<p>Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain.</p><p>In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. </p><p>Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent.</p><p>Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.</p>

Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain. In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent. Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands. The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins. We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

In the exploration into new therapeutic agents for human disease, medicinal herbs offer an enormous resource due to their wide range of biologically active components. However, because of these biologically active components, medicinal herbs can also have toxic side effects. The focus of this thesis is the effect of herbal therapies, both good and bad, on chronic kidney disease (CKD) and tubulointerstitial fibrosis. Tubulointerstitial fibrosis is considered one of the defining characteristics of CKD. In Chapter 1, the literature regarding the pathogenesis of tubulointerstitial fibrosis is reviewed, beginning with the mechanisms of its development, the main structural and functional features, and the molecular mediators. The structural features include activation of resident fibroblasts and transition of tubular epithelial cells into myofibroblasts, deposition of extracellular matrix proteins, increased apoptosis of normal cells of the renal nephron and development of tubular atrophy, increased renal oxidative stress, and hypoxia of renal tissues. Molecular mediators that are explored include angiotensin II, transforming growth factor-ß1 and numerous other cytokines and growth factors. Pharmacological manipulation of these features and their molecular mediators for regression of tubulointerstitial fibrosis is then discussed. Currently, the gold standard of therapy for people with CKD is blockade of the renin-angiotensin-aldosterone system with angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs). Because of the complexity of the pathogenesis of renal fibrosis and the multiple mechanisms by which ACEIs and ARBs work, this portion of the thesis focuses on the qualities that additional agents should have to complement their actions. These additional agents could work by decreasing oxidative damage, by decreasing fibroblast numbers through apoptosis, through the interruption of inflammatory, fibrotic mediators, by increasing anti-inflammatory mediators or through other mechanisms. The literature review therefore continues with a discussion of the historical use of medicinal herbs in genitourinary pathologies and the known contributions that medicinal herbs can make to the treatment or development of tubulointerstitial fibrosis and CKD. From this review, a large number of herbs were identified as having traditional use in renal disorders or as being of interest, currently, to researchers of renal pathologies. However, much information is lacking regarding the mechanisms by which the hypothesised benefits occur, making it impossible to assess which herb(s) may offer valuable additive support or alternative treatments to the therapies currently given to people with CKD. Furthermore, there is a lack of information regarding toxicity of these herbs; many herbs have never been assessed in cell culture or in animal toxicity studies. It was apparent that preliminary in vitro work was necessary before in vivo pharmacological work could be undertaken. This thesis, therefore, aimed to test the following hypotheses: (1) That medicinal herbs used currently for treatment of renal dysfunction have high anti-oxidant properties that can be further enhanced by specific extraction processes; (2) That the in vitro testing of selected extracts from medicinal herbs, identified in (1), will reveal some anti-oxidant benefits or indications of toxicity that need careful analysis in animal studies; (3) That careful in vivo testing of specific toxic medicinal herbs identified in these leadup studies will define specific pathological processes that predict an outcome of CKD; and (4) That careful in vivo testing of selected medicinal herbs, used in conjunction with more conventional medicines for CKD, will show an additive benefit when used to ameliorate development of CKD induced using an established animal model. The subsequent laboratory work was designed to test the validity of these hypotheses and the results are then presented in Chapters that each comprise a publication. The aim of Chapter 2 was to present a systematic analysis of the oxidant properties of 55 medicinal herbs that have been used traditionally to treat kidney and urinary disorders or have been of recent interest to researchers of renal disorders. Since different extraction processes yield different constituents, each of the herbs was sequentially extracted with three solvents of decreasing polarity. An assay was performed on each of the fractions to determine the oxygen radical absorbance capacity. The aim of Chapter 3 was to test the benefit or otherwise of each of the three extracts of the chosen herbs using an in vitro cell study. Each extract was tested for potential toxic, apoptotic, mutagenic and antioxidant activity on normal mammalian renal tubular epithelial cells (NRK-52E). The effect of the extracts on renal fibroblasts (NRK-49F) was also analysed. Several specific hypotheses arose from the combination of the systematic analyses and the literature review regarding benefits and toxicities of a number of the extracts. The subsequent in vivo work was designed to test the validity of two of these hypotheses. The aim of Chapter 4 was to test the hypotheses developed from the results of the previous Chapter. The herb Dioscorea villosa had demonstrated extreme cytotoxicity to mammalian renal epithelial cells and had caused transdifferentiation of epithelial cells into fibroblasts. An in vivo rodent model was used to test chronic dosage with this herb and its toxicity and predisposition for induction of CKD verified. The aim of Chapter 5 was to determine whether a herbal preparation (Angelica sinensis and Astragalus membranaceus) that had some support from the literature and the results from Chapters 2 and 3, could complement the actions of ACEIs in a rodent model of renal fibrosis (unilateral ureteral obstruction). The combination of herbal medicines and the ACEI was significantly more effective than the ACEI alone in ameliorating several characteristics of CKD development. To conclude the thesis, Chapter 6 provides an overview discussion of the results and a critical analysis of the methods used. Further, Chapter 6 looks towards future experiments that are planned to further resolve issues of concern about effects on renal health from use of medicinal herbs. .

La fibrose pulmonaire (FP) idiopathique est une maladie sans traitement efficace caractérisée par une prolifération de myofibroblastes et par un départ sous-pleural suggérant un rôle de la plèvre. Le transforming growth factor (TGF)-ß1 induit un processus de transformation des cellules mésothéliales pleurales (CMP) en cellules de type myofibroblaste. Les protéines de choc thermique régulent la voie du TGF-ß1. L’importance de l’axe caspase-1/IL-1ß, a été décrite dans les modèles animaux de FP.La protéine de stress AlphaB-crystallin a été étudiée dans la FP au niveau des CMP et l’importance de l’axe caspase-1/IL-1ß a été recherchée au niveau des cellules structurales pulmonaires dans la toxicité de la bléomycine (BLM).aB-crystallin est surexprimée dans la FP idiopathique au niveau des CMP. Son inhibition empêche la transformation et la migration des CMP dans la fibrose pleuro-pulmonaire. Dans un modèle de FP induite par la BLM chez la souris, la voie caspase-1 est activée dans les régions pleurales. In vitro, la caspase-1 a un rôle crucial dans la transformation des CMP. Son activation induit une réaction fibrosante chez la souris. Dans une seconde partie, nous montrons qu’une forme déglycosylée de la BLM, incapable d’activer la caspase-1, n’induit pas de FP mais a une activité anti-cancéreuse. La déglyco-BLM n’entraine pas la pyroptose, mort caspase-1 dépendante, chez les cellules épithéliales alvéolaires. Nos résultats suggèrent qu’AlphaB-crystallin et la voie caspase-1/IL-1ß pourraient être des cibles thérapeutiques dans la FP idiopathique ou induite par la BLM. Nous apportons aussi une preuve de concept de l’utilisation de la déglyco-BLM comme alternative non toxique à la bléomycine. / Idiopathic Pulmonary Fibrosis (PF) is a rare and devastating disease without efficient treatment at this time. Idiopathic FP is characterized by accumulation of myofibroblasts and has a typical sub-pleural onset suggesting a role of the pleura in the disease. Transforming Growth Factor (TGF)-ß1 induces transformation of pleural mesothelial cells (PMC) into active cells exhibiting myofibroblast phenotype. Heat shock proteins can act as regulator of the TGF-ß1 signaling. A role for caspase-1/IL-1ß axis has already been described in animal models of PF.The heat shock protein AlphaB-crystallin has been studied in PF at the PMC level and the importance of caspase-1/IL-1ß axis has been investigated specifically in lung structural cells in the context of bleomycin (BLM) toxicity.AlphaB-crystallin is overexpressed by PMC during idiopathic PF. Its inhibition in mice interferes with PMC transformation and subsequent migration in pleuro-pulmonary fibrosis. In BLM-induced PF in mice, caspase-1 is activated in sub-pleural areas. In vitro, caspase-1 has a crucial role in the transformation process of PMC. Activation of caspase-1 triggers fibrotic response in mice. In a second part, we show that a deglycosylated form of BLM, which failed to promote caspase-1 activation, is unable to trigger PF but stills have an anti-tumor activity. Deglyco-BLM does not induce pyroptosis, a caspase-1 dependent cell death, in alveolar epithelial cells.Our data suggest that AlphaB-crystallin and caspase-1/IL-1ß could represent interesting therapeutic targets in idiopathic as well as BLM-induced PF. We also bring a proof of concept for the use of deglyco-BLM as a less toxic alternative to BLM in cancer therapy.

Submitted by TATIANA EMY KOIKE null (tatikoike@yahoo.com) on 2016-03-10T11:09:02Z No. of bitstreams: 1 Encadernaçao Mestrado UNESP- Tatiana Emy Koike.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-10T20:19:49Z (GMT) No. of bitstreams: 1 koike_te_me_prud.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) / Made available in DSpace on 2016-03-10T20:19:49Z (GMT). No. of bitstreams: 1 koike_te_me_prud.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) Previous issue date: 2016-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Muscle injuries are often cause due to the practice of sports and recreational activities. Because of its high incidence, it is important to research the treatments that promote quality in the muscle and shorter repair process. To evaluate the effect of the combination of the therapeutic resources of Cryotherapy and Therapeutic Ultrasound in the treatment of muscle damage by impact. 55 Wistar rats was separate into groups, Acute Injury (AI), Injury (I), Cryotherapy (CR), Therapeutic Ultrasound (TU), Cryotherapy and Therapeutic Ultrasound (CRTU). All animals were anesthetize and muscle damage due to impact by the release of a load of 200 g at 30 cm. Then received treatments as allocated group and CR protocol using ice bag for 20 minutes and TU for five minutes with an intensity of 0.5W / cm2 and the frequency of 1MHz. Euthanasia was performed by intraperitoneal administration of overdose of Xylazine and Ketamine. The collection of the gastrocnemius muscle for the Body and Muscle mass analysis, histological analysis and fractal dimension of inflammation and collagen gene quantification of mRNA (TNF-α and TGF-β1). Data analysis was performed using SPSS for Windows 22. The Shapiro-Wilk test to verify the normality of the data was performed. When data showed normal, we used t test for paired samples test and one-way ANOVA followed by Tukey’s post-test. When it violated the normality of the data, followed by the Kruskal- Wallis test with Dunn’s post-test. For all analyzes was adopted the significance level of 5%. Among all groups, the CRTU lose less body and muscle mass, improved morphometry, besides presenting collagen reduction by DF compared to AI and CR (p <0.05). With regard to the inflammatory process CRTU group showed a significant reduction of DF in relation to the AI groups (p = 0.001), I (p = 0.001) and CR (p = 0.007), and TU reduced the DF significantly relative to AI groups (p = 0.001), I (p = 0.001) and CR (p = 0.036). The reduction of TNF-α was significant in TU group compared with AI groups (p = 0.008); I (p = 0.032) and CR (p = 0.046) and TGF- β1 in the CR group compared to AI (p = 0.001) and I (p = 0.006), in the TU group compared to AI (p = 0.049) and CRTU compared to AI (p = 0.023). The combination treatment was superior to the results presented by the isolated treatments in the muscle repair process. Observed by reducing the loss of body and muscle mass, improved histological appearance and reduction of collagen. / As lesões musculares são frequentemente ocasionadas em decorrência da prática de atividades esportivas e recreativas. Devido sua alta incidência, é importante pesquisar os tratamentos que promovam qualidade no processo de reparação muscular e menor duração. Avaliar o efeito da combinação dos recursos terapêuticos de Crioterapia e Ultrassom Terapêutico no tratamento de lesão muscular por impacto. 55 ratos wistar foram separados em Grupos Lesão Aguda (LA), Lesão (L), Crioterapia (CR), Ultrassom Terapêutico (US), Crioterapia e Ultrassom Terapêutico (CRUS). Todos os animais foram anestesiados e submetidos à lesão muscular por impacto pela liberação de uma carga de 200g a 30 cm de altura. Em seguida receberam os tratamentos conforme grupo alocado, sendo o protocolo de CR por meio de bolsa de gelo durante 20 minutos e o US durante cinco minutos com intensidade de 0,5W/cm2 e frequência de 1MHz. A eutanásia foi realizada por administração intraperitoneal de superdosagem de Xilazina e Ketamina, para subsequente coleta do músculo gastrocnêmio destinado às análises de massa Corporal e Muscular, análises Histológica e Dimensão Fractal do processo inflamatório e de colágeno, Quantificação gênica de RNAm (TNF-α e TGF-β1). A análise dos dados foi realizada utilizando o programa estatístico SPSS 22 for Windows. Foi realizado o teste de Shapiro-Wilk para verificação da normalidade dos dados. Quando os dados apresentaram normalidade, foi utilizado teste T para amostras pareadas e teste de Anova one-way, seguido pelo pós-teste de Tukey. Quando violada a normalidade dos dados, seguiu-se com o teste de Kruskall-Wallis, com pós-teste de Dunn. Para todas as análises foi adotado o nível de significância de 5%. Dentre todos os grupos, o CRUS perdeu menos massa corporal e muscular, melhora da morfometria, além de apresentar redução de colágeno pela DF em comparação aos LA e CR (p < 0,05). Com relação ao processo inflamatório, grupo CRUS apresentou redução significante da DF em relação aos grupos LA (p = 0,001), L (p = 0,001) e CR (p = 0,007), e o US reduziu a DF significativamente em relação aos grupos LA (p = 0,001), L (p = 0,001) e CR (p = 0,036). A redução de TNF-α foi significante no grupo US comparado com grupos LA (p = 0,008); L (p = 0,032) e CR (p = 0,046), e TGF-β1 no grupo CR em comparação aos LA (p = 0,001) e L (p = 0,006), no grupo US em comparação ao LA (p = 0,049), e CRUS em comparação aos LA (p = 0,023). A associação de tratamentos foi superior aos resultados apresentados pelos tratamentos isolados no processo de reparação muscular. Observado pela redução da perda de massa corporal e muscular, melhora do aspecto histológico e redução de colágeno.

Background Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and Methods In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. Results Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. Conclusions Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease worldwide and occurs in up to 40% of patients with diabetes. The standard of care for DKD treatment has not kept up with the current health epidemic, which has led to a heavy economic toll and substantial health burden. Targeting either cell surface (cs)GRP78, activated α2-macroglobulin (α2M*) or preventing their interaction may provide a novel anti-fibrotic therapeutic target for the treatment of DKD and potentially non-diabetic chronic kidney disease (CKD) as well. Previously our lab has shown that HG-induced csGRP78 is a mediator of PI3k/Akt signaling and downstream extracellular matrix (ECM) protein production in glomerular mesangial cells (MC). However, the ligand responsible for activating high glucose (HG)-induced csGRP78 had not yet been determined. We have shown thus far that α2M is endogenously produced, secreted, and activated (denoted α2M*) in HG by MC, which leads to its binding to and activation thereof csGRP78. Further, α2M knockdown or α2M* neutralization attenuated Akt activation, the production of the profibrotic cytokine connective growth tissue factor (CTGF) and ECM proteins fibronectin and collagen IV. We have also shown that integrin β1 (Intβ1), a transmembrane receptor, associated with csGRP78 under HG conditions and likely acts as a tether to present csGRP78 completely extracellularly on MC. Interestingly, Intβ1 activation, even in the absence of HG, was sufficient to induce csGRP78 translocation. Further, inhibition of either csGRP78 or Intβ1 prevented synthesis, secretion and signaling of TGFβ1. This data implicates a role for Intβ1 as a required signaling partner for csGRP78-mediated profibrotic signaling. To further our understanding of csGRP78/ α2M*’s role in DKD, we investigated their ability to mediate TGFβ1 signaling through its non-proteolytic activator thrombospondin-1 (TSP1). Here, HG-induced TSP1 expression, ECM deposition, and activation of TGFβ1 was regulated by the PI3k/Akt pathway via csGRP78/α2M* in MC. Furthermore, we assessed whether this csGRP78/ α2M* axis is relevant to promoting profibrotic signaling in other renal cell types, including proximal tubule epithelial cells (PTEC) and renal fibroblasts (RF), that contribute to the pathogenesis of both later stage DKD and non-diabetic CKD. We show evidence here that HG and direct treatment with TGFβ1, a key pathologic regulator of kidney fibrosis, induce GRP78 surface translocation as well as the endogenous production and activation of α2M in both PTEC and RF. Inhibition of either csGRP78 or α2M* prevented TGFβ1 signaling measured as Smad3 activation as well as downstream ECM production. Interestingly, inhibition of this pathway under direct TGFβ1 treatment did not prevent Smad3 activation, implicating a role for Smad-independent TGFβ1 signaling through this axis. We identified the known noncanonical TGFβ1 signaling partners, yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are mediated by csGRP78 and α2M*. Lastly, we evaluated the potential therapeutic benefit of inhibiting csGRP78/α2M* interaction in the kidney fibrosis model, unilateral ureteral obstruction (UUO). Here, we show evidence that inhibition of this signaling axis using an inhibitory peptide can prevent renal fibrosis. Whether this peptide also prevents fibrosis in DKD is currently being assessed. Together, these studies strongly implicate targeting csGRP78/α2M* interaction as a novel anti-fibrotic therapeutic intervention for early and late stage DKD, as well as a potential role in non-diabetic CKD. / Thesis / Doctor of Philosophy (Medical Science) / Diabetic kidney disease is the leading cause of kidney failure in developed nations. This progressive disease leads to the loss of kidney function due to an accumulation of scar proteins in the kidney over time. High glucose is a major factor that causes this to occur. Our lab studies specific kidney cells called mesangial cells, proximal tubule epithelial cells, and fibroblasts that produce scar proteins in the presence of high glucose. We have shown that when these cells are treated with high glucose, this causes the movement of a protein called GRP78 that normally resides inside the cell to move to the cell’s surface where it can interact with other proteins. My research has established that the proteins alpha 2-macroglobulin (ɑ2M), integrin β1 (Intβ1), and thrombospondin-1 (TSP1) can bind to GRP78 on the cell surface and cause cells to make scar proteins. Preventing ɑ2M or Intβ1 from binding to GRP78 or preventing TSP1 production prevents mesangial cells from making scar proteins when exposed to high glucose. In a mouse model that overproduces these scar proteins, we showed that preventing cell surface GRP78 and α2M interaction prevents scar protein production and is thus a novel potential treatment option for kidney disease.

Patients with classical vitiligo lose partially their protecting inherited pigment. The cause of the disease is still unknown. Despite massive epidermal oxidative / nitrative stress and signs for DNA-damage in the skin and in the plasma, these patients have no higher prevalence for sun induced non-melanoma skin cancer and increased photo-damage. Protection and DNA-repair have been attributed to a functioning up-regulated wild type p53 / p21 cascade in association with up-regulated p76 MDM2. As some patients with cutaneous melanoma develop depigmentations away from their primary tumour site post surgical excision, it became of our interest, whether this melanoma associated leucoderma (MAL) is the same as classical vitiligo. The purpose of this thesis was two-fold. In part I, we wanted to further substantiate the reasons behind the constantly up-regulated wild-type functioning p53 / p21 cascade in classical vitiligo utilising a panel of proteins with direct and / or indirect action on p53 regulation, including p21, p76MDM2, MDM4/MDM4phospho, SPARC, VEGF-A and TGF-β1. In part II, we wanted to characterize MAL and compare this peculiar leucoderma with classical vitiligo using the same protein panel and methodologies. To achieve our goals, we used in vivo FT-Raman spectroscopy, in vitro cell cultures, in vitro and in situ immuno-fluorescence labelling, Western blot, dot blot and computer modelling techniques. Our data showed distinct differences between classical vitiligo and MAL. Our results in MAL exhibited a concentration dependent protein expression gradient between the basal / suprabasl layers and the upper layers of the epidermal compartment using catalase, ONOO-, p53, p21, MDM4, p76MDM2, TGF-β1 and VEGF-A expression gradient. Moreover, we document for the first time the presence of a nitrated non-fuctional SPARC protein in classical vitiligo which is absent in MAL. Although we show in vivo considerable ROS / RNS- mediated stress in MAL and classical vitiligo documented by FT-Raman spectroscopy, Western blot and in situ immuno-fluorescence, our results prove that MAL and classical vitiligo are two distinct entities.