Programmed function of the tammar mammary gland; the role of milk proteins in gut development

 This thesis aimed to exploit the unique reproductive strategy of marsupials such as the tammar wallaby to prove that milk may regulate postnatal growth and development of organs such as the stomach.

Comparative analysis of caveolins in mouse and tammar wallaby: role in regulating mammary gland function.

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.

Innate immunity in the mammary gland and the role of the milk peptidome

 This research investigated how the mammary gland responds to disease states such as mastitis and how the milk plays a protective role. An in vitro mammary model was developed and shown to replicate the same responses to the breast when challenged with bacterial proteins demonstrating the usefulness of this model for future investigation of therapeutic interventions.

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

The relationship between milk, mammary adipocytes and ECM in regulating murine mammary gland involution

 This thesis investigated the role of milk, extracellular matrix and mammary adipocytes in regulating mammary gland function during involution in mice and explored the use of an in vitro culture model, the mammosphere model system to study the same.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxa, Brycon cephalus Gunther 1869 (Teleostei-Characidae)

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus, the head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column, the parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located, Such cells were interpreted as noradrenaline cells, Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

Third eyelid gland protrusion in dogs: an experimental model proposal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão imuno-histoquímica da calponina em glândula parótida de rato após obstrução do ducto excretor principal

Glandular atrophy is one of several alterations which can aflict the salivary glands, caused generally by obstructive lesions such as sialo1ithiasis, infections or compression by neoplastic processes arnong others. In this work, a morphological and immunohistochemical study was carried out in rat parotid glands, which were submitted to obstruction of the main excretory duct suffering atrophy at varied time intervals, with the aim of appraising the behavior of myoepithelial cells during the process of glandular atrophy. It was analized the immunohistochemical expression of calponin which detects myoepithelial cells in the parotids of 28 animaIs, which were divided into 7 groups, each one made up of 4 rats, afier the ductal ligature procedure, in the following time intervals: zero hour (control), 24 hours, 7, 15, 21, 30 and 60 days. Analysis of the immunohistochemicaI profile was carried through in which the calponin expression was veritied through its distribution pattem and numericaI index. All specimens exhibited positivity for calponin in myoepithelial cells which were distributed around the acini and the ductaI structures, a small number of positiveIy marked cells being detected in the controI group and in the 24-hour group when compared to subsequent ones, where it was perceived a Iarge increase in the number of positiveIy marked cens, mainly surrounding the ductiform structures which originated during the obstruction time. Upon application of statistical tests it was verified that the rise in the number the myoepithelial positive cells for calponin, when the control groups (zero hour) was compared to the 7, 15,21, 30 and 60-day groups afier obstruction, was statistica1ly significant. It was concluded then that the detected rise probably carne about due to an elevation in the rate of proliferation of the myoepitheliaI cells subsequent to the ductal obstruction, associated with a growing resistance of these cells to glandular atrophy.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sebaceous metaplasia in a canine mammary gland non-infiltrative carcinoma with myoepithelial component

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Absence of DNA damage in multiple organs (blood, liver, kidney, thyroid gland and urinary bladder) after acute fluoride exposure in rats

Fluoride has been widely used in dentistry as a caries prophylactic agent. However, there has been some speculation that excess fluoride could cause an impact on genome integrity. In the current study, the potential DNA damage associated with exposure to fluoride was assessed in cells of blood, liver, kidney, thyroid gland and urinary bladder by the single cell gel (comet) assay. Male Wistar rats aging 75 days were distributed into seven groups: Groups 1 (control), 2, 3, 4, 5, 6 and 7 received 0 (deionized water), 10, 20, 40, 60, 80 and 100 mgF/Kg body weight from sodium fluoride (NaF), respectively, by gastrogavage. These groups were killed at 2 h after the administration of the fluoride doses. The level of DNA strand breaks did not increase in all organs evaluated and at all doses of NaF tested, as depicted by the mean tail moment. Taken together, our results suggest that oral exposure to NaF did not result in systemic genotoxic effect in multiple organs related to fluoride toxicity. Since DNA damage is an important step in events leading to carcinogenesis, this study represents a relevant contribution to the correct evaluation of the potential health risk associated with chemical exposure.

Clinical, histological and immunophenotypic findings in a mare with a mammary lymphoma associated with anaemia and pruritus

An 18-year-old Quarter Horse mare presented with an 18 week history of dermatological lesions characterised by recurrent urticaria and recent signs of enlarged mammary glands, pyrexia, alopecia and intense pruritus. Haematological and serum biochemical analyses revealed anaemia, leucocytosis, thrombocytopenia and hyperfibrinogenaemia. Mammary discharge was evaluated and revealed neutrophilia. Cytological examinations of fine needle aspirates from the parotid lymph nodes were normal. Common causes of anaemia were ruled out by specific tests. The initial diagnosis was mastitis and 2 weeks later the animal returned to the hospital with worsening of clinical signs, intermittent fever, intense pruritus and generalised alopecia. Blood samples were collected for haematological and serum biochemical analyses, and the mammary gland and parotid lymph nodes were biopsied. Rectal palpation revealed a large irregular mass near the left kidney with adhesions of surrounding structures. The animal died before the laboratory results were ready. This report details the clinical, histological and immunophenotypic findings of a case of large lymphoma in a mare.