Failure of the embryo to implant into the uterine lining results in infertility and is also the rate limiting step in FVF. The tethered, glycoprotein MUC1 is a protective cell surface receptor that has been associated with infertility. Evidence suggests MUC1 and other endometrial mucins regulate embryo implantation. Endometrial epithelia must be shielded from infection whilst permitting the recognition and implantation of the embryo. The protein backbone of MUC1 is thought to act as a scaffold for L-selectin ligands and may be integral to the initial tethering of the embryo during early implantation. Objectives The remit of this thesis was to model the expression of MUC1 and other mucins in the human endometrium using endometrial cell lines and to use atomic force microscopy to understand the role played by these proteins in shaping the non-specific and embryo specific adhesive characteristics of endometrial monolayers. If possible the proposed L-selectin implantation mechanism was to be identified and functionally characterised. Methodology This project successfully married the traditional molecular tools of quantitative PCR and immunocytochemistry with novel application of INCELL analyzer high content screening protein analysis, peak force quantitative nano-mechanical mapping atomic force microscopy and single molecule force spectroscopy atomic force microscopy to characterise the specific and non-specific surface adhesion on live endometrial monolayers. Results Firstly, immunocytochemistry and qRTPCR revealed that basal MUC1 expression was significantly higher in Hec-1-B relative to Hec-IA, Ishikawa and Hec50. Secondly, INCELL analysis qualified a distinct heterogeneous expression of MUC1 across the endometrial monolayer and delineated altered patterning following treatment with estradiol and progestins. Thirdly, we have shown a direct and proportional correlation between MUC1 expression and adhesion in live Hec-IA and Hec-IB cell monolayers. Fourthly, this work has confirmed and characterised binding of recombinant L-selectin to the endometrial epithelial cell surface. Fifthly, it is shown that L-selectin surface binding decreases following a reduction in MUC1 surface presentation. Conclusions The results implicate MUC1 as a key component of endometrial adhesion and an initial mediator of implantation with a functional patterned expression suggesting areas of altered receptivity exist across endometrial monolayers. Abnormal MUC1 expression has been shown in endometrial pathologies and unexplained infertility. The current investigation suggests MUC1 protein may assist embryo attachment by retarding it sufficiently through mechanical impedance to allow specific L-selectin binding further securing the embryo. A non-receptive endometrium may contribute towards the infertile phenotype despite repeated IVF treatments, thus novel examination of potential embryo adhesion molecules such as MUC1 may aid understanding of endometrial characteristics which prevent embryo implantation and contribute towards IVF failure.

No description available.

Streptococcus mutans is a member of the dental plaque and is the principal causative agent of dental caries. It can metabolize a wide array of sugars which results in the production of acid that causes demineralization of the tooth surface. S. mutans can also persist for extended periods of starvation, which may occur in different niches within the oral cavity. Previous studies have shown that mucin prolonged the survival of S. mutans in batch cultures and biofilms. Our laboratory has shown that the pyruvate dehydrogenase (pdh) operon is upregulated in stationary phase and is important for prolonged survival during nutrient starvation in chemically defined medium (CDM). In this study, I found that mucin enabled S. mutans to grow in a minimal chemically defined medium (MCDM) containing glucose and lacking all amino acids. A pdh mutant was found to be impaired in survival in CDM or MCDM supplemented with mucin. Additionally, I have shown that a galactose utilization mutant was impaired in survival in CDM with mucin and no exogenous glucose; galactose is the most abundant utilizable sugar residue in mucin. Mucin, supplemented in minimal medium with sucrose, also enabled S. mutans to form biofilms. Survival of the biofilms was pdh independent. Although biofilm formation was not seen in the absence of sucrose, cells did adhere to the glass coverslip; their survival was found to be pdh dependent. Using a previously constructed reporter strain, pdh expression was observed in a majority of cells in this condition. The expression of the pdh operon was also monitored in batch cultures. The starting inoculum size affected the percentage of fluorescent cells and the outcome of survival in media (CDM or MCDM) supplemented with mucin and no sugar. Greater than 50% of the stationary phase population expressed pdh in CDM with galactose. Less than 1% expressed pdh in CDM with glucose. However, galactose was unable to prolong survival of S. mutans in batch cultures in contrast to the effect of mucin. These results show that mucin may have a metabolic role in promoting long term survival of S. mutans. However it is possible that different components of mucin can be utilized in different conditions. / Microbiology and Immunology

Mucins are heavily glycosylated epithelial proteins. Under or overexpression of mucins may lead to several different types of dysfunctions. Mucins are under investigation as possible diagnostic markers for malignancies and other disease. This study examined the over/under expression of Mucin-3 in the presence of IP6. Three groups of six mice were fed varying levels of phytate (inositol 6-phosphate, IP6) for six days before being subjected to carbon dioxide asphyxiation. Sections of the duodenum, jejunum, ileum, stomach, and colon were collected, lysed, and the tissue proteins were prepared and analyzed by Western Blot to determine Mucin-3 expression. The hypothesis was that the presence of phytate in the digestive tract would modulate the expression of Mucin 3. Results showed that on average, Mucin 3 expression in untreated tissue was higher in areas of the duodenum and jejunum and lower in the ileum and stomach. Presence of IP6 decreased expression of Mucin 3 in the stomach by one percent in the 1g/kg phytate group while increasing in the 2g/kg phytate group by one percent. The expression of Mucin 3 in the duodenum increased fifty-five and thirty-nine percent in the 1g/kg and 2g/kg phytate groups, respectively. Jejunum Mucin 3 expression was increased by ninety percent in both 1g/kg and 2g/kg phytate groups. The ileum 0g/kg and 2g/kg phytate groups had the same expression of Mucin 3, but the 1g/kg group had a thirty-six percent decrease.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 174-188).

Immune-orchestrating biomaterials that precisely modulate the immune reac-tion to the host could lead the way for improving the implantation outcomein the transplantation field, in comparison to passive biomaterials. The lab-oratory of Dr. Thomas Crouzier has shown hydrogels derived from mucinsare capable of orchestrating the immune response mediated by foreign bodyresponse (FBR), as a result of evading fibrosis. Further, a recent study fromhis group showed sialic acid on mucin hydrogels is essential for the immuno-logical activity of those materials. Mucin glycans transiently activated thendampened macrophages, important orchestrators for material-mediated FBR,in a sialic acid-dependent manner for the majority of cytokines followed. Thematerial properties such as rheological properties, self-healing capacity, andstability, can be governed by the crosslinking chemistry used and have a drasticimpact on the functionalities of the materials. In this project, various cross-linking strategies are applied to tune the hydrogel properties. We show thatthe robust cross-linking formed mucin hydrogels having a 1.5% (wt/v) bettersupported insulin-secreting cells form islet-like organoids, compared to 2.5%mucin hydrogels. We then investigate the self-healing properties of the newmucin hydrogels and their interactions with various cell systems. / Immunologiska biomaterial som specifikt modulerar en immunologisk reak-tion hos värden kan vara vägen till att förbättra resultatet av en transplantationi jämförelse med att använda passiva biomaterial. Inom laboratoriet lett av Dr.Thomas Crouzier har det kunnat påvisas att hydrogels erhållna från mucinerär kapabla till att förändra en immunlogisk respons, Foreign Body Respon-se (FBR) vilket resulterat i att kringgå fibrosis. Utöver detta har ytterligareen studie utförts av hans grupp vilken kunde påvisa att sialinsyra på mucinhydrogel är essentiellt för den immunologiska aktiviteten för dessa biomateri-al. Mucin glykaner aktiveras tillfälligt för att dämpa bildandet av makrofager,vilket är viktigt vid biomaterial FBR i ett sialinsyra-beroende för bildandet avmajoriteten av cytokiner. Materialegenskaperna såsom reologiska egenskaper,självläkande kapacitet, och stabilitet kan regleras genom en krosslänkad kemisom har en drastisk impakt för funktionen för biomaterialen. I detta projekt, ärflera krosslänkande strategier tillämpade för att förstå egenskaperna för hyd-rogelerna. Vi har kunnat påvisa att robusta krosslänkande system av mucinhydrogel bestående av 1.5% (wt/v) ger bättre support till insulin sekreterandeceller att bilda islet liknande organider, att jämföra med 2.5% mucin hydrogel.Vi undersöker sedan den självläkande egenskaperna av nya mucin hydrogeleroch deras interaktioner med ett flertal olika cellsystem.

O estudo da relação entre os aditivos utilizados e os nutrientes é fundamental para o aprimoramento da utilização desses insumos e o máximo desempenho dos animais. O objetivo do presente estudo foi avaliar o desempenho e saúde intestinal (morfometria, microbiota do intestino e produção de mucina) em frangos de corte desafiados com Eimeria spp. alimentados com rações com níveis crescentes de treonina digestível e a inclusão de uma combinação de óleos essenciais. Os tratamentos foram arranjados em esquema fatorial 3x2, com três níveis de treonina digestível (100, 110 e 120% das recomendações) e suplementação ou não de óleos essenciais na ração, em um experimento no delineamento inteiramente casualizado. As rações baseadas em milho e farelo de soja foram formuladas para atender as necessidades de todos os nutrientes e fornecidas à vontade. Cada tratamento tinha seis repetições com 45 aves cada, totalizando 1620 animais. Aos 14 dias de idade, todas as aves foram desafiadas com Eimeria spp. via oral. O desempenho das aves foi avaliado semanalmente até 40 dias e, aos 21 e 40 dias de idade, foram coletadas amostras dos intestinos para análise de morfometria das vilosidades. Aos 21 dias de idade foram coletadas amostras do conteúdo do intestino delgado e cecos para análise da microbiota. Aos 40 dias, o conteúdo do íleo foi coletado para a determinação da produção de mucina. Os dados foram submetidos à análise de variância a 5%, avaliando a interação e os efeitos dos fatores principais. Aos 40 dias de idade, a inclusão do blend de óleos essenciais na dieta diminui o consumo de ração dos animais, sem alterar o ganho de peso e a conversão alimentar. Aos 21 dias, houve efeito significativo (p<0,05) no nível de 120% de treonina digestível comparado ao nível de 110%, onde os animais que receberam um maior nível de treonina na dieta obtiveram uma maior altura de vilos no íleo. Não houve efeito dos tratamentos sobre a quantidade de mucina no conteúdo ileal de frangos de corte aos 40 dias de idade. A inclusão de níveis crescente de treonina digestível e óleos essenciais na dieta, não modifica a população microbiana, podendo ser observado apenas tendências de alterações nas frequências de alguns gêneros no ceco dos animais. / The study of the relationship between the additives and nutrients is essential for improving the use of these substances and the maximum animal performance. The purpose of this study was to examine the effects of levels of threonine and the inclusion of blends of essential oils on the performance and intestinal health (morphology, intestinal microbiota and mucin production) in broilers challenged with Eimeria spp. The treatments followed a 3x2 factorial arrangement with three levels of digestible threonine (100, 110,120% of the Brazilian Tables recommendations), and supplementation or not of a blend of essential oils in the feed in a completely randomized design experiment. The diets based on corn and soybean meal to meet the nutritional requirements were fed ad libitum. Each treatment had six replicates of 45 birds each, totaling 1620 animals. On day 14, all birds were orally gavaged with 10 times the recommended dose of a commercial coccidial vaccine with sporulated live oocysts of Eimeria spp. . Performance was evaluated weekly until 40 days of age. At 21 and 40 days of age, samples of the intestines were collected for morphometry. At 21 days of age samples of the contents of the small intestine and the cecum were collected for analysis of microorganisms. At 40 days of age, the contents of the ileum were collected for determining the mucin production. The data were submitted to analysis of variance at 5%, studying the interaction and the effects of the main factors. At 40 days of age the inclusion of essential oils in the diet decrease the feed intake of the animals, without changing growth and feed conversion . At 21 days of age, 120% of digestible threonine in the diet increased the height of villus in the ileum when compared with 110% of digestible threonine (p<0,05). There was no effect of treatments on the production of mucin in the contents of the ileum. The increasing levels of digestible threonine and essential oils in the diet did not modify the microbiota.It is possible to observe a tendency of change on the frequency of some genera in the cecum of the animals.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well recognized tumor antigen, contributing to tumorigenesis and immune evasion. While MUC1/TM has been correlated with malignancy, it appears that a secreted splice variant of MUC1 (MUC1/sec) has antitumor properties and prevents tumor development. It was discovered that MUC1/sec expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase plasminogen activator (uPA) relative to the parental tumor line, and tumor cells expressing MUC1/TM (DA-3/TM). The serine protease uPA, has been found to be involved in growth promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Furthermore, the tumor suppressive and interferon responsive Stat1 transcription factor is dramatically upregulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned media containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the anti-proliferative effects of IFN-g. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a downregulation of uPA, and delays tumor progression. Since myeloid-derived suppressor cells (MDSC) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by DA-3/sec and DA-3/TM cells. DA-3/sec tumor cells recruit dramatically lower levels of MDSC, relative to DA-3/TM cells. Since MUC1/sec down-regulates tumor expression of uPA, its potential role in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSC, and correlates with tumor development. In addition to diminishing recruitment of MDSC, the effect of MUC1/sec on MDSC suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide (IEP), is capable of blocking expression of arginase 1 and production of reactive oxygen species (ROS) in MDSC, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting both tumor cells and MDSC. Furthermore, it was discovered that MDSC home to the liver in addition to the tumor, bone marrow, blood, and spleen of tumor bearers, as previously described. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly, via interactions with a variety of immune cells.

MUC4 is a heterodimeric mucin glycoprotein expressed in the epithelia of tissues. Previous studies in our laboratory demonstrated that MUC4 protein expression is regulated by exogenous growth factors and that MUC4 is found in complex with the receptor tyrosine kinase ErbB2. MUC4 protein expression in airway epithelia was evaluated using molecular biology techniques. The impact of the protein on ErbB2 activation was evaluated post mechanical wounding of airway epithelia, and upon MUC4 RNA silencing. MUC4 levels were increased with exposure to the differentiating agent retinoic acid and decreased upon exposure to epidermal growth factor, a proliferative agent. In the absence of MUC4, ErbB2 phosphorylation was diminished. These results support the hypothesis that MUC4 expression is enhanced during differentiation of epithelia. Furthermore these findings provide evidence for an additional level of ErbB regulation in airway injury and subsequent epithelial wound healing.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>