Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.

L’objectif de cette étude est d’évaluer si trois souches de levure Saccharomycescerevisiae (Lv01, Lv02, Lv03) exercent in vitro des propriétés immuno-modulatrices suite àl’exposition de cellules épithéliales intestinales porcines à Escherichia coli entérotoxinogène(ETEC). Ensuite, nous avons évalué si ces propriétés sont associées à une protection des porceletscontre une infection colibacillaire au post-sevrage et à une stimulation de l’immunité maternellepassive chez la truie.In vitro, nous avons montré que Lv01 exerce une activité anti-inflammatoire suite àl’exposition de cellules épithéliales intestinales porcines à ETEC. Cette activité réside dans 1) lasécrétion des facteurs solubles et est associée 2) à la diminution de la phosphorylation des protéinesERK1/2, p38 et 3) à l’agglutination des ETEC par Lv01. Cependant, Lv01 ne prévient pasl’altération de la barrière épithéliale induite par ETEC.In vivo, à la différence d’expérimentations antérieures, les levures Lv01 et Lv02 neprotègent pas les porcelets contre une colibacillose expérimentale au post-sevrage. Par ailleurs, lestrois souches de levure stimulent l’accroissement des IgG et IgA dans le colostrum et le lait destruies suggérant la transmission d’une meilleure protection immunitaire aux porcelets. / The objective of this work is to investigate whether three Saccharomyces cerevisiaeyeast strains (Lv01, Lv02, Lv03) induce in vitro immuno-modulatory properties in pig intestinalepithelial cells exposed to enterotoxigenic Escherichia coli (ETEC). Thereafter, we assessedwhether these properties are associated to a piglet protection against post-weaning colibacillosisand to a stimulation of maternal passive immunity in sows.In vitro, we have shown that Lv01 induces an anti-inflammatory activity in pig intestinalepithelial cells exposed to ETEC. This activity is induced by 1) secreted soluble factors and isassociated with 2) a decrease of both ERK1/2 and p38 phosphorylation and 3) an agglutination ofETEC by Lv01. However, Lv01 does not prevent the alteration of epithelial barrier integrityinduced by ETEC.In vivo, we have shown that in contrast to previous data, Lv01 and Lv02 do not protectweaned piglets against an experimental ETEC infection. Elsewhere, the three yeast strainsstimulate the immunoglobulin levels (IgG, IgA) in colostrum and milk of sows suggesting thetransfer of a better immune protection to piglets.

Made available in DSpace on 2014-06-11T19:28:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-08Bitstream added on 2014-06-13T20:58:01Z : No. of bitstreams: 1 delgado_tfg_me_jabo.pdf: 1053821 bytes, checksum: 151e3e8ed68856f56009d9bdde539d85 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A seleção de animais mais precoces e produtivos, e a melhoria da qualidade do leite são os principais objetivos dos caprinocultores. Com o objetivo de avaliar a interação de dois importantes hormônios, o Cortisol (CORT) e o Fator de Crescimento Análogo à Insulina -1 (IGF-1) na lactação, e suas possíveis influências no desenvolvimento da glândula mamária, produção e qualidade do leite, 38 cabritas (na 1ª fase do experimento) e 24 cabras (na 2ª e 3ª fase do experimento) Saanen divididas em quatro grupos (G1- tratadas com GH e desafiadas com ACTH; G2 – tratadas com GH e desafiadas com placebo; G3 – tratadas com placebo e desafiadas com ACTH; e G4 – tratadas com placebo e desafiadas com placebo). Ao longo de todo experimento foram realizadas coletas de sangue pontuais e durante os desafios, medidas morfométricas internas e externas de úbere e tetos foram realizadas e análises da composição do leite. Não houve diferença no desenvolvimento de úbere e tetos entre animais tratados e não tratados com GH, porém houve maior produção de leite pelos animais tratados, confirmando a eficiência do hormônio em promover maior persistência de lactação. Também houve uma resposta antagônica do IGF-1 ao CORT, durante o desafio das cabras na 3ª fase do experimento, resultado que pode indicar que animais adultos tratados com GH são menos suscetíveis ao estresse que os animais não tratados. O desafio com ACTH e o tratamento com GH não alteraram a composição do leite e a contagem de células somáticas. / The selection of early animals and more productives, and improving the quality of milk are the main goals of goat breeders. With the objective of evaluating the interaction of two important hormones, cortisol (CORT) and the Insulin-Like Growth Factor -1 (IGF-1) in milk, and their possible influences on the development of the mammary gland, yield and quality of milk, 38 kid goats (the 1st. phase of the trial) and 24 goats (in the 2nd and 3rd phase of the trial) Saanen divided into four groups (G1- treated with GH (growth hormone) and challenged with ACTH (adrenocorticotropic hormone), G2 - treated with GH and challenged with placebo; G3 - treated with placebo and challenged with ACTH, and G4 - treated with placebo and challenged with placebo). Throughout experiment samples of blood were collected, internal and external morphometric measures of udder and teats reviews were performed and were performed composition of milk. There were not differences in the development of udder and teats of animals treated and not treated with GH, but treated animals showing higher production than non treated goats, confirming the effectiveness of treatment with GH in promoting increase on persistence of lactation. There was also a response of IGF- 1 antagonist to CORT, the challenge of goats during the 3rd phase of the experiment, result that may indicate that adults treated with GH are less susceptible to stress that the animals not treated. The challenge with ACTH and treatment with GH did not alter the composition of milk and somatic cell count.

Abstract Peroxisomes are small oxidative organelles found in all eukaryotes. They contain a matrix which is surrounded by a single membrane and consists mainly of soluble proteins. Peroxisomal enzymes are involved in a broad spectrum of metabolic pathways including conversion of lipids, amino- and hydroxyacids, purines and reactive oxygen species. The carbon fluxes through peroxisomal pathways require a continuous metabolite crossing of the peroxisomal membrane. A long-standing and still unresolved problem of the physiology of mammalian peroxisomes is the role of the membrane of these organelles as a permeability barrier to solute molecules. In this study, we have shown that the peroxisomal membrane represents a type of biomembrane where channel-forming proteins coexist with solute transporters. Disruption of the mouse Pxmp2 gene, encoding the peroxisomal integral membrane protein Pxmp2 also known as PMP22, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations revealed that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2-/- mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Hence, Pxmp2 is the first peroxisomal pore-forming protein identified, and its existence suggests that the mammalian peroxisomal membrane is permeable to small solutes, while transfer of bulky metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters. In addition, the phenotypic characterisation of Pxmp2-/- mice has revealed a role for Pxmp2 during the development of the epithelia in the mammary glands of female mice. The disruption of Pxmp2 leads to the impairment of ductal outgrowth of mammary glands at puberty, which is followed by the inability of Pxmp2-/- mice to nurse their offspring.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19021号 / 農博第2099号 / 新制||農||1030(附属図書館) / 学位論文||H27||N4903(農学部図書室) / 31972 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 久米 新一, 教授 祝前 博明, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Lactating dairy cows heavily rely on mammary gland functionality to maximize milk production. The number and activity of secretory mammary epithelial cells (MEC) plays a pivotal role in defining the synthesis potential of the gland. This dissertation aimed to investigate the effects of increased milking frequency (IMF), heat stress (HS), and cell heterogeneity as key contributors to the regulation of mammary gland milk synthesis capacity in lactating Holstein cows. The first study evaluated the implementation of IMF with 2x and 4x udder halves at early and mid-lactation for 21 and 20 d on milk yield (MY) and its association with changes in cistern and alveolar capacity. Results showed that udder halves milked 4x produced 2.27 kg more MY. Additionally, cows milked during early and mid-lactation had increased cistern capacity, while alveolar capacity remained unaffected. This suggests that increased cistern capacity may support MY enhancement through possible systemic responses caused by IMF. The second study examined the effects of 4 days of HS on mammary gland tissue structure, MEC number, and activity using a pair feeding model. Heat stress reduced MY of 4.3 kg/d. At the tissue level, HS decreased alveolar area and increased alveoli number and nucleated MEC per area. Gene expression analysis revealed unaffected activity-related targets but showed reduced phosphorylation of protein synthesis (pSTAT5) and cell survival (pS6K1) markers, as well as upregulation of an autophagosome-related protein (LC3 II). These findings indicate impaired pathways that could explain the reduction in MY after acute HS. The final study utilized single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of epithelial and immune cell subpopulations in milk. Analysis revealed multiple subpopulations with distinct gene expression profiles, including different subtypes of mammary epithelial cells expressing representative marker genes (CSN3, CSN2, CSN1S1, CSN1S2, and LALBA) and immune cell types such as T cells, granulocytes (including neutrophils), macrophages, and B cells. Understanding the populations of hematopoietic cells in milk provides valuable insights into mammary gland function during lactation. The investigation of factors influencing cell number and activity in MEC is crucial for optimizing milk production and maintaining udder health. By identifying and addressing these factors, dairy farmers and researchers can implement strategies to enhance mammary gland function, improve milk production efficiency, and ensure the overall well-being of dairy cows. / Doctor of Philosophy / Milk production capacity in dairy cows relies on specialized cells in the mammary gland called secretory mammary epithelial cells (MEC). This study investigated how management practices, environmental factors, and individual cow factors affect the regulation of milk synthesis in Holstein cows. In the first study, we compared milking frequency in udder halves milked two times or four times per day during early and mid-lactation. The cows that were milked four times produced 2.27 kg/d of additional milk. This perhaps happened because the mammary gland's storage capacity increased with more frequent milking. Next, we studied the effects of short-term heat stress on the structure of the mammary gland tissue and the number and activity of MEC. Heat stress lowered milk production by 4.3 kg/d. We observed changes in the size and number of certain cells in the mammary gland, which likely affected the observed milk production findings. We also noticed differences in the activity of proteins related to protein production, cell survival, and the recycling of cell materials. In the final part of the study, we used single-cell characterization techniques to examine the different types of MEC and immune cells in milk. We found that there are various subgroups of MEC, as well as different types of immune cells such as T cells, neutrophils, macrophages, and B cells. Understanding the variety and abundance of these cell populations helps us learn more about how the mammary gland works during milk production. Studying the factors that influence the number and activity of MEC is essential for optimizing milk production in dairy cows. By identifying and addressing these factors, dairy farmers and researchers can develop strategies to enhance mammary gland function, improve milk production efficiency, and ensure the overall well-being of dairy cows.

Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8α. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines. / Master of Science

Mastitis is often bacterial in origin. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), endotoxins from gram-positive and gram-negative bacteria, respectively, are potent inducers of mammary gland inflammation. Inflammation can alter expression of transporters responsible for transport of substrates important in synthesis of milk constituents and cellular metabolic energy. Since, gram-positive and gram-negative bacterial infections cause a different clinical course of mastitis, I investigated whether LTA and LPS differentially alter proton-coupled (MCT1) and sodium-coupled monocarboxylate transporter (SMCT1, SMCT2) expression and functional outcomes of altered expression. Human mammary epithelial cells (MCF-12A) were incubated with 1 microgram/mL LPS or LTA for 6, 12 and 24 hours and mRNA expression of TNF-alpha, IL-1β, IL-6, MCT1, SMCT1, and SMCT2 were measured using Quantitative RT-PCR. LPS decreased SMCT1, but increased SMCT2 expression after 6 h, while LTA increased MCT1 expression at 6 h, followed by gradual decrease in expression until 24 h. To know whether such differential changes in transporter expression by LPS and LTA could cause changes in cellular energy production, I quantified creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) and oxygen consumption rates using HPLC and Hansatech oxygen electrode, respectively. At 12 h, LPS increased concentrations of Cr, CrP, ATP and ADP, whereas LTA caused changes in CrP and ADP concentrations relative to control. Both LPS and LTA decreased oxygen consumption rates after 12 h. Furthermore, to know whether changes in transporter expression lead to differences in substrate availability, I performed uptake studies for carnitine using radiolabelled tritium L-carnitine. LPS and LTA challenge did not affect the affinity, but caused a 2-3-fold increase in maximal activity (Vmax) of carnitine transport. Although increases in Vmax were not significant, the increase in Vmax after 12 h exposure by LPS and LTA corresponds to changes in mRNA expression of the OCTN2 transporter (previously reported in the laboratory). In conclusion, LPS and LTA differentially alter mRNA expression of transporters, which leads to changes in cellular energy levels and oxygen consumption rates and possibly to changes in the functional activity of transporters. Whether such differences contribute to the different clinical course of mastitis warrants further investigation.

The mammary gland cell growth and differentiation are under the control of both systemic hormones and locally produced growth factors. Among all these important hormones and growth factors, estrogen plays a central role in mammary gland development. The biological function of estrogen is mediated by estrogen receptor α (ERα) and estrogen receptor β (ERβ). Both ERα and ERβ are expressed in the mammary gland, but with distinct expression patterns. In the mammary gland, ERα has been proved to be the estrogen receptor that mediates the mitogenic function of estrogen. However the function of ERβ in mammary cell proliferation is less understood and there remains some controversy. Accumulating evidence indicates that ERβ, unlike ERα, is a negative regulator of mammary epithelial cell proliferation. In this dissertation, ERα and ERβ were evaluated for their expression patterns in the mammary gland. In the proestrus phase, ERα was detected in about 20% of mammary epithelial cells; in the diestrus phase, no ERα staining was detected in the mammary gland. ERβ was expressed in more than 50% of mammary epithelial cells and ERβ staining was detected in some stromal cells in the proestrus phase. In the diestrus phase, ERβ staining cells were very limited and the staining intensity was very weak. These data suggest that the expression levels of both ERα and ERβ undergo dynamic changes during the estrous cycle. In the ovariectomised (OVX) rats, both ERα and ERβ were detected in more than 50% of mammary epithelial cells. Compared with the ovary-intact rats, the mammary gland of the OVX rats showed more cells with ERα expression, but the staining intensity was weaker. Taken together, the expression of ERα and ERβ is regulated by estrogen in normal mammary gland, while without estrogen stimulation in the OVX rats, more mammary cells showed ERα expression, but at a lower level in these cells. The effects of ERα and ERβ on mammary cell proliferation were studied by two different approaches, activation of endogenous ERα and ERβ via selective agonists, and overexpression of ERα and ERβ via lentiviral infection. In the first approach, we used ERα and ERβ selective agonists, propylpyrazole-triol (PPT) and diarylpropionitrile (DPN) respectively, to activate endogenous ERα and ERβ in the OVX rats. We found that ERβ selective agonist DPN counteracts the proliferative effect of ERα selective agonist PPT in the mammary gland. In the second approach, ERα and ERβ were ectopically overexpressed in the mammary gland of mature virgin rats by lentivirus infection. We found that ERβ overexpression significantly decreased mammary cell proliferation rate in both the proestrus and diestrus phases, indicating that ERβ, unlike ERα, is a negative regulator for mammary cell proliferation. Collectively, these data supports that in contrast to ERα, ERβ activation or overexpression is able to inhibit mammary cell proliferation.