E-&ndashcadherin (CDH1) is a cell adhesion molecule that coordinates key morphogenetic processes regulating cell growth, cell proliferation and apoptosis. Loss of CDH1 is a trademark of the cellular event epithelial to mesenchymal transition (EMT), which increases the metastatic potential of malignant cells. PTEN is a tumor suppressor gene commonly mutated in many human cancers, including endometrial cancer. In the mouse uterus, ablation of Pten induces epithelial hyperplasia, leading to endometrial carcinomas. However, loss of Pten alone does not affect longevity until around 5 months. Similarly, conditional ablation of Cdh1 alone does not predispose mice to cancer. We characterized the impact of dual Cdh1 and Pten ablation using Pgr-Cre (Cdh1d/d Ptend/d) in the mouse uterus. We observed that Cdh1d/d Ptend/d mice died at postnatal day 15-&ndash19 with massive blood loss from their reproductive tract (abnormal metrorrhagia) with prevalent vascularization in both the endometrium and myometrium. Their uteri were abnormally structured with curly horns, disorganized epithelial structure, and increased cell proliferation. Co-&ndashimmunostaining of KRT8 and ACTA2 showed invasion of epithelial cells into the myometrium. Further, the uteri of Cdh1d/d Ptend/d mice had prevalent vascularization in both the endometrium and myometrium. We also observed reduced expression of estrogen and progesterone receptors, loss of cell adherens and tight junction molecules (CTNNB1 and claudin), as well as activation of AKT in the uteri of Cdh1d/d Ptend/d mice. However, complex hyperplasia was not found in the uteri of Cdh1d/d Ptend/d. Collectively, these findings suggest that ablation of Pten with Cdh1 in the uterus accelerates cellular invasiveness and angiogenesis, and causes early death. Thus, this model does not allow sufficient time for the emergence of advanced, clinically over aggressive endometrial tumorigenesis and metastasis. Additionally, we looked at a new Cre system to ablate Pten and Cdh1 only in the epithelial cells of the uterus. Sprr2f, an estrogen dependent gene that is found highly expressed in the uterus, helps with structure and barrier function of epithelial cells. Prg-Cre turns on at postnatal day 3-5 before development of the uterus; whereas, Sprr2f-Cre is active around 3 weeks which is after uterine development. We have driven the ablation of Cdh1d/d Ptend/d using the Sprr2f-Cre. The Sprr2f-Cre Cdh1d/d Ptend/d mice successfully lived to 2 months. The Sprr2f-Cre Ptend/d mice displayed hyperplastic epithelial cells, most prominently in the glandular like structures of the uterus. Lack of cellular structure was observed in the Sprr2f-Cre Cdh1d/d Ptend/d mice. We also developed a model of orthotopic tumor transplantation to study further tumor development including cell invasion, dissemination and metastasis. The uteri of control, Cdhd/d, Ptend/d and Cdhd/d Ptend/d mice were collected and dissected to approximately ~1 mm in diameter. Then, the tissue fragments were orthotopically implanted into the uterine wall (endometrium) of wild-type syngeneic host mice. We have observed successful implantation and sustainability of the tissue through this technique. The tissue viability was successfully verified by implanting donor uterine pieces under the kidney capsule of recipient wild type mice. This study has shown that the ablation of Cdh1 and Pten in the mouse uterus initiates a more aggressive form of type I endometrial carcinoma when using Pgr-Cre as well as Sprr2f-Cre. However, neither conditional ablation approaches allowed us to fully observe the progression of the carcinoma to a metastatic disease. Our intrauterine endometrial/myometrial implantation technique proved to be an incomplete method to further study the metastatic potential of the PgrCre/+ Cdh1f/f Ptenf/f mice.

This study aimed to investigate the effects of commercially available natural (sugar cane molasses, white sugar and brown sugar) and artificial (Canderel™, Equal™, Natreen™, Sweetex™, Splenda™ and Swheet™) sweeteners on various physiological systems. The artificial sweeteners tested in this study may be categorised into their respective groups based on their primary ingredient. The brands Canderel™ and Equal™ contain aspartame, Natreen™ and Sweetex™ consist of saccharin and Splenda™ and Swheet™ are composed of sucralose. The inclusion of artificial or natural sweeteners in the human diet has been continually debated and their implication in the development of certain diseases has raised concern regarding their safe use. Therefore, it is necessary that these food products be subjected to a battery of tests to determine adverse effects on human health.

This study aimed to investigate the effects of commercially available natural (sugar cane molasses, white sugar and brown sugar) and artificial (Canderel™, Equal™, Natreen™, Sweetex™, Splenda™ and Swheet™) sweeteners on various physiological systems. The artificial sweeteners tested in this study may be categorised into their respective groups based on their primary ingredient. The brands Canderel™ and Equal™ contain aspartame, Natreen™ and Sweetex™ consist of saccharin and Splenda™ and Swheet™ are composed of sucralose. The inclusion of artificial or natural sweeteners in the human diet has been continually debated and their implication in the development of certain diseases has raised concern regarding their safe use. Therefore, it is necessary that these food products be subjected to a battery of tests to determine adverse effects on human health.

Philosophiae Doctor - PhD / This study aimed to investigate the effects of commercially available natural (sugar cane molasses, white sugar and brown sugar) and artificial (Canderel, Equal, Natreen, Sweetex, Splenda and Swheet) sweeteners on various physiological systems. The artificial sweeteners tested in this study may be categorised into their respective groups based on their primary ingredient. The brands Canderel and Equal contain aspartame, Natreen and Sweetex consist of saccharin and Splenda and Swheet are composed of sucralose. The inclusion of artificial or natural sweeteners in the human diet has been continually debated and their implication in the development of certain diseases has raised concern regarding their safe use. Therefore, it is necessary that these food products be subjected to a battery of tests to determine adverse effects on human health. / South Africa

Orientador: Eunice Oba / Resumo: As substâncias desreguladores endócrinas de diversas origens e propriedades químicas acomentem uma diversidade de mamíferos, animais silvestres e seres humanos, que podem interagir com o sistema reprodutivo feminino e prejudicar a homeostase hormonal. Dentro dessa classificação, os parabenos podem ser utilizados como conservantes em diversos produtos comerciais. Seus efeitos se tornam cada vez mais complexos em relação à idade, história reprodutiva, ambiente endócrino da espécime no momento da exposição, como também não é sabido todos os reais mecanismos desses compostos quando associados. Sendo assim, o objetivo deste estudo foi avaliar os efeitos dos parabenos em ratas Wistar durante um período de 90 dias consecutivos de exposição. Assim, as ratas foram distribuídas em quatro grupos experimentais: controle, que recebeu somente óleo de milho como veículo, e tratados com propil e buti-parabeno nas doses de 10 mg/kg, 100 mg/kg, ou 200 mg/kg e expostas via subcutânea. Foi utilizada a técnica de citologia vaginal para investigar a ciclicidade das fêmeas, bem como a caracterização das fases do ciclo estral pelo método Shorr. Além desses parâmetros, foram analisados aspectos morfológicos de ovários e útero, quantificação das estruturas ovarianas e avaliação ultraestutural de folículos pré-ovulatórios. Foram avaliados a concentração plasmática de estrógeno, peso corporal bem como o peso dos órgãos reprodutivos. Os resultados revelaram que o método Shorr, foi eficaz para identificar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The endocrine disruption substances of many origins and chemistry properties affect a diversity of mammals, wild animals and human beings. These substances can interact with the female reproductive system and damage the hormonal homeostasis. In this classification, the paraben could be used as preservatives in different commercial products. Its effects get even worst and complex with the age, reproductive history, and endocrine environment of the specie in the moment of exposure of the drug. In complement, it is not known the actual mechanisms of those compounds when they are associated. Thus, the goal of this experiment was evaluate the effects of parabens in females Wistar during 90 consecutive days of exposure. Therefore, the females were divided in four experimental groups: Control group – Corn oil; and a mixture of propil and butyl paraben with 10 mg/kg, 100 mg/kg and 200 mg/kg and exposed through the subcutaneous pathway. The vaginal cytology was performed to investigate the cyclicality as well as the characterization of the estrous phases by the Shor method. Besides that, morphological aspects of the ovary and uterus, counting of ovaries structure and ultrastructural of pre-ovulatory follicle. To complete the evaluation, the plasmatic estrogen, body weight and reproductive organ weight were assessed. The Shor method was effective to characterize and identify the cells types of the vaginal epithelium and identify the estrous cycle phase. Moreover, the relative and absol... (Complete abstract click electronic access below) / Mestre

Worldwide, more than half of all HIV-infected individuals are women. Since mucosal surfaces are the primary gateway for HIV entry, maintaining the integrity of the female reproductive tract (FRT) is essential for preventing infection. The FRT employs many immune mechanisms that serve as the first line of defense against HIV transmission. Among these are vaginal fluid secretions rich in antimicrobial peptides, and commensal bacteria that colonize the vagina and prevent infections. We sought to study vaginal fluid as an innate immune component of the FRT in the prevention of HIV infection. Additionally, we investigated the anti-HIV microbicide candidate RC-101 as a possible treatment against pathogenic bacteria that disrupt the healthy microbiota of the FRT and create a suboptimal immune state that increases host susceptibility to viruses, such as HIV. Here we report that vaginal fluid collected from healthy females inhibits HIV infection. Moreover, our studies reveal that vaginal fluid collected from Black and White women exhibit disparate anti-HIV activity, possibly rendering Black women more susceptible to HIV infection. In addition, we show that RC-101, which is active against HIV, can also inhibit pathogenic bacteria that compromise FRT innate immunity, providing a dual mechanism of protection against HIV acquisition. Overall, these findings show that vaginal fluid is an important part of female innate immunity that protects the host from heterosexual HIV acquisition. Furthermore, the microbicide RC-101 may prevent HIV infection by both directly preventing viral entry, and by restricting the growth of pathogenic bacteria that disrupt the protective commensal vaginal flora. Together, innate mechanisms and bolstered protection present a multifaceted approach to maintaining effective host immunity.

Human immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can increase susceptibility to HIV. Accordingly, one common risk factor for HIV acquisition is the microbial shift condition known as bacterial vaginosis (BV), which is characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. Our model showed good agreement with clinical BV trends; we observed a distinct cytokine and human β- defensin-2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria. These iii data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. We next evaluated the effect of this interaction on HIV infection by investigating the soluble effectors secreted when FRT epithelial cells were cocultured with A. vaginae. We observed increased proviral activity mediated by secreted low molecular weight effectors, and determined that this activity was not likely mediated by cytokine responses. Instead, we identified a complex mixture containing several upregulated host proteins. Selected individual proteins from the mixture exhibited HIV-enhancing activity only when applied with the complex mixture of proviral factors, suggesting that HIV enhancement might be mediated by synergistic effects. In addition to characterizing the immune interactions that mediate the enhanced HIV acquisition associated with BV, we also evaluated the safety and efficacy of RC- 101, a candidate vaginal microbicide being developed for the prevention of HIV transmission. RC-101 has been effective and well tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation. Here, we treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate compatibility of this microbicide with FRT tissue and microflora. RC-101 was well tolerated by host tissues and commensal vaginal bacteria, while BV-associated bacteria were inhibited by RC-101. By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection.

The most common neoplasm found in young to middle-aged men is testicular cancer (TCa). This disease not only poses a risk of early death, but can also affect a male's fertility and testosterone levels and can diminish one's mental health and/or quality of life. One particular line of research that is emerging in the field is a possible genetic association of TCa with female reproductive cancers. We employed a systematic review to assess the methodological quality of articles that met the inclusionary criteria. To be selected for this review, articles had to go through a primary, secondary, and tertiary screening procedure using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines. Four studies were selected, and the Newcastle-Ottawa Scale (NOS) was used to measure the quality of each nonrandomized, case-control or cohort study. Two articles received perfect scores, one case-control study received a near-perfect score of 8 out of 9 stars, and only one out of the four included studies received 5 out of 9 stars. Upon organizing and analyzing the data, we found a significantly increased risk (20%) of female reproductive cancer among women who had a father with TCa. Also, we found that men were 12% more likely to develop TCa if they had a sister with female reproductive cancer, and 16% more likely if their mother had ovarian, endometrial, breast or cervical cancer. The goal of this review was to assess the overall strength of association, or lack thereof, between TCa and female reproductive cancers. Findings of this review suggest that an association exists between these discordant forms of cancer. There were significant risks found between mothers and sons, backed by substantial evidence of an X-linked inheritance pattern. This information has the potential to improve our knowledge of cancer etiology and treatment.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Seminal fluid proteins (SFPs) have been shown to affect the physiology,behaviour and immune responses of mated females in some species. Thisopen window for manipulation of female’s fitness allows the possibility forcomplex evolutionary dynamics between the SFPs and proteins of femalesthat would counter the effects of the former, the female reproductive proteins (FRPs). Also, the bean beetles of the Bruchinae subfamily are pests to pre-ferred species of plant hosts. The hosts have a great variety of secondary defensive metabolites between them and to detoxify those compounds, each beetle species is expected to have a well adapted arsenal of digestive proteinsfor a specific host. I carried out a comparative study with four species of bean beetles with the aim to identify patterns of selection in the proteins mentioned. Expression data for one of those species, Callosobruchus maculatus, has allowed to identify its SFPs, FRPs and digestive proteins and with orthology inference I identified their orthologues in the other three species. Then I estimated theratio of non-synonymous to synonymous substitution rates (ω) for each protein by using codeML of the PAML package and used them as a proxy for estimating selection. FRPs had about the same ω values as conserved genes found across the Arthropod phylum, while the SFPs and digestive proteins hadhigher ω values, indicating more relaxed purifying selection. I also performed tests of positive selection and have identified 92 digestive proteins, 9 FRPs and 26 SFPs as potential targets for future functional work. Finally, I examined the scenario of co-evolution between SFPs and FRPs because of direct interaction. By correlating branch-specific ω values for each possible pairs of proteins I found that SFPs are associated on average more with FRPs than with digestive or conserved genes, as expected. The same was true for the FRPs. Also I examined the possibility of factors contributing to the association such as expression levels, sex-biased expression and protein function. Using linear regression models I found that expression levels and proteinfunction do predict in some degree the ω estimates and could thus also affectthe correlations examined. High gene expression levels reduce the overall ωvalues of genes, also known as E-R anticorrelation. Sex-biased expression does not affect the overall ω values, but does affect the intensity of the E-R anticorrelation, with it being less prominent in male-biased genes and more prominent towards female-biased genes.