Previous research has shown that dairy cows require a dry period of at least 40 d for maximal milk yield in the subsequent lactation. Reducing the dry period requirement could prove beneficial to animal health and dairy profitability if subsequent milk yield was not reduced. Studies were conducted to evaluate the effects of continuous milking (CM) and hormonal treatments on milk yield, mammary epithelial cell (MEC) turnover during late gestation and early lactation, and mammary gene expression in dairy cows. A commercial trial using primiparous and multiparous cows demonstrated equal milk yields in bST-supplemented, CM and 60-d dry (CTL) multiparous cows, but lower milk yields in bST-supplemented CM, primiparous cows treated with bST. Subsequent experiments evaluated mammary development requirements during the dry period in primiparous cows and methods of rescuing milk yield. MEC growth was lower in CM glands during most of late gestation. Maintenance of lactation in CM glands resulted in a marked reduction in the MEC turnover process that occurs in the early dry period. In the last 20 d of gestation, MEC growth remained reduced in CM glands. By the last week of gestation, MEC growth was 50% less in CM tissue vs. CTL tissue. MEC apoptosis was unaffected by CM during the last 20 d of gestation, but a premature decrease in early lactation apoptosis occurred in CM glands at 7 d postpartum. Mammary gene expression demonstrated bax and insulin-like growth factor binding protein 5 are involved in apoptosis and cyclin D1, CCAT/enhancer binding protein-β, and bcl2 are involved in mammary development. Ultrastructure of CM tissue revealed large populations of resting or involuting alveoli by d 20 postpartum, whereas CTL glands had a homogenous population of secretory alveoli. Collectively, these data suggest that a 40-53% reduction in milk yield in CM glands is caused by reductions in MEC renewal and reduced secretory capacity. Treatments (bST, prostaglandin E2) to stimulate milk synthesis or MEC growth in CM primiparous glands were unsuccessful. In conclusion, primiparous cows continue to require a 60-d dry period, but multiparous cows are good candidates for short dry periods, and potentially no dry period.

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The centrosome is the main microtubule-organising centre in animal cells; important to assemble a bipolar mitotic spindle ensuring proper chromosome segregation and genomic stability. Whereas correct centrosome number (1-2) is tightly maintained in normal cells, cancer cells usually have an increased number of centrosomes (>2), termed centrosome ampli cation. Centrosome ampli cation has been correlated with aneuploidy, increased tumour grade, chemoresistance and overall poor prognosis. Cancer cells primarily adapt to supernumerary centrosomes by clustering them into two poles resulting in a `normal' pseudo-bipolar mitosis. Undermining centrosome clustering is a potential target for cancer-speci c treatment. Indeed, depleting the kinesin HSET has already been shown to speci cally kill cancer cells by impairing the centrosome clustering mechanism. However, it is unclear whether this process requires adaptation or it is inherent to all cell types. Using a panel of non-transformed cell lines, we observed that cells expressing Ecadherin have ine cient clustering mechanisms compared to cell lines without E-cadherin. Loss of E-cadherin (siRNA/CRISPR) promotes centrosome clustering and survival of epithelial cells with multiple centrosomes. In addition, loss of DDR1, involved in regulating cortical contractility downstream of E-cadherin, increases centrosome clustering in epithelial cells. Using Atomic Force Microscopy we con rmed that indeed loss of E-cadherin leads to increased cortical contractility in mitotic cells. Inhibition of actomyosin contractility prevents e cient clustering in cells that do not express E-cadherin, further suggesting that it is important for this process. Loss of E-cadherin and DDR1 is strongly correlated with high levels of centrosome ampli cation in breast cancer cell lines suggesting that these changes are an important adaptation mechanism to centrosome amplification.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands. / Ph. D. / The mammary gland is a heterogeneous tissue with two main cell types - mammary epithelial cells and fibroblasts. These two cells types produce the majority of the extracellular matrix (ECM) within the mammary gland. The ECM is important for regulation of cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands.

The genetic mechanisms underlying the specification of thymic epithelial (TE) lineage cells are poorly understood. Foxn1 is an early specific marker of thymic epithelial cells (TECs) in the third pharyngeal pouch (3PP) and is required for development of all mature TE lineage cells but does not specify the TE lineage. The upstream regulators of Foxn1 are currently unknown, however evidence points to a potential role for Pax1 and Pax9. While the thymus phenotypes of the Pax1-/- and Pax9-/- mutant mice have been investigated in detail and TECs in these mice are known to express Foxn1, the possibility of functional redundancy exists and the compound mutants of these genes have not been studied. Therefore, the aim of this thesis was to test the hypothesis that Pax1 and Pax9 are required for TE lineage specification and regulation of Foxn1 expression. This hypothesis was addressed by analysis of thymus development and TEC function in Pax1/Pax9 compound mutant mice. The data presented in this thesis indicates that prenatally, Pax1 and Pax9 cooperatively regulate thymus organogenesis, such that the size, structure and location of the thymus is affected in a Pax1/Pax9 gene dosage-dependent manner, and the Pax1unex/unexPax9lacZ/lacZ embryo is functionally athymic. Furthermore, they establish that the thymic rudiment of Pax1unex/unexPax9lacZ/lacZ embryos does not express Foxn1, establishing that Pax1 and Pax9 are required together for the initiation of Foxn1 and suggesting they are required to specify the TEC lineage. Postnatally, enlarged blood vessels observed in the Pax1unex/unex thymus suggested a role for Pax1 in vascularisation of the thymus. In addition, the effect of loss of one or more Pax1/Pax9 alleles on the expression of Foxn1 and other genes known to regulate TEC development or function was assessed. These data demonstrate that Pax1 and Pax9 co-operate to regulate Foxn1 in a dosage-dependent manner. Furthermore, Pax1 and Pax9 appear to negatively regulate both Hoxa3 and Vegfa, providing a possible explanation for the enlarged blood vessels in the postnatal Pax1unex/unex thymus. Finally, an inducible and reversible recombinase-mediated cassette exchange system that will allow the knockdown of Pax1 and Pax9 at defined time points during development has been established, that has the potential to test the function of these genes during thymus organogenesis and in the postnatal thymus.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. Previous studies have demonstrated that the sexually transmitted pathogen, Neisseria gonorrhoeae, can induce or inhibit host cell death. N. gonorrhoeae is a mucosal pathogen and, in females, initiates infection in epithelial cells of the ectocervix and endocervix. Mucosal epithelial cells of the female genital tract are the first line of defense, and thus their cellular fate can alter the early immune response to invading pathogens such as N. gonorrhoeae. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In the present study, we demonstrate that N. gonorrhoeae stimulation induces a transient increase in cIAP2 protein levels in human cervical epithelial cells. High intracellular protein levels were observed during early N. gonorrhoeae stimulation and were followed by a marked intracellular decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes, which are nano-sized vesicles that carry protein and coding RNA as cargo from one cell to another. We also observed that depletion of cIAP2 in N. gonorrhoeae stimulated cells resulted in cell death resembling necroptosis, an inflammatory form of cell death. Furthermore, inhibition of cIAP2 led to an increase in interleukin-1β production. Exosomes have been found to have important roles in cell communication during microbial infection. Here, we demonstrate that N. gonorrhoeae induces exosome production and alters exosome content. We also demonstrate that exosomes elicit cytokine responses in uninfected naïve cells. Collectively, these studies highlight an additional mechanism for epithelial cells to orchestrate the immune response in the female genital tract during N. gonorrhoeae infection.

Les protéases sont impliquées dans de nombreux processus biologiques et ont des origines très variées (cellules immunitaires, cellules épithéliales...). Leur activité est régulée par des inhibiteurs de protéases. Cette étude s'intéresse a` la balance entre les protéases épithéliales et leurs inhibiteurs dans le contexte de pathologies affectant l'intégrité´ de l'épithélium intestinal. Les conséquences d'un déséquilibre de la balance protéolytique ont été étudiées dans deux maladies chroniques aux composantes différentes, les Maladies Inflammatoires Chroniques de l'Intestin (MICI) (cycles de poussées inflammatoires) et le Syndrome du Côlon Irritable (SCI) (cycles de douleur). La muqueuse colique de patients atteints de SCI sécrète une activité de type trypsine dont l'origine et les fonctions sont mal définies. Cette étude s'intéresse à la source de cette activité trypsique dans le côlon des patients atteints de SCI, sa nature et son rôle dans l'activation neuronale. L'activité trypsique chez les patients atteints de SCI est augmentée majoritairement dans les cellules épithéliales (CEI). La stimulation de monocouches de cellules épithéliales avec du LPS ou de l'épinéphrine induit une augmentation de la quantité de trypsine-3 et de sa sécrétion au pôle basal de la cellule épithéliale stimulée, en corrélation avec une augmentation de l'activité trypsique. L'hyperactivité de la trypsine-3 au pôle basal entraine une perte de la fonction barrière de l'épithélium, anomalie retrouvée aussi chez les patients atteints de SCI. De plus, nous avons mis en évidence que la trypsine-3 est capable d'activer les neurones humains myentériques et les neurones sensitifs murins. In vivo, son administration intra-rectale à des souris induit une hypersensibilité viscérale dépendante du récepteur PAR2 (Protease Activated Receptor-2). Ainsi, les cellules épithéliales intestinales des patients atteints de SCI produisent et sécrètent de la trypsine-3 spécifiquement au pôle basal. Cette activité trypsique active des neurones sensitifs, participant à l'hypersensibilité viscérale, symptôme majeur dont souffrent les patients atteints de SCI. Les pathologies inflammatoires peuvent aussi être source d'un dérèglement protéolytique. Les patients atteints de MICI présentent une dérégulation de la balance élastolytique colique. Notre équipe a montré que l'apport d'ELAFINE (un inhibiteur d'élastase) par la bactérie génétiquement modifiée L.lactis au contact de la muqueuse inflammatoire protège les souris traitées en réduisant l'inflammation intestinale. Cependant les mécanismes protecteurs médiés par L'Elafine restent encore à déterminer. L'ELAFINE est un inhibiteur d'élastases mais possède aussi des propriétés antimicrobiennes. Afin de mettre en lumière quelles fonctions de L'Elafine portent les propriétés anti-inflammatoires des mutants de L'Elafine ont été générés et insérés dans la bactérie L.lactis: une mutation pour annuler sa fonction inhibiteur de protéase, un second mutant de son domaine antimicrobien et un dernier mutant ayant perdu ces 2 fonctions. Dans des monocouches de cellules intestinales épithéliales, l'apport d'ELAFINE protège de l'inflammation médiée par un déséquilibre de l'activité élastolytique : on observe une restauration de la fonction barrière de l'épithélium et une diminution des cytokines pro-inflammatoires (CXCL8 et IP10). La mutation du domaine antimicrobien n'affecte pas ces propriétés. Cependant, l'absence de la boucle inhibitrice annihile les propriétés anti-inflammatoires de L'Elafine. Ces travaux ont pu mettre en évidence l'importance de l'équilibre protéolytique au sein de la cellule épithéliale dans les pathologies intestinales. La balance entre les protéases et leurs inhibiteurs joue donc un rôle dans l'homéostasie épithéliale et les pathologies inflammatoires. / Proteases are involved in some biologic processes and their origins are variable (immune cells, epithelial cells...). Their activity is regulated by antiproteases. This study investigates the balance between proteases and their inhibitors in pathologies which modify epithelium integrity. Consequences of an unbalance in proteolytic activity was studied in two chronic pathologies with different components: Inflammatory Bowel Disease (IBD) (cycles of inflammatory boost) and Irritable Bowel Syndrome (IBS) (cycles of pain symptoms). Colonic mucosa from IBS patients releases trypsin activity. The origin and the functions of this activity are not well defined. This study investigated the source of this trypsin activity in the côlon of IBS patients, its nature and its role in neuronal activation. Trypsin activity from IBS patients is increased mostly in epithelial cells. Stimulation of epithelial cell monolayers with LPS or epinephrine induces an increase of trypsin-3 quantity and its secretion specifically in the basal side of epithelial cells. This is in correlation with the increase of trypsin activity. Trypsin-3 hyperactivity at the basal side provokes a loss of epithelium barrier function, which is also found in colons of IBS patients. Then, we have highlighted that trypsin-3 is able to activate human myenteric neurons and murine sensitive neurons. In vivo, its intra-rectal administration to mice induces a visceral hypersensitivity dependent of PAR2 (Protease Activated Receptors 2). Thus, intestinal epithelial cells from IBS patients produce and release trypsin-3 specifically on their basal side. This trypsin activity activates sensitive neurons which participate to visceral hypersensitivity, a major symptom of IBS patients. Inflammatory pathologies could be a source of proteolytic malfunction. IBD patients have a dysregulation of elastolytic balance in the colon. Our team has shown that ELAFIN (an elastase inhibitor) delivered by the bacteria genetically modified L.lactis near the inflamed mucosa, protects mice from intestinal inflammation. However, the protective mechanisms induced by ELAFIN need to be investigate. ELAFIN is an elastase inhibitor but have also antimicrobial properties. With the aim to highlight what function of ELAFIN owns anti-inflammatory properties, mutants of ELAFIN have been generated and were insered into L.lactis: a first mutant lacked its antiprotease function, a second lacked antimicrobial properties and a last mutant lacked both properties. In intestinal epithelial monolayers, ELAFIN delivered by L.lactis protects against inflammation: a restauration of epithelial barrier function and a decrease of pro-inflammatory cytokines (CXCL8 and IP10) are observed. Mutation of antimicrobial domain doesn't affected these properties. Nevertheless, the absence of inhibitory loop annihilates anti-inflammatory functions of ELAFIN. This work highlights the importance of proteolytic balance inside the epithelial cell in intestinal pathologies. The balance between proteases and antiproteases plays an important role in epithelial homeostasis.