Despite of all the research going on for the treatment of ocular diseases, age-related macular degeneration (AMD) remains one of the serious vision threatening disease worldwide. Choroidal neovascularization, a pathophysiological characteristic of wet AMD, is the growth of anomalous blood vessels in the eye choroidal layer. Neovascularization is a key factor in AMD and thus anti-angiogenic therapy is beneficial in reducing the development of new abnormal blood vessels to prevent progression of AMD. Axitinib, multi-receptor tyrosine kinase inhibitor, is a small molecule that works by blocking vascular endothelial growth factor receptors (VEGFR) and platelet derived growth factor receptors (PDGFR) responsible for developing neovascularization. Thus, goal of this study was to develop and characterise a sustained release formulation of Axitinib loaded poly (lactic-co-glycolic) acid (PLGA) nanoparticles. The nanoparticles were characterized for particle size and zeta potential as well as using DSC, TEM and in vitro drug release profile. The cytotoxicity of the formulation was evaluated on human retinal pigmented epithelium ARPE19 cells by MTT assay. The cellular uptake, anti-migration assay, and VEGF expression levels were found out in vitro using cells. The optimized formulation was 131.33 ± 31.20 nm in size with -4.63± 0.76 mV zeta potential. Entrapment efficiency was found to be 87.9 ± 2.7%. The cytotoxicity of ARPE19 cells was less than 12% for nanoparticles suggesting the in vitro compatibility at 10 µM concentration of drug. Cellular uptake, anti-migration assay and VEGF expression levels for the nanoparticles had greater uptake, had significant anti-angiogenic potential and exhibited inhibition of VEGF activity. The results showed successful development of axitinib loaded PLGA nanoparticles as an alternative potential treatment option for AMD.

Neural stem cells are capable of differentiating into three major neural cell types, but the underlying molecular mechanisms remain unclear. Here, we investigated the mechanism by which integrin β4 modulates mouse neural stem cell differentiation in vitro. Inhibition of endogenous integrin β4 by RNA interference inhibited the cell differentiation and the expression of fibroblast growth factor receptor 2 but not fibroblast growth factor receptor 1 or fibroblast growth factor receptor 3. Overexpression of integrin β4 in neural stem cells promoted neural stem cell differentiation. Furthermore, integrin β4-induced differentiation of neural stem cells was attenuated by SU5402, the inhibitor of fibroblast growth factor receptors. Finally, we investigated the role of integrin β4 in neural stem cell survival: knockdown of integrin β4 did not affect survival or apoptosis of neural stem cells. These data provide evidence that integrin β4 promotes differentiation of mouse neural stem cells in vitro possibly through fibroblast growth factor receptor 2.

Most existing studies of receptor signaling are qualitative, which can lead scientists to misinterpret or overlook key information about the extent and timing of key events. To overcome these shortcomings, we have applied quantitative approaches to characterize receptor activation and signaling events. Most signaling studies focus on events occurring at a particular level in the system (e.g., on the membrane, at the level of phosphorylation of intracellular signaling molecules, or at the level of transcription). Instead, we are interested in taking a longitudinal view of signaling by achieving a quantitative understanding of a single signaling pathway from initial stimulation of the receptor by its growth factor (GF) ligand, through to gene expression, and functional cellular responses. As a model system for our studies, we used the growth factor receptor tyrosine kinase, REarranged during Transfection (RET), which requires a ligand and a glycosylphosphatidylinositol-anchored co-receptor for activation. RET mediates the response of cells to members of the glial cell-line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. We have characterized the molecular mechanisms of RET activation and signaling by pursuing the following four aims: 1) We developed a sensitive and robust luciferase reporter gene assay for RET signaling. 2) We characterized the dynamic relationship between receptor activation and downstream signaling events, including gene transcription and translation of three target genes. 3) We used the reporter gene assay, and other detection approaches, to test and quantify crosstalk between RET and other GF receptors. 4) We developed a FRET reporter system to enable monitoring of the assembly of the activated RET receptor complex on cells, as a means to distinguish between ligand-induced oligomerization and pre-associated oligomer mechanisms. Through these four aims, we have established new methods to quantitatively elucidate mechanisms of GF receptor activation, new insights into how signals are propagated from the receptor to the nucleus and into a functional response, and have established crosstalk between RET and other GF receptor pathways.

The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Recent studies show that CaSR suppresses the malignant phenotype through a variety of pathways that inhibits growth and promotes differentiation. CaSR also promotes cytotoxic response to fluorouracil. These studies, taken together, have led me to formulate the following working hypotheses: (i), CaSR is a robust inducer of differentiation by virtue of its ability to activate and integrate diverse growth and differentiation control signals; (ii), loss of CaSR expression enable cellular escape from CaSR control and (iii), loss of CaSR expression is an underlying mechanism of malignant transformation, progression and drug resistance in colon cancer. Previous studies showed that there are endogenous small subpopulations that do not express CaSR in colon carcinoma cell lines. These cells are highly drug resistant. Indeed, immunocytochemical analyses of CaSR showed that the expression of CaSR in both the CBS and HCT116 colon carcinoma cell lines are heterogeneous. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). In order to further test my hypotheses, the isolation and characterization of CaSR null cells are required. Here, I report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional (EMT) molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while the expression of oncogenic micro RNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. I conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer. I discovered that CaSR null cells, cultured in defined human embryonic stem cell culture medium, can be induced to differentiate and acquire CaSR expression when the medium of the null cells was changed to conventional cell culture medium containing fetal bovine serum. I hypothesize that expression of CaSR can alter the phenotype of the CaSR null cells. The objectives of this study were then three folds: (i), determine if induction of CaSR expression could circumvent the molecular phenotype of the CaSR null cells; (ii), determine if CaSR was required in altering the null phenotype and (iii), determine the underlying mechanism of CaSR induction. I hypothesize that if CaSR is a strong promoter of differentiation, then without CaSR, the constraint exerted by CaSR will not be functional and pathways normally inhibited by CaSR will be activated. I found that induction of CaSR expression led to a more indolent phenotype which includes the acquisition of epithelial morphology, down-regulated expression of cancer stem cell markers, down-regulated expression of thymidylate synthase and survivin and increased sensitivity to fluorouracil. Molecular profiling also revealed that the induction of CaSR expression was linked to a down-regulated expression of EMT molecules, EMT associated transcription factors and oncogenic miRNAs with a concurrent up-regulated expression of tumor-suppressive molecules. With the exception of the cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, was directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. I further report that demethylation of the CaSR gene promoter underlie CaSR induction. I conclude that induction of CaSR expression in CaSR null cells resulted in a more indolent phenotype concurrent with a variety of molecular changes and that these changes (with the exception of stem cell markers) are dependent on the expression and function of CaSR. I further conclude that methylation of the CaSR gene promoter is an underlying mechanism of maintaining the CaSR null phenotype while promoter demethylation is an underlying mechanism responsible for CaSR induction. CaSR null is a phenotype of the rapidly proliferating, undifferentiated crypt stem cells at the base of colonic crypts. Differentiation of crypt stem cells toward the apex of a crypt (in the direction of the lumen), on the other hand, is tightly linked to CaSR expression. What induces CaSR expression as the crypt stem cells migrate up the crypts is unknown. I hypothesize that as the colonic crypt stem cells migrate up the crypt, they become increasingly exposed to the colonic fluid in the lumen and components in the colonic fluid can trigger the induction of CaSR expression. Both Ca2+ and vitamin D are good candidates because either Ca2+ or vitamin D can stimulate CaSR expression in the parental CBS and HCT116 human colon carcinoma cells. Certainly, Ca2+ and vitamin D are not the only components involved in regulating CaSR expression. A variety of minerals in the colonic fluid may also serve as good candidates in the induction of CaSR. Of interest is Aquamin, a calcium-rich mineralized extract from the red marine algae, Lithothamnion calcareum, which has been shown to induce differentiation in colon carcinoma cells and possess chemopreventive properties against colon polyp formation in mice fed a high fat diet. CaSR null cells cultured in defined human embryonic stem cell culture medium were used to test this hypothesis because they offer an in vitro model in determining the triggers and the underlying mechanisms of CaSR induction that may resemble that of the colonic crypt stem cells in vivo. I found that all three agonists (Ca2+, vitamin D and Aquamin) induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental cells which express a heterogeneous mixture of cells with different level of CaSR expression. These agonists also induced CaSR mRNA and protein expression and inhibited cellular proliferation in the homogeneous isolated CaSR null cells. In both cases, Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists in the CaSR null cells resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. Induction of CaSR expression resulted in a down-regulated expression of tumor inducers and up-regulated expression of tumor suppressors in the CaSR null cells. Again, Aquamin was found to be most potent in this regard. Taken together, I conclude that nutrients are good candidate in the induction of CaSR and differentiation in colonic epithelia cells. Similar to CaSR, transforming growth factor β (TGFβ) is also a robust promoter of differentiation in the colonic epithelium. The expression profile of both CaSR and TGFβ in the colonic epithelium is tightly linked to differentiation. Both CaSR and TGFβ expression progressively increases as the undifferentiated crypt stem cells migrate and differentiate toward the apex of a crypt in the direction of the lumen. Similar to the loss of CaSR in cancer cells, loss of TGFβ responsiveness has long been considered an underlying mechanism of early colon carcinogenesis. I hypothesize that there is functional linkage between CaSR and TGFβ function. Human colonic epithelial CBS cells originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer an opportunity to determine the functional linkage (if any) between CaSR and TGFβ. I found that knocking down CaSR expression in the CBS cells abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca2+ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca2+ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR, there was no up-regulation of TGFβ receptor. I conclude that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function. In summary, my research has revealed the important role of CaSR in controlling differentiation. CaSR also function as a robust tumor suppressor. My study clearly discerns the multifarious molecular signaling cascades involved in CaSR function and that methylation and demethylation regulates CaSR expression. My work has also established the importance of CaSR in the chemoprevention of colon cancer. My thoughts in regard to future studies and the potential role that CaSR could play in the management of colon cancer are given in the perspective section of this dissertation.

No description available.

No description available.

The objective of the study reported here was to document the distribution of epidermal growth factor receptor (EGFr) and quantitate receptor density in normal as well as ulcerated equine gastric squamous mucosa. Fifteen horses with endoscopically normal stomachs were divided into three equal groups. Group 1 was a normal control. A protocol that alternated 24 hour periods of free-choice hay with 24 hours of feed deprivation was utilized to induce squamous mucosal gastric ulceration in Group 2 (48 hours total off-feed) and Group 3 (96 hours total off-feed). Gastric tissue was collected from 3 stomach locations at post-mortem examination and an avidin-biotin immunoperoxidase technique was developed to stain the formalin-fixed tissue for EGFr. A computerized image analysis system was used to measure EGFr area and mean intensity values at four sites within the epithelium from the basal cell layers to the lumen in the ulcer/erosion margin, erosion bed, and 10-14 mm distant from the lesion. / Master of Science

There is a growing interest in using naturally-occurring compounds as cancer chemopreventive or chemotherapeutic agents. This study investigated the anticancer potential of the graviola fruit extract (GFE) on specific human breast cancer (BC) cells. GFE was found in our preliminary screening to selectively inhibit the growth of certain human BC cells (MDA-MB-468) but did not affect non-transformed breast epithelial MCF-10A cells. GFE treatment was very effective against the growth of MDA-MB-468 BC cells with an IC50 of 4.8 µg/ml. In vitro, effects of GFE treatment on MDA-MB-468 BC cells were further examined for apoptosis and cell proliferation. Apoptosis, determined qualitatively and quantitatively, was enhanced and accompanied by caspase-3 activation. GFE treatment also induced cell cycle arrest at the G1 cell cycle phase and significantly reduced the percentage of MDA-MB-468 cells in S-phase following 24h of exposure. Moreover, the results from analysis of the mRNA expression of epidermal growth factor receptor (EGFR), which plays an important role in regulating cell development and death, by qRT-PCR, suggested that GFE-induced selective growth inhibition of MDA-MB-468 BC cells is associated with a significant inhibition of EGFR gene expression in the cells. In vivo, dietary treatment with GFE significantly inhibited MDA-MB-468 tumor growth implanted in mice by reducing tumor wet weight and significantly reduced EGFR and p-ERK protein expression in tumors. Overall, GFE attenuated cell proliferation, induced apoptosis, modulated cell cycle regulation and downregulated EGFR gene expression both in vitro and in vivo. These discoveries support the further studies to fully elucidate the antitumor potential of GFE and its components as a dietary agent for BC. / Master of Science in Life Sciences

A neuraminidase 1 (Neu1) é a enzima que regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência da Neu1 é a base da sialidose, doença grave associada a um amplo espectro de manifestações, incluindo hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem degeneração muscular caracterizada principalmente por atrofia, invasão das fibras musculares por fibroblastos e expansão da matriz extracelular. A Neu1 controla a proliferação de fibroblastos de pacientes por meio da desialilação dos receptores de PDGF e IGF. Além disso, há enzimas lisossomais que são moduladas pela Neu1, tais como as catepsinas, que são capazes de degradar componentes musculares e estariam excessivamente ou erroneamente ativas (sialiladas) em decorrência da deficiência de Neu1. O objetivo deste trabalho foi identificar se o fenótipo da musculatura esquelética de camundongos Neu1-/- poderia estar associado à atividade do IGF-1R, PDGFR e/ou à sialilação de catepsina B, através da análise histológica e proteica de músculos esqueléticos e fibroblastos de camundongos Neu1+/+ e Neu1-/- tratados com inibidores de IGF1-R e PDGFR. O estudo da expressão proteica de catepsina B foi realizado nos músculos tratados com os inibidores de IGF-1R e PDGFR, e nas frações citosólica e lisossomal de fibroblastos tratados com neuraminidase exógena. Em comparação com camundongos Neu1+/+, os músculos de animais Neu1-/- apresentam menor área de fibra, peso corporal, expressão de pAkt e maior expressão de catepsina B; e os fibroblastos Neu1-/- exibem maior proliferação e expressão de pAkt. A inibição do IGF-1R em camundongos Neu1-/- aumentou a área das fibras musculares, expressão de pAKt e diminuiu a expressão de catepsina B; em relação aos fibroblastos Neu1-/-, entretanto aumentou a proliferação celular com diminuição de pAkt. A inibição do PDGFR em músculos de camundongos Neu1-/- levou ao aumento da expressão de pAkt, da área das fibras, com diminuição de pERK e catepsina L, quando comparados com os controles Neu1-/-; a mesma inibição in vitro conduziu à diminuição da expressão de pAkt, pERK e proliferação. A catepsina B encontra-se bastante ativa na fração lisossomal e o tratamento com neuraminidase foi eficaz na correção de seu peso molecular e compartimentalização lisossomal. De forma geral, o fenótipo muscular de camundongos Neu1-/- parece estar relacionado com a atividade de IGF-1R e PDGFR, e a catepsina B hipersialilada é potencialmente deletéria para o músculo esquelético / Neuraminidase 1 (Neu1) is an enzyme that regulates the catabolism of sialoglycoconjugates in lysosomes. Neu1 deficiency is the basis of sialidosis, a severe disease associated with a broad spectrum of manifestations, including hypotonia and muscle weakness. Neu1 deficient mice develop muscular degeneration characterized by atrophy, invasion of muscle fibers by fibroblasts, and expansion of the extracellular matrix. Neu1 controls the proliferation of fibroblasts from patients through the desialylation of PDGF and IGF receptors. In addition, lysosomal enzymes are modulated by Neu1, such as cathepsins, which degrade muscle components and are excessively or erroneously active (sialylated) as a result of Neu1 deficiency. The aim of this study was to identify whether skeletal muscle phenotype of Neu1-/- mice may be associated with IGF-1R, PDGFR and/or sialylation of cathepsin B, through protein and histological analysis of skeletal muscles and fibroblast from Neu1+/+ and Neu1-/- mice treated with IGF-1R and PDGFR inhibitors. The study of cathepsin B protein expression was performed in skeletal muscles treated with IGF-1R and PDGFR inhibitors, and in the cytosolic and lysosomal fractions of fibroblasts treated with exogenous neuraminidase. Compared with Neu1+/+ animals, Neu1-/- muscles showed smaller muscle fiber area, body weight, pAkt expression and higher cathepsin B expression; and Neu1-/- fibroblasts exhibited increased proliferation and expression of pAkt. The inhibition of IGF-1R Neu1-/- mice increased the area of muscle fibers, expression of pAkt and decreased expression of cathepsin B; but, considering Neu1-/- fibroblasts, there was increased cell proliferation with reduction of pAkt. The inhibition of PDGFR in muscles of Neu1-/- mice led to increased expression of pAkt, muscle fiber area, with decreased expression of pERK and cathepsin L, when compared with the Neu1-/- controls; the same inhibition in vitro led to reduced expression of pAkt, pERK and cell proliferation. Cathepsin B presented high activity in the lysosomal fraction and the treatment with neuraminidase was effective in the correction of its molecular weight and lysosomal compartmentalization. In general, the muscular phenotype of Neu1-/- mice is possibly related to IGF-1R and PDGFR activity, and oversialylated cathepsin B is potentially deleterious for the skeletal muscle

A neuraminidase 1 (Neu1) é a enzima que regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência da Neu1 é a base da sialidose, doença grave associada a um amplo espectro de manifestações, incluindo hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem degeneração muscular caracterizada principalmente por atrofia, invasão das fibras musculares por fibroblastos e expansão da matriz extracelular. A Neu1 controla a proliferação de fibroblastos de pacientes por meio da desialilação dos receptores de PDGF e IGF. Além disso, há enzimas lisossomais que são moduladas pela Neu1, tais como as catepsinas, que são capazes de degradar componentes musculares e estariam excessivamente ou erroneamente ativas (sialiladas) em decorrência da deficiência de Neu1. O objetivo deste trabalho foi identificar se o fenótipo da musculatura esquelética de camundongos Neu1-/- poderia estar associado à atividade do IGF-1R, PDGFR e/ou à sialilação de catepsina B, através da análise histológica e proteica de músculos esqueléticos e fibroblastos de camundongos Neu1+/+ e Neu1-/- tratados com inibidores de IGF1-R e PDGFR. O estudo da expressão proteica de catepsina B foi realizado nos músculos tratados com os inibidores de IGF-1R e PDGFR, e nas frações citosólica e lisossomal de fibroblastos tratados com neuraminidase exógena. Em comparação com camundongos Neu1+/+, os músculos de animais Neu1-/- apresentam menor área de fibra, peso corporal, expressão de pAkt e maior expressão de catepsina B; e os fibroblastos Neu1-/- exibem maior proliferação e expressão de pAkt. A inibição do IGF-1R em camundongos Neu1-/- aumentou a área das fibras musculares, expressão de pAKt e diminuiu a expressão de catepsina B; em relação aos fibroblastos Neu1-/-, entretanto aumentou a proliferação celular com diminuição de pAkt. A inibição do PDGFR em músculos de camundongos Neu1-/- levou ao aumento da expressão de pAkt, da área das fibras, com diminuição de pERK e catepsina L, quando comparados com os controles Neu1-/-; a mesma inibição in vitro conduziu à diminuição da expressão de pAkt, pERK e proliferação. A catepsina B encontra-se bastante ativa na fração lisossomal e o tratamento com neuraminidase foi eficaz na correção de seu peso molecular e compartimentalização lisossomal. De forma geral, o fenótipo muscular de camundongos Neu1-/- parece estar relacionado com a atividade de IGF-1R e PDGFR, e a catepsina B hipersialilada é potencialmente deletéria para o músculo esquelético / Neuraminidase 1 (Neu1) is an enzyme that regulates the catabolism of sialoglycoconjugates in lysosomes. Neu1 deficiency is the basis of sialidosis, a severe disease associated with a broad spectrum of manifestations, including hypotonia and muscle weakness. Neu1 deficient mice develop muscular degeneration characterized by atrophy, invasion of muscle fibers by fibroblasts, and expansion of the extracellular matrix. Neu1 controls the proliferation of fibroblasts from patients through the desialylation of PDGF and IGF receptors. In addition, lysosomal enzymes are modulated by Neu1, such as cathepsins, which degrade muscle components and are excessively or erroneously active (sialylated) as a result of Neu1 deficiency. The aim of this study was to identify whether skeletal muscle phenotype of Neu1-/- mice may be associated with IGF-1R, PDGFR and/or sialylation of cathepsin B, through protein and histological analysis of skeletal muscles and fibroblast from Neu1+/+ and Neu1-/- mice treated with IGF-1R and PDGFR inhibitors. The study of cathepsin B protein expression was performed in skeletal muscles treated with IGF-1R and PDGFR inhibitors, and in the cytosolic and lysosomal fractions of fibroblasts treated with exogenous neuraminidase. Compared with Neu1+/+ animals, Neu1-/- muscles showed smaller muscle fiber area, body weight, pAkt expression and higher cathepsin B expression; and Neu1-/- fibroblasts exhibited increased proliferation and expression of pAkt. The inhibition of IGF-1R Neu1-/- mice increased the area of muscle fibers, expression of pAkt and decreased expression of cathepsin B; but, considering Neu1-/- fibroblasts, there was increased cell proliferation with reduction of pAkt. The inhibition of PDGFR in muscles of Neu1-/- mice led to increased expression of pAkt, muscle fiber area, with decreased expression of pERK and cathepsin L, when compared with the Neu1-/- controls; the same inhibition in vitro led to reduced expression of pAkt, pERK and cell proliferation. Cathepsin B presented high activity in the lysosomal fraction and the treatment with neuraminidase was effective in the correction of its molecular weight and lysosomal compartmentalization. In general, the muscular phenotype of Neu1-/- mice is possibly related to IGF-1R and PDGFR activity, and oversialylated cathepsin B is potentially deleterious for the skeletal muscle