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The Role of Exogenous Somatotropin, Ovariectomy and Extracellular Matrix in Bovine Mammary Gland DevelopmentHuderson, Brandy Patrice 09 March 2010 (has links)
The highly regulated maturation of the mammary gland is poorly understood. Our studies were designed to further characterize the role of ovarian hormones, growth hormone (GH)/IGF axis proteins and extracellular matrix (ECM) in the growth and development of prepubertal mammary glands. Prepubertal heifers were injected with either exogenous GH or subjected to ovariectomy (OVX). Mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested for DNA, protein, lipid, and western blot analysis. Remaining tissues were preserved for histological staining or snap frozen for quantitative real-time PCR. We examined 13 genes that work in conjunction with the extracellular matrix to regulate mammary proliferation and morphogenesis. Administration of GH, while impacting composition of MFP, had no effect on expression of the selected genes; there was a decrease in expression of fibronectin in PAR. Ovariectomy had no effect on gene expression in MFP but decreased expression of epimorphin, a potent regulator of morphogenesis, in PAR. In both experiments, the presence of a 55 kDa band corresponding to androgen converting enzyme aromatase was detected but its expression was unaffected. In another study, we used in vitro cell culture to evaluate the role of ECM in mammary gland maturation and employed quantitative real-time PCR to evaluate gene expression profiles of select genes involved in proliferation and differentiation. Expression of Rac1 was decreased in response to bovine insulin (BI) but increased on collagen I (Col). Expression of aldehyde dehydrogenase was decreased in BI and serum on plastic and on Col in the presence of BI. Expression of IGF binding proteins (BP) 3, -4, and -6 were decreased in the presence of serum on laminin (LM). Also, IGF-BP2 expression was decreased on Col while IGF-BP6 was increased on LM with BI. Clusterin, a ubiquitous non-adhesive ECM protein was not affected by ECM substrate but did increase over time. In conclusion, we propose that the mammary gland is not able to respond to GH at this age and that while OVX did effect the expression of some genes, the presence of aromatase maintained local estrogen concentrations. Furthermore, ECM alone is insufficient to regulate mammary gland development and growth. / Ph. D.
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Potentiel probiotique des bactéries lactiques de l'écosystème mammaire bovin contre les mammites à Staphylococcus aureus / Probiotic potential of lactic acid bacteria from bovine mammary ecosystem against Staphylococcus aureus mastitisBouchard, Damien 14 November 2013 (has links)
Staphylococcus aureus est un des pathogènes majeurs impliqué dans les mammites chez les ruminants. Il engendre des tableaux cliniques très variable, allant des mammites subcliniques aux mammites gangréneuses, mais les mécanismes de cette variabilité restent encore mal connu. La volonté de réduire l'utilisation d'antibiotiques en médecine vétérinaire ainsi que leurs faibles efficacités contre les mammites staphylococciques soulignent la nécessité de développer de nouvelles stratégies alternatives. L'une des particularités des infections intramammaires à S. aureus est leur pouvoir chronique et persistent dont l'une des principales causes est la capacité de S. aureus à adhérer et internaliser dans les tissus de l'Hote. L'un des concepts ayant fait ses preuves chez l'Homme et l'animal se base sur le concept de la lutte biologique en utilisant les propriétés inhibitrices des bactéries dites « probiotique » d'un écosystème pour réduire, prévenir ou traiter les infections. Cette étude vise à mieux caractériser le microbiote mammaire et notamment la composante lactique qui par analogie avec les autres écosystèmes jouent un rôle positif sur la santé de l'Hôte et de déterminer son potentiel à moduler l'adhésion et l'internalisation de S. aureus. L'ensemble de ce travail a été réalisé avec deux souches de S. aureus très différentes au niveau des tableaux cliniques engendrés. La souche RF122, très virulente et la souche Newbould 305, induisant des infections chroniques. Deux messages clairs ressortent des analyses indépendantes du pathogène et du microbiote : d'une part, l'édute comparative in silico et in vitro révèle que l'invasion cellulaire semble être majeur pour la chronicité de la souche Newbould 305 et d'autre part que l'écosystème de la glande mammaire présente une composante lactique ayant des capacités probiotiques intéressantes. Dans notr! e modèle d'interaction tripartite, trois acteurs ont été retenus, S. aureus, Lactobacillus casei et les cellules épithéliales mammaires MAC-T. Dans ce système, L. casei affecte l'adhésion et surtout l'internalisation de S. aureus sans modifier la viabilité et la morphologie des cellules en culture. Les mécanismes identifiés dans l'inhibition semblent dépendant d'un contact direct avec des L. casei viables et permettent de poursuivre la caractérisation des bases moléculaires de ce phénomènes impliquant une modulation des gènes de virulence et cellulaire lors d'interaction avec une flore compétitrice. Ces résultats ouvrent de nouvelles perspectives dans l'application de bactéries lactiques en tant que probiotique mammaire. / Staphylococcus aureus is one of the main pathogen involved in ruminant mastitis. Severity of bovine S. aureus mastitis is highly variable, from subclinical to gangrenous mastitis. Nevertheless S. aureus factors that may explain this variability still poorly documented. The need to reduce antibiotics in veterinary medicine as well as their low efficiencies against staphylococcal mastitis underline the necessity to develop new alternative strategies. One of the particularity of S. aureus infections is his ability to persist within the mammary tissue and induce chronicity of the infection. One of the main causes is the ability of S. aureus to adhere to and internalize into host cells. The concept of biological control, using natural inhibitory properties of lactic acid bacteria as probiotics, are now well etablished in human and animal ecosystem for their ability to reduce, prevent or treat bacterial infections. This study aims to better characterize the mammary microbiota in p! articular the lactic component which by analogy with the other ecosystems play a positive role in the host health and to determine his potential to modulate adhesion and internalization of S. aureus. This work was realized with two S. aureus strains with different severity degree. The virulent strain RF122 and the Newbould 305 one, involved in chronic mastitis. Two main messages came out from the independent analyses of the pathogen and the microbiota: on one hand, the in silico and in vitro comparison of the two strains reveals that the cellular invasion propress seems to be major for the chronicity of the Newbould 305 strain and on the other hand that the bovin mammary ecosystem presents a lactic biodiversity with interesting probiotic capacities. In our model of tripartite interaction, three actors were retained, S. aureus, Lactobacillus casei and the mammary epithelial cells MAC-T. In this system, L. casei affect the adhesion and internalization of S. aureus without mod! ifying the viability and the morphology of cells in culture. Mechanisms identified in the inhibition seem dependent on a direct contact with live L. casei and allow to continue the molecular basis of the virulence and cells genetic expression modulation during interaction with a competitive flora. These results open new perspectives in the application of lactic acid bacteria as mammary probiotic.
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Growth Hormone Alters Components Related to Differentiation, Metabolism and Milk Synthesis and Secretion in MAC-T CellsJohnson, Tasha Lynn 01 June 2010 (has links) (PDF)
The mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin and is extensively used to study bovine mammary epithelial cell function. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and induce cytoskeletal rearrangement in 3T3 fibroblast cell line and a Chinese hamster ovary (CHO) cell line. Few studies have focused on identifying the mechanisms involved in differentiated MAC-T cells’ response to GH. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations in milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. To identify mechanisms that are involved in MAC-T cells’ response to GH, global protein was assessed through two-dimensional gel electrophoresis and differentially expressed proteins were identified through mass spectrometry. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium increased production of α-S1 casein and α-lactalbumin mRNA. Proteins that were differentially expressed are related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, detoxifying and calcium metabolism. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and supports GHs involvement in differentiation, while altering cell metabolism in preparation for synthesis and secretion of milk components.
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Microphysiometry Studies of Rapid Binding of Insulin-Like Growth Factor I by Parental and Transfected Mammary Epithelial Cell LinesRobinson, Rose Marie 13 November 1998 (has links)
Breast cancer is a leading cause of cancer death of women in the U.S. today. Members of the family of insulin-like growth factors (IGFs) are proposed to play a major role in the development and subsequent uncontrolled proliferation of breast cancer cells. Insulin-like growth factor-I (IGF-I) is known to be a potent mitogen for mammary epithelial cells. IGF-I acts by binding to cell surface receptors, thereby stimulating a cascade of events leading to cell division. In the interest of interrupting the effect of IGF-I on cancerous mammary epithelial cells, an understanding of how IGF-I behaves in the presence of other extracellular components is needed. This study examines the IGF-I response of SV40-IGF-I, an immortalized bovine mammary epithelial cell line which secretes IGF-I constitutively.
The microphysiometer allows real-time sampling of cellular activity by measuring the excretion of protons from a sample of cells stimulated by IGF-I binding. The contributions of other factors in enhancing or suppressing stimulation can be compared by examining the pH response of cells exposed to IGF-I in the presence of these factors. We present data showing the stimulatory effect of IGF-I in a dose dependent manner on the SV40-IGF-I cell line.
In addition, we compare IGF-I stimulation with stimulation by long R3IGF-I, a substituted analogue of IGF-I having a reduced binding affinity for the IGF binding proteins. We examine the effect of insulin-like binding protein-3 (IGFBP-3) both in the presence and absence of IGF-I, finding no IGF-I independent effect in the rapid binding experiment and no effect on stimulation of IGFBP-3 pre-incubated cells by subsequent IGF-I challenge. This is of particular interest due to recent work demonstrating an IGF-independent IGFBP-3 response in a number of cell lines. Binding studies to correlate with the rapid binding stimulation show binding of the IGFBP-3 molecule with high affinity to a small number of surface receptors on the SV40-IGF-I cell.
Analysis of the extracellular environment and the components contributing to the binding of IGF-I to the cell membrane receptor will provide information for the development of interventions to slow or interrupt the process of IGF-I binding and therefore cancer growth. Optimization of the Cytosensor(r) Microphysiometer System for the (transfected) SV40-IGF-I and the (parental) MAC-T cell lines was achieved to continue comparison studies of autocrine and paracrine stimulation of bovine mammary epithelial cells by IGF-I.
This work was supported by the Whitaker Foundation Biomedical Engineering Grant. / Master of Science
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In Vitro Binding and Transport Regulation by Endothelial Cells: Preliminary Studies looking at FIX and IGF-ISutton, Amanda 13 April 2005 (has links)
Endothelial cells separate the bloodstream from the underlying tissue and play a crucial role in vascular homeostasis. They also form an important barrier for vascular drug delivery. This thesis contains preliminary studies targeted at understanding the mechanisms of binding and transport across endothelial cells cultured in vitro. Specifically, the first study investigates how the recombinant source of Factor IX (FIX), a blood coagulant protein used in the treatment of Hemophilia B, impacts surface ligand binding (FIX to its specific receptors) to bovine aortic endothelial cells (BAECs). Competitive binding experiments between 125I-FIX and FIX were undertaken to quantify the interaction of recombinant and transgenic FIX with BAECs and human collagen IV and determine if there was a measurable difference in binding affinity. Results indicate limited specific binding of 125I-FIX to BAECs and no binding to human collagen IV. Concrete conclusions were not drawn from this data due to technical issues during the experimental process. The second study investigates insulin-like growth factor-I (IGF-I) transport across both BAEC and MAC-T cells, a mammary epithelial cell line, cultured on tissue culture inserts. IGF-I is a circulatory growth factor implicated in the regulation of cell division and tissue proliferation. Competitive binding experiments between 125I-IGF-I and unlabeled protein (IGF-I, Y60L-IGF-I, a mutant of IGF-I, and IGF Binding Protein-3 (IGFBP-3)) were undertaken to quantify the binding and transport of IGF-I under various experimental conditions. Results confirmed earlier work from the Williams' laboratory indicating that 125I-IGF-I transport was enhanced by incubation with its non-receptor-binding analog, Y60L-IGF-I, but cell surface associated 125I-IGF-I was decreased by its presence. Other studies were undertaken but conclusive results could not be drawn. / Master of Science
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Regulation of Bovine Mammary Epithelial Cell Response by Autocrine IGF-I and by Collagen IRobinson, Rose Marie 24 August 2006 (has links)
Understanding how insulin-like growth factor-I (IGF-I) signaling in mammary epithelial cells may be modified or interrupted by modifications in the cellular environment may lead to 1) methods to increase the growth and proliferation of normal mammary epithelial cells for an increase in the amount of milk produced on a per animal basis or to 2) the development of medical interventions to disrupt the growth and proliferation of cancerous mammary epithelial cells. IGF-I, a signaling protein provided by stromal cells and through the bloodstream, stimulates the proliferation of mammary epithelial cells and is crucial for mammary development. Collagen I is an extracellular matrix protein (ECM) found in skin and in other connective tissues throughout the body. The guiding question in this dissertation was how IGF-I signaling and how binding protein profile were influenced by autocrine IGF-I and by collagen I. The MAC-T cell line was chosen as the cell model utilized in these investigations because it is an immortalized bovine mammary epithelial cell line known to retain hormonal responsiveness to IGF-I.
It was hypothesized that the production of IGF-I by mammary epithelial cells (autocrine secretion) would alter the response of these cells to additional IGF-I by de-sensitizing the IGF-I receptor on the cell surface. The normal mammary epithelial cell does not produce IGF-I and responds to IGF-I supplied either by stromal cells (paracrine pathway) or through the bloodstream (endocrine pathway). The IGF-I secreting bovine mammary epithelial cell line was investigated for the response of the cells to autocrine IGF-I, and the response was compared to the normal, parental cell line. To examine the effect of autocrine IGF-I on the cells, IGF-I was added both to MAC-T cells and to cells transfected to secrete IGF-I (SV40-IGF-I). The cell response of the two cell lines was compared using microphysiometry, a tool that measures IGF-IR stimulation by detecting resultant extracellular acidification. It was found that the SV40-IGF-I cell line retains IGF-I receptor sensitivity, yet, unlike the parental cell line, does not proliferate in response to IGF-I. Both cell lines exhibited increased protein synthesis in response to IGF-I as measured by amino acid uptake (AIB incorporation), but the lack of a proliferation response to additional IGF-I in the SV40-IGF-I cell line suggested that the autocrine cell line exhibited an un-coupling of IGF-IR stimulation with downstream cell proliferation. Both autocrine IGF-I and added IGF-I increased the amount of IGFBP-3 secreted by the cells into growth media.
Additionally, it was hypothesized that the presence of collagen I, an important ECM protein, would alter the cell production of insulin-like binding protein-3 (IGFBP-3), a protein that modulates IGF-I interaction with the IGF-I receptor (IGF-IR). The literature reports that surface substrate can affect the phenotypic expression of cells, presumably via interaction with integrins, the cell surface receptors that connect cells to ECM proteins and that are responsible for cell adhesion and for cell migration. It was hypothesized that the MAC-T cells would interact with a collagen I surface (possibly via the a2b1 integrin) and that the stimulation of this transmembrane signaling molecule would in turn impact the IGF-I signaling pathway. Comparison studies on tissue culture plastic, collagen I BIOCOAT, and collagen I gel were performed. It was found that collagen I gel increased IGFBP-3 secretion and decreased insulin-like binding protein-2 (IGFBP-2) secretion in MAC-T cells. The collagen I BIOCOAT did not induce this response.
Additional studies were performed to determine if there were differences in IGF-IR phosphorylation, exogenous IGF-I utilization, and IGFBP mRNA production by cells cultured on the three different substrates. IGF-IR phosphorylation was only evident following the addition of IGF-I to MAC-T cells on all three substrates. Measurement of residual IGF-I present in the cultured media of cells on all three substrates by radioimmunoassay did not reveal any differences in the amount of IGF-I present. Northern blot analysis revealed that the addition of IGF-I caused an increase in detected IGFBP-3 mRNA and a decrease in detected IGFBP-2 mRNA across all three surfaces. As measured by ligand blot analysis, cells cultured on all three surfaces showed an increase in IGFBP-3 protein in the media with IGF-I addition, and the collagen I gel showed more IGFBP-3 protein than the other two surfaces. However, cells cultured on collagen I gel showed a decrease in IGFBP-2 protein expression compared to cells cultured on tissue culture both with and without the addition of IGF-I. Cells cultured on tissue culture plastic and on collagen I BIOCOAT did not show a decrease in IGFBP-2 to correspond with the decreased IGFBP-2 mRNA detected in the presence of IGF-I on all three substrates. DNA assays to detect cell proliferation revealed no differences in cell DNA content in the absence of exogenous IGF-I and revealed similar increases in response to IGF-I addition on all three substrates.
In conclusion, it was found that autocrine IGF-I un-couples increased IGF-IR stimulation by exogenous IGF-I from a downstream cell proliferation response. IGFBP-3 inhibits the ability of IGF-I to interact with the IGF-IR in MAC-T cells and inhibits subsequent cell proliferation. Collagen I gel increases IGFBP-3 secretion and decreases IGFBP-2 secretion by MAC-T cells.
The relevance of this work is that it adds to the body of knowledge in understanding cellular function in mammary epithelial cells. It is known that the growth and the maintenance of living tissue are dependent on an intricate system of intercellular and intracellular responses which are orchestrated by the movement and secretion of proteins and other molecules. Goals of understanding mammary epithelial cell function include having the means to find ways to increase cell functionality via bioengineering and having the means to find ways to restore cells to normal function in disease processes such as cancer. / Ph. D.
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