first drug with survival benefits. Although sorafenib was originally designed as a specific Raf kinase inhibitor, we and other investigators have found many off-target effects of sorafenib that may have significant implications regarding its anti-tumor activity and resistance mechanisms of sorafenib in HCC cells. Our laboratory has found, for example, that sorafenib can down-regulate Cyclin E1 expression in mRNA and protein levels in various HCC cell lines. The resistance of HCC cells to sorafenib is associated with higher basal levels of Cyclin E1 expression and failure by sorafenib to cause down-regulation. Knockdown of Cyclin E1 expression by siRNA reversed the resistance of HCC cells to sorafenib in terms of cell apoptosis induction. In the present study, we sought information for a more in-depth discussion of the role Cyclin E1 plays in sorafenib-resistant HCC, with particular attention to feasibility and clinical applications. In the study, I promoted the over-expression of Cyclin E1, and this was effectively able to prevent apoptosis induction by sorafenib in HCC. In exploring the clinical application of a combination of sorafenib and flavopiridol, a cyclin-dependent kinase (CDK) inhibitor, I found that it synergistically inhibited cell growth and induced apoptosis in HCC cells. The synergistic efficacy was associated with the suppression of Bcl-XL and Mcl-1 expression in HCC cells. Finally, sorafenib combined with flavopiridol inhibited tumor growth significantly better than either agent did individually in the xenograft models. In conclusion, the results indicate that Cyclin E1 might be a potential target for cancer therapy and a candidate marker for disease diagnosis or prognosis. In addition, sorafenib combined with flavopiridol could be of potential therapeutic value in the future.

博士
國立臺灣大學
臨床牙醫學研究所
103
Transforming growth factor-β (TGF-β) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) is induced by TGF-β1 and sustain the pro-fibrotic response in GO. CCN2 expression is positively related to the degree of fibrosis in GO. Previous studies have shown that JNK and Smad3 activation is required for TGF-β1-induced CCN2 expressions in human gingival fibroblasts (HGFs). In this study, we found pretreatment with two Src kinase inhibitors (PP2, Src inhibitor-1) significantly reduced TGF-β1-induced CCN2 synthesis and JNK and Smad3 activation in HGFs. Src is an upstream signaling transducer of JNK and Smad3 with respect to TGF-β1-stimulated CCN2 expression in HGFs. We also found TGF-β1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGF-β1-induced ROS production; Src, JNK, and Smad3 activation; CCN2, and type I collagen protein expression in HGFs. NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of both canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Even under meticulous periodontal maintenance, the recurrence rate after surgical excision in 18 mounths was still up to 34%. The potential mechanism for rapid recurrence is the early recruitment of platelets and coagulation factors to the surgical wound. We found thrombin induced CCN2 expression in HGFs through the activation of PAR1. Pretreatment with antioxidant N-acetyl-L-cysteine (NAC), and c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 significantly reduced thrombin-induced CCN2 expression in HGFs. Thrombin induced CCN2 via PAR1/ ROS/ JNK in HGFs. Furthermore, we found thrombin induced Smad3 phosphorylation and increased activated TGF-β1 levels in HGFs. Pretreatment with Src inhibitor PP2, ROCK inhibitor Y27632, actin/myosin destablizer blebbistain, integrin αv and β1 blocking antibody significantly reduced thrombin-induced activated TGF-β1 levels in HGFs. Thrombin induced CCN2 via PAR1/ Src/ ROCK/ integrin αvβ1/ TGF-β1 in HGFs. Epithelium-mesenchymal transition (EMT) is involved in the pathogenesis of GO. We found thrombin significantly increased vimentin、snail and slug expression in two human gingival epithelial cell lines OECM-1 and Ca9-22. Furthermore, we found thrombin induced Smad3 phosphorylation and increased activated TGF-β1 levels in OECM-1 and Ca9-22 cells. Pretreatment with Src inhibitor PP2, ROCK inhibitor Y27632, actin/myosin destablizer blebbistain, Integrin αvβ6 blocking antibody significantly reduced thrombin-induced activated TGF-β1 levels in OECM-1 and Ca9-22 cells. These results suggested that thrombin induced EMT via PAR1/ Src/ ROCK/ integrin αvβ6/ TGF-β1 in OECM-1 and Ca9-22 cells. We further found curcumin significantly abrogated the TGFβ1-induced CCN2 cell migration and myofibroblast transdifferentiation in HGFs through inhibiting NOX4 protein expression. Furthermore, curcumin inhibited thrombin-induced the activated TGFβ1 level in HGF, OECM-1 and Ca9-22. Curcumin potentially qualifies as a useful agent for the control of GO.

博士
國立臺灣大學
臨床牙醫學研究所
100
Connective tissue growth factor (CCN2) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesos of oral submucous fibrosis (OSF). We first immunohistochemically examined the expression of CCN2 protein in 20 cases of OSF and found positive CCN2 staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed the arecoline, a main alkaloid found in AN, stimulated CCN2 synthesis in a dose- and time- dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CCN2 during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-κB inhibitor Bay 11-7082、JNK inhibitor SP600125、p38 MAPK inhibitor SB203580、antioxidatn N-acetyl-L-cycteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CCN2 synthesis. Curcumin completely inhibited arecolined-induced CCN2 synthesis and the inhibition is dose-dependent. Arecoline also induced Smad 2 and Smad 3 activation. TGF-β neutralizing antibody、activin receptor-like kinase 5 (ALK5) inhibitor SB431542、Smad 3 inhibitor SIS3 significantly reduced arecoline-induced CCN2 synthesis. Furthermore, arecolined-induced CCN2 synthesis was inhibited by diphenyleneiodonium (DPI), a NADPH oxidase (NOX) inhibitor, NOX4 inhibitor plumbagin, but not muscarinic receptor antagonist atropine and NOX2 inhibitor apocynin. These results indicate that arecoline induced CCN2 expression in buccal mucosal fibroblasts via TGF-β signaling pathway. Curcumin may serve as a useful agent in controlling OSF.

Lysyl oxidase like-2 (LOXL2) was found to be present extracellularly in primary human gingival fibroblast cells. This project has been primarily focused on investigating our hypothesis that LOXL2 may play a critical role in regulating cell proliferation and collagen accumulation in primary human gingival fibroblast cells, which may contribute to the development of fibrotic changes in human gingival tissue. LOXL2 shRNA lentivirus reduced the LOXL2 mRNA and protein expression by 90 – 95%. Knockdown of LOXL2 or inhibition of LOXL2 enzymatic activity strongly inhibited both basal and CCN2/CTGF-stimulated collagen accumulation (p<0.05). Proliferation assays demonstrated a marked decrease in cell proliferation in both the short and long term in LOXL2 shRNA knockdown cells with minimal or no stimulation of cell apoptosis. Pharmacologic inhibition of LOXL2 enzyme activity reduced basal and CCN2/CTGF-stimulated cell proliferation (40% and 50%) in short term cultures. Furthermore, there was 15-20% inhibition seen in long term assays. Recombinant active LOXL2 significantly increased collagen accumulation and cell proliferation (p<0.05). Thereby, our investigation in vitro by loss and gain of function experiments confirmed that LOXL2 is critically required for both gingival fibroblast proliferation and for collagen accumulation in the presence or absence of CCN2/CTGF. LOXL2 stimulation is critical for both proliferation and collagen accumulation in primary human gingival fibroblasts. Lastly, we found that the presence of LOXL2 extracellularly and LOXL2 may regulate cell proliferation by enhancing the phosphorylation of PDGFR.

博士
國立臺灣大學
臨床牙醫學研究所
101
Transforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) is required for most of the increased ECM production and other profibrotic activities generally observed in response to TGFβ. Arecoline induces CCN2 and cyclooxygenase-2 (COX-2) expressions in human buccal mucosal fibroblasts (BMFs). CTGF/CCN2 and COX-2 have been found to overexpress in OSF. However, the molecular mechanisms are not entirely clear. The aims of this study were to investigate the molecular mechanism underlying the TGFβ-induced CTGF/CCN2 expressions in human buccal mucosal fibroblasts (BMFs) and to identify the potential targets for drug intervention or chemoprevention of OSF. Pretreatment with activin receptor-like kinase 5 (ALK5) inhibitor SB431542, Rac1 inhibitor NSC23766, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, antioxidant N-acetyl-L-cysteine (NAC), NOX inhibitor diphenylene iodonium (DPI), NOX4 inhibitor plumbagin, and Src inhibitors (PP2, Src inhibitor-1, and Src si-RNA) significantly reduced TGFβ1-induced CTGF/CCN2 synthesis in BMFs, but not extracellular signal-regulated kinase (ERK) inhibitor PD98059, or NOX2 inhibitor apocynin. Furthermore, the phosphorylations of Src were inhibited by NAC and DPI but not NSC23766. The phosphorylations of JNK were inhibited by NAC, DPI, and PP2 but not NSC23766. The phosphorylations of p38 MAPK were inhibited by NAC and DPI but not NSC23766 or PP2. The phosphorylations of SMAD3 were inhibited by NAC and PP2 but not NSC23766 or DPI. These results revealed that multiple parallel signal transduction pathways together mediated the TGFβ1-induced CTGF/CCN2 expression in BMFs. At least four major pathways TGFβ1/ALK5/Rac1/CTGF(CCN2), TGFβ1/ALK5/ROS/Src/SMAD3/CTGF, TGFβ1/ALK5/NOX4/ROS/Src/JNK/CTGF, and TGFβ1/ALK5/NOX4/ROS/p38/CTGF were identified. Epigallocatechin-3-gallate (EGCG) blocked TGFβ1-induced CTGF/CCN2 synthesis by inhibiting the phosphorylation of Src, JNK, and p38 MAPK. Prostaglandin E2 (PGE2) inhibited the TGFβ1-induced CTGF/CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs. The exceptional signal transduction pathways of TGFβ1-induced CTGF/CCN2 production in BMFs contribute to the resistance of PGE2 downregulation of CTGF/CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. In addition, EGCG inhibits the expression of TGFβ1-induced type I procollagen, reduces the excessive soluble collagens produced by TGFβ1-treated BMFs, and attenuates the fibroblast-mediated gel contraction stimulated by TGFβ1. Therefore, EGCG may serve as a useful agent in controlling OSF.

碩士
國立臺灣大學
口腔生物科學研究所
103
Oral submucous fibrosis (OSF) is a chronic inflammatory disease. Its clinical sign and symptoms include stiffness of oral mucosa, difficulty in mouth opening, swallowing and speech. The regular use of areca nut (AN) is the major etiological factor of OSF. Previous study indicated that arecoline, TGF-β1 and microtrauma play an important role in the pathogenesis of OSF. We previously showed TGF-β1 induced CCN2 through ALK5/JNK p38 pathway in human buccal mucosal fibroblasts (BMFs). Thrombin induced CCN2 expression through PAR1/ROS/ASK1/JNK pathway in BMFs. Both of them can be inhibited by epigallocatechin-3-gallate (EGCG). The aim of this study was to investigate whether thrombin induced CCN2 expression in BMFs is via TGF-β1 activation. We first found thrombin induced Smad3 activation in BMFs. Pretreatment with TGF-β1 neutralizing antibody, ALK5 inhibitor SB431542 and Smad3 inhibitor SIS3 significantly reduced thrombin induced CCN2 expression. These results indicate that thrombin-induced CCN2 expression in BMFs is via TGF-β1-dependent pathway. Pretreatment with RGD blocking-peptide and integrin neutralizing antibodies significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2, suggest that integrin is involved in thrombin activate TGF-β1 pathway. Furthermore, ROCK inhibitor Y27632, actin/myosin destabilizing agent blebbistatin significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2. In addition, EGCG completely inhibited thrombin-induced activated TGF-β1 level. These results indicated that thrombin-induced CCN2 expression is via TGF-β1 pathway and mediated by PAR1/ROCK/Actin polymerization/integrin cascade.

碩士
國立臺灣大學
口腔生物科學研究所
99
Thrombin has been implicated in fibrotic disorders of several organs such as kidney, liver, and lung. Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues and was found to overexpress in oral submucous fibrosis (OSF). OSF is the result of persistent chemical irritation from areca nut (AN) constituents and the mechanical trauma to the oral mucosa from the coarse fibers of AN. Mechanical trauma could lead to activation of the coagulation cascade. In this study, we showed that thrombin stimulated CTGF synthesis in human buccal mucosal fibroblasts (BMFs). The effect of thrombin could be mimicked with a selective proteinase activated receptors 1 (PAR1)-activating peptide and was completely abolished with the specific thrombin serine protease inhibitor PPACK, indicating that thrombin mediated this effect via the proteolytic activation of PAR1. These results suggested that thrombin produced by microtrauma during AN chewing may contribute to the pathogenesis of OSF by up-regulating CTGF expression in BMFs. Pretreatment with antioxidant N-acetyl-L-cysteine, ASK1 inhibitor thioredoxin, JNK inhibitor SP600125, but not PI3K inhibitor LY294002, ERK inhibitor PD98059, p38 MAPK inhibitor SB203580, significantly reduced thrombin induced CTGF synthesis. Furthermore, epigallocatechin-3-gallate completely inhibited thrombin-induced CTGF synthesis. These results suggested that thrombin-induced CTGF synthesis could be mediated by reactive oxygen species, ASK1 and JNK pathways in BMFs and EGCG may serve as a useful agent in controlling OSF.

碩士
高雄醫學大學
牙醫學系碩士班
104
Oral squamous cell carcinoma (OSCC) is one of the most diagnosed malignancy of head and neck regions with growing morbidity, and mortality, while RIF (radiation induced fibrosis) is one of the most severe long-term effects of radiotherapy for oral cancer. Like other fibrotic diseases, which may be exaggerated for years even whole life. Present study was to investigate TGFβR1 (transforming growth factorβ receptor 1) and its’ potential downstream mediators in field of OSCC and fractionated RIF in DMBA (7, 12-dimetheyl benz[a]anthrance) induced hamster buccal pouch squamous cell carcinoma model. Thirty-six male golden Syrian hamsters were divided into five groups: DMBA + Radiation (DR), DMBA (D), Radiation (R), mineral oil (MO) and no treatment (N). Hamsters were induced buccal pouch cancer by topical application of DMBA for 12 weeks, and all the hamsters in the DR and RT groups received whole head irradiation by the linear accelerator with a total dose of 42Gy divided into 6 fractions, twice weekly. Immunohistochemistry and western blotting studies showed that TGFβR1 (transforming growth factorβ receptor 1), Smad3 (drosophila mothers against decapentaplegic protein 3), Erk1/2 (extracellular signal-regulated kinase 1/2), CCN2 (connective tissue growth factor) were highly expressed in cancer cells. The gross pouch tissues in the D & DR groups showed thickened and significant volume size shrinkage, histopathology/immunohistochemistry studies showed that D and DR groups were highly fibrotic, TGFβR1, Smad3, p-Smad3, Erk1/2, p-Erk1/2, CCN2 and α-SMA (α-smooth muscle actin) expressions were all statistically significantly increased when compared with N and R groups. Only Smad3, p-Smad3, and Erk1/2 were higher in DR group than in D group with marginally statistical significant (p=0.051). In conclusion, present study indicated TGFβR1, Smad3, Erk1/2, and CCN2 may be crucial in hamster buccal pouch OSCC and RIF. Though there was no direct evidence of association within upregulation of above proteins in our experiment, further study may be conducted toward these targets. DMBA-induced hamster buccal pouch cancer model could be employed to the study of OSCC and its radiation effects.