Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-19T13:51:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_RayanaLuna_2016 (1).pdf: 8156604 bytes, checksum: 38605ef2e80adea3732612e363d4a7d2 (MD5) / Made available in DSpace on 2016-09-19T13:51:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_RayanaLuna_2016 (1).pdf: 8156604 bytes, checksum: 38605ef2e80adea3732612e363d4a7d2 (MD5) Previous issue date: 2016-06-15 / FACEPE / O desenvolvimento de uma gestação saudável depende de uma adequada circulação uteroplacentária. Abortos recorrentes (AR), óbito fetal, partos prematuros, pré-eclâmpsia (PE) e trombofilia materna (TM) são algumas das patologias que interferem nessa rede vascular. São muitos os fatores que podem influenciar na tênue interface entre o parto de um feto saudável, um parto prematuro ou até mesmo um aborto. Uma variedade de moléculas como citocinas, proteínas de adesão e fatores de crescimento podem ter um papel importante durante o desenvolvimento fetal assim como em todo processo gestacional. Desta forma, desequilíbrio entre vias de sinalização assim como as vias inflamatórias têm sido relacionadas com o desencadeamento do processo abortivo. O fator de crescimento placentário (PGF) é um marcador importante para identificar deficiência placentária que pode ser ligada a PE. Mulheres com PE tendem a ter níveis inferiores de PGF circulantes quando comparadas a mulheres com gestações saudáveis. A inflamação aguda é comumente associada com injúria fetal assim como a inflamação crônica tem sido associada com a morte fetal e aborto. Estudos epigenéticos e epidemiológicos têm demostrado que indivíduos nascidos de mães que tiveram complicações durante a gravidez possuem maior risco de desenvolverem patologias diversas na vida adulta como acidente vascular cerebral, cardiopatias, obesidade e infertilidade. O Sildenafil, um inibidor de fosfodiesterase tipo-5 com características vasodilatadoras tem ajudado na terapia de restrição de crescimento fetal (RCF). Recentemente Sildenafil, tem sido demostrado por apresentar-se como anti-inflamatório em diversos modelos animais de patologias humanas como esclerose múltipla, hiperplasia prostática, doenças cardiovasculares, pulmonares entre outras. Por outro lado a Heparina de baixo peso molecular tem sido utilizada como tratamento para casos de AR quando o diagnóstico é associado ou não com TM, apesar deste tipo de esquema terapêutico não ser considerado eficaz em muitos casos. O presente estudo se propôs a investigar alvosterapêuticos em modelos animais de AR e PE, através da avaliação do tratamento com Sidenafil ou/com Heparina para a prevenção do processo abortivo assim como a investigação da influência da sinalização via-PGF na formação da vascularização cerebral fetal. Tratamento com Sildenafil sozinho ou tratamento conjunto com Heparina protegeu contra a mortalidade fetal, diminuiu a sinalização inflamatória na placenta assim como melhorou a saúde dos fetos de camundongo expostos a altas doses de Lipopolissacarídeos (LPS) como modelo para perda gestacional. PGF foi identificado em áreas importantes do cérebro de fetos geneticamente normais, no período de gestação média assim como também foram identificados níveis compatíveis do receptor 1 para fator de crescimento vascular endotelial (VEGF): Flt-1. A via que envolve o PGF mostrou-se essencial para o desenvolvimento vascular cerebral saudável. A proteção placentária e por consequência manutenção da saúde do feto é de suma importância para que a gestação chegue a termo e resulte no parto de um indivíduo saudável e sem riscos à saúde da mãe. Esse trabalho traz informações detalhadas de como acontece a modulação durante a vascularização cerebral durante o desenvolvimento em animais geneticamente normais assim como em camundongos PGF-/-. Adicionalmente, demostra como a terapia utilizando Sildenafil isoladamente ou em combinação com Heparina pode diminuir o perfil inflamatório e/ou trombótico do sistema fisiológico gravídico e desta forma prevenir o processo abortivo, mantendo a saúde fetal. / The development of a healthy pregnancy depends on proper uteroplacental circulation. Recurrent miscarriages (RM), fetal death, pre-term labour, preeclampsia (PE) and maternal thrombophilia (MT) are some of the pathologies that may affect with arrangements of this circulation network. There are many factors that can influence the maternal-fetal interface and aberrant gene expression at the site of this interface can result in premature birth or even miscarriage. A variety of molecules including a variety of cytokines, adhesion proteins and growth factors play an important role during fetal development as well as throughout gestation. Indeed, an imbalance of signaling and inflammatory signaling pathways have been associated with the initiation of the abortive process, regardless of the origin of this event. Placental growth factor (PGF) is an important marker to identify placental defects that can be linked to PE. Women with PE tend to have lower levels of circulating PGF when compared to women with healthy pregnancies. Acute inflammation is often associated with fetal injury and chronic inflammation has been associated with abortion and fetal death. Additionally, epigenetic and epidemiological studies have shown that individuals born to mothers who have complications during pregnancy have a higher risk of developing various diseases in adulthood such as: stroke, heart disease, obesity and infertility. Sildenafil, a phosphodiesterase type-5 inhibitor with vasodilating characteristics has proved beneficial in fetal growth restriction (FGR) pathologies. Recent studies have shown that Sildenafil plays an anti-inflammatory agent in a wide range of pathologies in animal models of human diseases such as: multiple sclerosis, prostatic hyperplasia, cardiovascular, and pulmonary diseases among others. Low molecular weight heparin (LMWH) has been used as a treatment for RM cases when diagnosis is associated or not with MT. Despite these facts, recently published meta-analyses have demonstrated that treatments using LMWH are not superior. This study proposed to investigate new therapeutic approaches in animal models of RM and PE. This was accomplished by evaluation of the effectiveness of Sildenafil and/or Heparin in the prevention of fetal mortality/abortion as well as the investigation of the influence of PGF signaling pathway in the formation of fetal brain vasculature. Treatment with sildenafil; alone or in combination with heparin protected against fetal mortality and decreased inflammatory signaling in the placenta, as well as improved the health of the mouse fetuses exposed to high doses of lipopolysaccharide (LPS). PGF was detected in important areas of the brain in genetically normal fetuses and the average period of gestation was also identified as compatible to vascular endothelial growth factor (VEGF) receptor: Flt-1. The pathway involving PGF was found to be essential for healthy vascular development in the brain. Therefore, early placental protection and therefore maintaining the fetus' health is of paramount importance if the pregnancy comes to term and results in the birth of a healthy individual and without risks to health of the mother. This work provides detailed information into critical events that occur during the development of the cerebral vasculature both in genetically normal as well as in PGF -/- mice. Additionally, this work demonstrates how therapies with sildenafil alone or in combination with heparin can reduce the inflammatory status and/or thrombotic physiological systems of pregnancy and thereby prevent fetal mortality, thus maintaining fetal health.

ABSTRACT Preeclampsia (PE) is a one of the more common pregnancy complications that affects 5-8% of pregnancies worldwide and produces significant morbidity and mortality for mother and fetus. Shallow trophoblast invasion and insufficient maternal spiral artery remodeling early in gestation is believed to lead to a relatively hypoxic placenta with inflammatory and trophoblast endoplasmic reticulum (ER) stress. These stresses cause an imbalance in trophoblast expression of angiogenic/anti-angiogenic molecules with decreased placental growth factor (PGF) and increased soluble fms like tyrosine kinase-1 receptor (sFlt-1) production. The decrease in trophoblast PGF seems to be mediated at the transcriptional level while increased expression of sFlt-1 is mediated by alternative splicing. Two variants known to be elevated in PE are the sFlt-1i13 and sFlt-1e15a isoforms. Both share the first 13 exons of Flt-1. A read through into intron 13 and utilization of an alternative poly (A) signal sequence produces the sFlt-1i13 variants protein, while sFlt-1e15a results from alternative splicing of exon 14 to exon 15a, rather than exon 15, and utilization of an alternative poly (A) signal sequence. This angiogenic imbalance contributes to the clinical manifestations of PE later in pregnancy including maternal hypertension and proteinuria. Currently, there are no pharmacological options available for the prevention of PE and the only way to reverse PE symptoms is by delivery. The overall goal of my project was (1) to investigate potential therapeutic mechanisms that could be used to relieve the maternal symptoms of PE by correcting the angiogenic imbalance, and (2) to gain a better understanding of the alternative splicing mechanisms responsible for producing sFlt-1 gene expression in human trophoblast. PE shares some similar pathophysiology and risk factors with cardiovascular diseases. This has prompted use of statins as a potential therapy for PE. However, existing preclinical investigations for statin use has been mostly restricted to PE animal models without elucidating the cell types that respond to statin treatment. Therefore, we sought to determine the effect of statins on angiogenic gene expression in cells that are in direct contact with maternal blood during pregnancy and could contribute to PE: primary trophoblast and endothelial cells. Placental tissue and isolated cells were cultured under hypoxic stress (1% O2) as a model for the hypoxic environment noted in PE. We compared the effectiveness of two types of statins (hydrophilic vs hydrophobic) on angiogenic and anti-angiogenic gene expression from the human tissues and cells. Human placenta villus explants, umbilical vein endothelial cells (HUVECs), and cytotrophoblast were isolated from normal term placentae and cultured under low oxygen tension (1% O2) with serial concentrations of statins. Expression of proangiogenic genes (VEGF and PGF) and the prominent anti-angiogenic sFlt-1 isoforms (sFlt-1i13 & sFlt-1e15a) were analyzed. In villus explants, hypoxia (1% O2) tended to alter angiogenic gene expression in their predicted fashion, by increasing VEGF mRNA (hypoxia marker), decreasing PGF mRNA, and increasing both sFlt-1i13 and sFlt-1e15a mRNA expression. However, the changes in gene expression were quite variable and statistically not significant. Hypoxia significantly increased both sFlt-1i13 and sFlt-1e15a mRNA and protein expression in primary trophoblast but had limited effects on expression in HUVECs. Hypoxia significantly decreased PGF mRNA and protein expression in primary trophoblast, yet significantly increased PGF mRNA and protein expression in HUVECs. Concentrations of pravastatin or simvastatin used had limited effects on altering PGF mRNA and protein expression in any of the cell types. In primary trophoblast, lower concentrations of pravastatin (100/500 µg/ml) had no significant effects on sFlt-1i13 or sFlt-1e15a mRNA expression while higher concentrations (1000 µg/ml) significantly decreased sFlt-1i13 and tended to decrease sFlt-1e15a mRNA expression. Secreted sFlt-1 protein from trophoblast decreased with increasing concentrations of pravastatin. Similarly, simvastatin had limited effects and did not significantly decrease sFlt-1i13 or sFlt-1e15a expression in hypoxic primary trophoblast. Both pravastatin and simvastatin significantly down-regulated sFlt-1i13 and sFlt-1e15a mRNA expression and sFlt-1 protein production in HUVECs. To overcome the effects of statin treatments on sFlt-1 expression, primary HUVECs were treated with farnesyl pyrophosphate ammonium salt (FPP), an intermediate in the cholesterol synthesis pathway. FPP partially restored sFlt-1i13 and sFlt-1e15a mRNA expression. Our data support that the angiogenic imbalance seen in PE can be medicated by hypoxia, and that statin could be a promising medication to limit PE symptoms. The effect of statins may be more evident on endothelial cells than on trophoblast, and the reduction in sFlt-1 expression by statins seems to be partially mediated through the cholesterol synthesis pathway in endothelial cells. The antiangiogenic protein, sFlt-1, plays a central role in the pathophysiology of PE. Excessive amounts of the sFlt-1 receptor in maternal circulation leads to maternal endothelial cell dysfunction and subsequent clinical symptoms of PE. However, the mechanism governing sFlt-1 mRNA expression in trophoblast remains unclear. Jumonji C domain containing gene 6 (JMJD6) has been shown to be involved in splicing of sFlt-1i13 in endothelial cells, although with conflicting outcomes as to whether it increases or decreases alternative splicing of sFlt-1i13. It is unknown if JMJD6 functions to regulate splicing in human primary trophoblast. Therefore, we assessed whether JMJD6 expression is altered in primary trophoblast under hypoxia or ER stress and its ability to regulate alternative splicing of sFlt-1. Human cytotrophoblast were isolated from normal term placentae and were cultured in the presence or absence of ER stress inducer (tunicamycin) or at 1% O2 to simulate trophoblast stressors during PE. Expression of JMJD6, C/EBP homologous protein (CHOP), and sFlt-1 (sFlt-1i13, and sFlt-1e15a) variants were analyzed. Hypoxic stress significantly increases JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression. ER stress also tended to increase JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression in primary trophoblast. Collectively, our results show that low oxygen tension (1% O2) or ER stress increase JMJD6 mRNA expression which may contribute to increased sFlt-1i13 and sFlt-1e15a variant expression in primary trophoblast. Similarly, JMJD6 knock down with siRNA tends to slightly decrease sFlt-1i13 and sFlt-1e15a mRNA expression in primary trophoblast. JMJD6 overexpression in HTR-8 cells (choriocarcinoma) tended to increase sFlt-1i13 and sFlt-1e15a mRNA expression; however, results using HTR-8 were inconsistent due to extremely low expression of endogenous Flt-1 mRNA. To overcome this, a Flt-1 minigene plasmid was transfected into HTR-8 cell line. Under 1%O2 these cells increased expression of the sFlt-1i13 isoform. To more directly confirm effects of JMJD6 and hypoxia on sFlt-1 expression, HEK293 and JEG3 stable clones harboring the Flt-1 minigene were generated. Preliminary results from selected single colony isolates show that several stable clones express the Flt-1 minigene products. HEK293 and JEG3 stable clones harboring the Flt-1 minigene, HEK293-Flt1#5 and JEG3-Flt1#5 respectively, were cultured at 1%O2 for 48 or 72 hours. Hypoxic stress had no significant on altering sFlt-1 variant production or JMJD6 mRNA expression in HEK293-Flt1#5 cells. However, hypoxic JEG3-Flt1#5 cells significantly increased sFlt-1i13 isoform mRNA expression (˜6 fold) and mFlt-1 mRNA expression (2.5 Fold) and also increased JMJD6 mRNA expression (1.8 Fold). In summary, these data suggest a role for statins as a potential therapeutic approach for the prevention and treatment of PE by decreasing systemic sFlt-1 expression in endothelial cells. This effect seems most significant in endothelial cells. If substantiated by clinical studies, use of statins would offer an affordable and easily accessible therapy to lessen PE symptoms. Moreover, our preliminary data suggest a potential involvement of JMJD6 in splicing process of sFlt-1i13. Confirming of JMJD6 role in splicing of Flt-1 may provide therapeutic strategies to treat Flt-1 associated disorder.

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Institute for Rural Development / MRDV

We have studied aspirin as a potential chemopreventive for colorectal cancer, completing Phase I studies on aspirin pharmacology and potential biomarker assays (prostaglandins, PGE2 and PGF(2α) and cyclooxygenase modulation) in normal human subjects. These studies have determined the optimal dose of aspirin for future Phase IIa and IIb chemopreventive trials in high-risk cohorts of patients for colon cancer. Aspirin's effects on rectal prostaglandins are prolonged, detectable even after aspirin and its metabolite are removed from the plasma. Aspirin-mediated inhibition of prostaglandin production in the human rectal epithelium may be related to direct suppression of cyclooxygenase transcription and not to enzyme inactivation by acetylation. A systematic method to monitor adherence (self- report, telephone contact, pill count, and microelectronic monitoring) has been established for future trials. Strategies to improve recruitment of high-risk cohorts have been developed. Phase IIa non-randomized studies with aspirin at 81 mg in high-risk cohorts (resected Duke's A colon cancer, Duke's C colon cancer treated with adjuvant therapy and disease-free at 5 years, history of colon adenomas > 1 cm, two or more first-degree relatives with colon cancer, and familial adenomatous polyposis and hereditary non-polyposis colorectal cancer syndromes) are currently being conducted for surrogate end- point biomarker (prostaglandins, cyclooxygenase, cellular mucins, and proliferation) modulation.

L’obiettivo di questo lavoro è guidare il docente attraverso la letteratura esistente, gli strumenti già in uso e si propone di fornire una modellizzazione per la pratica didattica delle lingue straniere ad alunni sordi. Nel primo e nel secondo capitolo si raccolgono, analizzano e definiscono le basi teoriche di riferimento per la didattica delle lingue a soggetti sordi. Nello specifico, si definiscono il quadro della glottodidattica speciale, i principi di bisogno speciale e specifico e le linee guida per la didattica in contesti di disabilità, procedendo con l’inclusione della sordità tra le esigenze a cui riferirsi con tali strumenti. Il terzo e il quarto capitolo rappresentano l’applicazione concreta dei modelli esposti con l’applicazione del modello SOMA al mondo della sordità e la costruzione del Profilo Glottomatetico Funzionale dell’alunno sordo. Nell’ultimo capitolo si presenta la proposta operativa di didattica dell’inglese ai sordi, partendo dall’analisi e la scelta dei materiali più accessibili, passando per la progettazione fino ad arrivare alla strutturazione specifica del lavoro di classe. / This work aims to guide the foreign language teacher trough the literature on special education needs and deafness, the available tools, and the modeling for everyday practice to encourage the inclusion of deaf students until now considered not teachable. In the first and second chapters, the theoretical approaches to deaf students learning are presented in the contexts of special language teaching, special education, and disabilities. The third and fourth chapters represent the application of those principles to include deaf students in the language classroom activities following the operational models usually used for different learning disabilities. The last chapter offers the concrete model that may guide teachers from their deaf students' needs analysis and the correct material choice for them, passing through the learning project management to get to the specific class lessons, in collaboration with support staff members.