Дисертації з теми "Placental proteins"

UVOD: Hipertenzivna oboljenja u trudnoći predstavljaju heterogenu grupu bolesti koja se javljaju kod 3-8% trudnica opšte populacije. Najteže forme ovih oboljenja preeklampsija, eklampsija i HELLP sindrom su vodeći uzročnici morbiditeta i mortaliteta majke i ploda u odnosu na sve druge komplikacije u trudnoći. Etiopatogeneza ovih oboljenja je još uvek nedovoljno razjašnjena ali se smatra da placenta ima ključnu ulogu u nastanku ovih komplikacija, odnosno da placentalna insuficijencija, koja nastaje kao posledica nedovoljne adaptacije decidualnih i intramiometrijalnih delova spiralnih arterija već u prvih nekoliko nedelja trudnoće, dovodi do redukcije utero-placentalne cirkulacije i lokalne placentalne hipoksije, što se nepovoljno održava i na majku i na plod. U cilju razjašnjenja patofizioloških mehanizama nastanka hipertenzivnih oboljenja u trudnoći i pronalaska dovoljno senzitivnih makera koji bi pomogli u ranom predviđanju nastanka najtežih formi ovih oboljenja, do sada su ispitivani brojni proteini koji učestvuju u procesima stvaranja i razvoja placentalne vaskularne mreže kao što su vaskularni endotelni faktor rasta (VEGF-A), placentalni faktor rasta (PlGF) i solubilni receptor fms-like tirozin kinaza receptor (sFlt-1). CILJ: Uporediti serumske koncentracije (sFlt-1, PlGF, VEGF-A, PAPP-a, freeßhCG, glukoze, ukupnog holesterola, HDL holesterola, LDL holesterola, triglicerida, apo-AI, apoB, AST, ALT, GGT, kreatinina, ureje, mokraćne kiseline, hsCRP, Na, K, Cl, P, Mg i Ca između grupe trudnica sa preeklampsijom, hroničnom i gestacijskom hipertenzijom i kontrolne grupe trudnica u prvom trimestru trudnoće između 11 i 14. nedelje gestacije. Ispitati da li se vrednosti odabranih laboratorijskih parametara (sFlt-1, VEGF-A i PLGF) kod ispitivanih trudnica statistički značajno razlikuju u odnosu na gestacijsku nedelju u trenutku porođaja, težinu i dužinu i APGAR skor bodovanja novorođenčeta. Ispitati da li se vrednosti angiogenih proteina:sFlt-1, VEGF-A, PlGF značajno razlikuju kod ispitivanih trudnica u odnosu na broj prethodnih trudnoća i starosti trudnica. MATERIJAL I METODE: Istraživanje je sprovedeno kao prospektivno analitička studija u Kliničkom centru Vojvodine, u periodu od juna 2012. do februara 2015. godine. U istraživanje je uključeno ukupno 143 trudnice starosti od 18 – 43 godine. Sve trudnice uključene u istraživanje podeljene su na dve ispitivane i jednu kontrolnu grupu. Prvu ispitivanu grupu činilo je 43 trudnice koje su po definisanim kriterijuma razvile preeklampsiju u aktuelnoj trudnoći. Drugu ispitivanu grupu činilo je 46 trudnica kojima je dijagnostikovana ili potvrđena hronična ili gestacijska hipertenzija u aktuelnoj trudnoći. Kontrolnu grupu činilo je 54 zdravih trudnica sa verifikovanim fiziološkim ishodom trudnoće u terminu, bez maternalnih i fetalnih komplikacija. Prilikom regrutovanja trudnica (između 11+0 i 13+6 nedelja gestacije) za učešće u istraživanju, uzeti su anamnestički podaci o faktorima rizika za pojavu hipertenzivnih oboljenja u trudnoći, i u okviru kliničkog i akušerskog pregleda urađena su antropometrijska merenja, merenje krvnog pritiska, i specijalizovani ultrazvučni pregled ploda radi utvrđivanja gestacijske starosti ploda i određivanja rizika za pojavu hromozomskih anomalija ploda. Trudnicama je nakon uzimanja anamnestičkih podataka i kliničkog i akušerskog pregleda i potpisanog pisanog pristanka pacijenta o dobrovoljnom učestvovanju u istraživanju izvađena krv radi određivanja odabranih laboratorijskih parametara. Serumske koncentracije sFlt1, VEGF-A i PIGF određivane su kvantitativnom ELISA tehnikom (R&D Systems Europe Ltd. Abingdon, UK), dok su: glukoza, ukupni holesterol, HDL holesterol, LDL holesterol, trigliceridi, apo-AI I apoB, AST, ALT, GGT, kreatinin, ureja, mokraćna kiselina, hsCRP, Na, K, Cl, Mg, P, Ca određivani na automatizovanim analizatorskim sistemima. Sve trudnice su kategorisane u 2 ispitivane i kontrolnu grupu na osnovu pojave ili isključenja hipertenzivnih oboljenja u aktuelnoj trudnoći. Statistička obrada podataka urađena je u statističkom programu STATISTICA 12 (StatSoft Inc.,Tulsa, OK, USA). Podaci su predstavljeni tabelarno i grafički, nivo statističe značajnosti p, je tumačen statistički značajnim ukoliko su vrednosti p<0,05. REZULTATI: Vrednosti serumskih koncentracija sFlt-1 se statistički značajno razlikuju u sve tri grupe ispitanica i značajno su više u grupama sa hipertenzivnim oboljenjima u odnosu na zdravu grupu ispitanica, p<0,001. Serumske koncentracije VEGF-A su značajno niže u grupi trudnica sa preeklampsijom u odnosu na zdrave trudnice kontrolne grupe (p<0,001), dok se nivoi serumskih koncentracija PlGF statistički značajno razlikuju između sve tri grupe trudnica tako da su najniže vrednosti uočene u grupi sa preeklampsijom (p<0,001) u odnosu na preostale dve grupe ispitanica. Nije uočeno postojanje statistički značajne razlike u nivoima PAPP-A, biohemijskih parametara (glukoze, AST, ALT, GGT kreatinina, ureje, mokraćne kiseline), lipidskih parametara (uk. holesterol, LDL, apo A-I, apo B), parametara inflamatornog (kompletna krvna slika, fibrinogen), hemostaznog (D-dimer, vWF-antigen) i elektrolitskog statusa (Na, K, Cl, P, Mg), p>0,05. Nivoi free ßhCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno više u grupi trudnica sa preeklampsijom u odnosu na grupu bez hipertenzivnih poremećaja u trudnoći. Serumske koncentracije sFlt-1 preko 865 pg/ml imaju senzitivnost od 93% i specifičnost od 81,5% u predviđanju nastanka preeklampsije, dok serumske koncentracije PlGF ispod 60 pg/ml senzitivnost od 88,4% i specifičnost od 79,6% u predviđanju pojave preeklampsije. Serumske koncentracije sFlt-1, VEGF-A i PlGF ne pokazuju statistički značajnu razliku u odnosu na godine života trudnice i broja prethodnih trudnoća p>0,05. Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na telesnu težinu novorođenčeta, tako da su niže vrednosti oba proteina detektovane u grupi novorođenčadi sa porođajnom težinom ispod 1500 gr. u odnosu na telesnu masu između 2800-3300 gr, p<0,001. Takođe su nađene niže vrednosti sFlt-1 i PlGF u grupi trudnica koje su se porodile pre 33. nedelje gestacije u odnosu na nedelju gestacije u trenutku porođaja preko 37 nedelje gestacije, p<0,001. Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na indeks telesne mase majke tako da su više vrednosti sFlt-1 i niže vednosti PlGF nađene u grupi trudnica sa indeksom telesne mase ispod 25 u odnosu na grupu trudnica sa indeksom telesne mase preko 30 kg/m2, p<0,001. Serumske koncentracije sFlt-1 u prvom trimestru trudnoće su značajno povezane sa parametrima inflamacije (hsCRP), vrednostima dijastolnog krvnog pritiska i nivoima free ßhCG. Takođe se uočava značajna povezanost koncentracije PlGF sa indeksom telesne mase, vrednostima sistolnog krvnog pritiska i koncentracijom hsCRP u prvom trimestru trudnoće. ZAKLJUČAK: Nivoi antiangiogenog proteina sFlt-1 su više u grupi trudnica sa preeklampsijom u odnosu na grupu sa hroničnom i gestacijskom hipertenzijom i grupu trudnica bez hipertenzivnih poremećaja trudnoći. Nivoi proangiogenog proteina VEGF-A su značajno niže u grupi trudnica sa preeklampsijom i hroničnom i gestacijskom hipertenzijom u odnosu na grupu trudnica bez hipertenzivnih poremećaja u trudnoći. Serumske koncentracije proangiogenog proteina PlGF su niže u grupi trudnica sa preeklampsijom u odnosu na grupu sa hroničnom i gestacijskom hipertenzijom i grupu trudnica bez hipertenzivnih poremećaja trudnoći. Serumske koncentracije placentalnog proteina free ßhCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno više u grupi trudnica sa preeklampsijom u odnosu na grupu bez hipertenzivnih poremećaja u trudnoći. Između trudnica sa hipertenzivnim poremećajima u trudnoći i zdravih trudnica nije uočeno postojanje značajne razlike u vrednostima placentalnog proteina PAPP-A, biohemijskih parametara (glukoze, AST, ALT, GGT kreatinina, ureje, mokraćne kiseline), lipidskih parametara (uk. holesterol, LDL, apo A-I, apo B), parametara inflamatornog (kompletna krvna slika, fibrinogen), hemostaznog (D-dimer, vWF-antigen) i elektrolitskog statusa (Na, K, Cl, P, Mg). Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na gestacijsku starost na porođaju i telesnu masu novorođenčeta i niže su kod trudnica koje su se prevremeno porodile kao i kod novorođenčati sa manjom porođajnom težinom. Serumske koncentracije sFlt-1 se značajno razlikuju u odnosu telesnu dužinu i APGAR skor novorođenčeta, tako da su više vrednosti sFlt-1 udružene sa većom telesnom dužinom novorođenčeta i boljim APGAR skorom. Serumske koncentracije sFlt-1, VEGF-A i PlGF se ne razlikuju značajno u odnosu na godine života trudnice i broja prethodnih trudnoća. Nivoi proteina angiogeneze sFlt-1 i PlGF predstavljaju dobre prediktore u predviđanju nastanka preeklampsije u prvom trimestru trudnoće.
INTRODUCTION: Hypertensive disorders in pregnancy are a heterogeneous group of diseases that occur in 3-8% of all pregnancies. The most difficult forms of these diseases: preeclampsia, eclampsia and HELLP syndrome are the leading causes of maternal and fetal morbidity and mortality in relation to all other pregnancy complications. Etiopathogenesis of these diseases is still insufficiently understood but it is thought that the placenta plays a key role in the development of these complications, and that placental insufficiency, which occurs as a result of insufficient adaptation of decidual intramiometrial and parts of the spiral arteries in the first few weeks of pregnancy, leading to a reduction of utero- placental circulation and local placental hypoxia, which adversely affects the mother and the fetus. In order to elucidate the pathophysiological mechanisms of hypertensive disorders in pregnancy and to find sufficiently sensitive makers for early prediction of the most severe forms of these diseases, so far have been investigated a number of proteins involved in the processes of creation and development of placental vascular network such as vascular endothelial growth factor (VEGF-A), placental growth factor (PlGF) and soluble fms-like receptor tyrosine kinase receptor (sFlt-1). OBJECTIVE: The aim of the study was to compare serum concentration of sFlt-1, PlGF, VEGF-A, PAPP-A, freeßhCG, glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apo-AI, apo B, AST, ALT, GGT, creatinine, urea, uric acid, hsCRP, Na, K, Cl, P, Mg and Ca between the group of pregnant women with preeclampsia, chronic and gestational hypertension and the control group of pregnant women in the first trimester of pregnancy between 11 and 14 weeks gestation. Also the aim was to examine whether the value of selected laboratory parameters (sFlt-1, VEGF-A and PlGF) differ in relation to gestational week at the time of birth, weight, length and APGAR scoring system of newborns. The aim was to examine whether the value of angiogenic proteins: sFlt-1, VEGF-A and PlGF differ significantly in relation to the number of previous pregnancies and age of the pregnant woman. MATERIALS AND METHODS: The study was conducted as a prospective analytical study in the Clinical Center of Vojvodina, in the period from June 2012 to February 2015. The study included a total of 143 pregnant women aged 18 - 43 years. All pregnant women included in the study were divided into two study and one control group. The first study group consisted of 43 pregnant women who developed preeclampsia during the current pregnancy. The second study group consisted of 46 pregnant women who are newly diagnosed or confirmed chronic or gestational hypertension during the current pregnancy. The control group consisted of 54 healthy pregnant women with verified physiological outcome of pregnancy at term without maternal and fetal complications. Patients were included in the study between 11 + 0 and 13 + 6 weeks of gestation. All patients had data about risk factors for developing hypertensive disorders in pregnancy. After clinical and obstetric examination all patients underwent anthropometric measurements, measurement of blood pressure, and specialized ultrasound examination to determine precise gestational age of the fetus and to determine the risk for fetal chromosomal abnormalities. All patients signed a written consent of the patient's voluntary participation in the study. Serum levels of sFlt1, VEGF-A and PlGF were determined by quantitative ELISA (R & D Systems Europe Ltd., Abingdon, UK), while glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apo-AI, apo B, AST, ALT, GGT, creatinine, urea, uric acid, hsCRP, Na, K, Cl, P, Mg, Ca were determined on automated analyzer systems. All pregnant women were categorized into 2 study and a control group on the basis of presence of hypertensive disorders in the current pregnancy. Statistical analysis was performed in 12 statistical program STATISTICA (StatSoft Inc., Tulsa, OK, USA). The data are presented in tables and graphs, the level of significance p is interpreted statistically significant if the p value was less than <0.05. RESULTS: Serum concentrations of sFlt-1 are statistically significantly different in all study groups and significantly higher in the groups with hypertensive disorders compared to healthy subjects p <0.001. Serum levels of VEGF-A are significantly lower in the preeclampsia group compared to healthy control group (p <0.001), while the levels of serum concentration of PlGF statistically significantly different between all groups so that the lowest values are observed in the preeclampsia group (p <0.001) compared to the other two study groups. There is no statistically significant differences in the levels of PAPP-A, biochemical parameters (glucose, AST, ALT, GGT creatinine, urea, uric acid), lipid parameters (total cholesterol, LDL, apo AI, apo B), inflammatory parameters (complete blood count, fibrinogen), hemostatic (D-dimer, vWF-antigen) and electrolyte status (Na, K, Cl, P, Mg, Ca), p> 0.05. Levels of free ßhCG and HDL cholesterol levels are significantly lower, while the value of hsCRP and triglycerides significantly higher in the group of women with preeclampsia compared to the healthy control group. Serum concentrations of sFlt-1 over 865 pg/ml have a sensitivity of 93% and specificity of 81.5% in predicting preeclampsia, while serum PlGF concentration below 60 pg/ml, a sensitivity of 88.4% and a specificity of 79.6% in predicting preeclampsia. Serum concentrations of sFlt-1, VEGF-A and PlGF do not show a statistically significant difference compared to the age of pregnant women and the number of previous pregnancies p> 0.05. Serum concentrations of sFlt-1 and PlGF are significantly different in relation to the weight of the newborn, so that the lower values of both proteins are in the group of infants with birth weight below 1500 gr. in relation to the body weight between 2800-3300 gr., p <0.001. There is also lower concentrations of sFlt-1 and PlGF in a group with deliveries before 33 weeks of gestation compared to the deliveries after 37 week of gestation, p <0.001. Serum concentrations of sFlt-1 and PlGF are significantly different in relation to the mother's body mass index so that the lower values of sFlt-1 and PlGF are in the group of women with a body mass index below 25 in relation to a group with a body mass index over 30 kg/m2, p <0.001. Serum concentrations of sFlt-1 in the first trimester of pregnancy were significantly associated with the parameters of inflammation (hsCRP), diastolic blood pressure and levels of free ßhCG. It is also observed a significant correlation between PlGF with a body mass index, systolic blood pressure and hsCRP concentration in the first trimester of pregnancy. CONCLUSION: The levels of anti-angiogenic protein sFlt-1 are higher in the group of pregnant women with preeclampsia than in the group with chronic and gestational hypertension and the control healthy group. Levels of proangiogenic VEGF-A protein are significantly lower in the preeclampsia group and group with gestational and chronic hypertension compared to the control group. Serum levels of proangiogenic PlGF protein are significantly lower in the preeclampsia group than in the group with chronic and gestational hypertension and the control group. Serum concentrations of placental protein free ßhCG and HDL cholesterol are significantly lower, while the value of hsCRP and triglycerides significantly higher in the preeclampsia group compared to the control group. Among pregnant women with hypertensive disorders in pregnancy and healthy pregnant women there are no significant differences in the values of placental PAPP-A protein, biochemical parameters (glucose, AST, ALT, GGT creatinine, urea, uric acid), lipid parameters (total cholesterol, LDL, apo AI, apo B), inflammatory parameters (complete blood count, fibrinogen), hemostatic (D-dimer, vWF-antigen) and electrolyte status (Na, K, Cl, P, Mg, Ca). Serum concentrations of sFlt-1 and PlGF are significantly different in relation to gestational age at delivery and newborn body weight and are lower in group with preterm delivery and newborns with lower birth weight. Serum concentrations of sFlt-1 are significantly different compared to body length and Apgar score, so that the higher values of sFlt-1 are associated with better outcome of newborns (greater body length and better APGAR score). Serum concentrations of sFlt-1, VEGF-A and PlGF are not different significantly with respect to age of pregnancy and the number of previous pregnancies. The levels of sFlt-1 and PlGF represents helpful markers in prediction of preeclampsia in the first trimester of pregnancy.

The placenta is responsible for mediating fetal growth and development, thereby influencing health across the lifespan. Physical activity (PA) confers benefits to mother and baby during pregnancy, but little is known about its impact on the placenta. There were two purposes of this study: i) to determine if maternal PA during pregnancy influences placenta mitochondrial protein content and function, and ii) to determine if there were differences in placenta mitochondrial protein content and function in different regions of the placenta, namely proximal or distal to the centre of the placenta. Healthy women between 12-28 weeks gestation were recruited, and free-living PA was objectively assessed at multiple time points during pregnancy using an accelerometer. Participants were grouped by minutes of moderate-to-vigorous PA (MVPA) per day. Placenta tissue samples were collected from central and distal placental regions immediately post-birth and were used for two separate analyses. Half of the samples were flash frozen in liquid nitrogen and used for western blot analysis of mitochondrial complex I-V proteins. Fresh mitochondria were isolated from the other half of the samples, and high-resolution respirometry was used to measure placental mitochondrial respiration. There were significant positive correlations between maternal PA and mitochondrial protein content in peripheral tissue samples, but protein content was significantly higher in central tissue compared to peripheral tissue samples. In addition, state 3 respiration was higher in central tissue samples of placentas from participants with high MVPA compared to participants with low MVPA. Finally, complex I protein was higher in central tissue samples of placentas from female offspring compared to placentas of male offspring. However, many of these results are underpowered and further study is warranted. This study provides new avenues to explore the relationship between PA and placenta mitochondria in healthy populations.

Apesar de sua importância clínica e epidemiológica, a fisiopatologia da préeclâmpsia ainda não foi completamente compreendida. Sabe-se que a doença constitui-se de uma fase pré-clínica e um estágio clínico. Durante a última década muito esforço tem se concentrado na identificação precoce da doença, ainda em sua fase pré-clínica. A literatura científica tem demonstrado claramente um desequilíbrio na regulação da angiogênese das gestantes com pré-eclâmpsia, marcado por níveis elevados do fator antiangiogênico soluble fms-like tyrosine kinase-1 (sFlt-1) e níveis diminuídos do fator pró-angiogênico placental growth fator (PlGF). Embora um número crescente de estudos em populações de alto risco tenha avaliado o papel desses biomarcadores no diagnóstico de pré-eclâmpsia, dados sobre sua utilização para a predição de pré-eclâmpsia superajuntada, cujo diagnóstico pode ser particularmente difícil, permanecem relativamente escassos e controversos. Com o presente estudo pretendemos avaliar o desempenho de medidas seriadas dos níveis maternos circulantes dos fatores sFlt-1 e PlGF, bem como da razão sFlt-1/PlGF, para predição de pré-eclâmpsia superajuntada e compará-lo ao seu desempenho na predição de pré-eclâmpsia em sua forma \"pura\", não superajuntada. Para este propósito, estudamos uma coorte prospectiva composta de dois braços, um de gestantes com hipertensão arterial crônica e outro de gestantes normotensas, e avaliamos os níveis séricos de sFlt-1 e de PlGF e a razão sFlt-1/PlGF nas idades gestacionais de 20, 26, 32 e 36 semanas, tendo como desfecho principal o diagnóstico de pré-eclâmpsia. Um total de 97 gestantes foram acompanhadas, 37 normotensas e 60 com hipertensão arterial crônica. Entre elas, 4 (10,8%) desenvolveram pré-eclâmpsia e 14 (23,3%) desenvolveram pré-eclâmpsia superajuntada. Para predição de pré-eclâmpsia, a análise ROC (Receiver Operating Characteristics) apresentou área sob a curva (AUC - area under curve) de 0,83 (IC 95% = 0,68-0,99, P = 0,035) para dosagem de PlGF com 20 semanas e AUC = 0,92 (IC 95% = 0,81 - 1,00, P = 0,007) para a razão sFlt-1/PlGF com 26 semanas de gestação. A variação percentual dos níveis de PlGF entre 26 e 32 semanas de gestação apresentou AUC = 0,96 (IC de 95% = 0,89-1,00, P = 0,003). Para a predição de pré-eclâmpsia superajuntada, a razão sFlt-1/PIGF na idade gestacional de 32 semanas apresentou AUC = 0,69 (IC de 95% = 0,53-0,85, P = 0,039). Entre 20 e 26 semanas de gestação, a variação percentual do PIGF e da razão sFlt-1/PlGF apresentaram, respectivamente, AUC = 0,74 (IC de 95% = 0,58-0,90, P = 0,018) e AUC = 0,71 (IC 95% = 0,52-0,91, P = 0,034). Por nossos resultados podemos concluir que, embora os níveis de PlGF e a razão sFlt-1/ PlGF tenham apresentado bons desempenhos na predição de pré-eclâmpsia, é preciso ter cuidado ao usá-los para a predição de pré-eclâmpsia superajuntada. Nessas gestantes, a dosagem dos fatores angiogênicos apresenta capacidade de predição menor e mais tardia. Avaliações seriadas dos fatores podem melhorar o desempenho dos testes para predição de pré-eclâmpsia superajuntada em idades gestacionais mais precoces
Despite being a major public health problem, the pathophysiology of preeclampsia is incompletely understood. Preeclampsia progression comprises a pre-clinical stage and a clinical stage. During the last decade much work has focused on identifying the pre-clinical stage of preeclampsia. Many researchers have clearly demonstrated an anti-angiogenic imbalance that is marked by higher levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and lower levels of placental growth factor (PlGF) in the subjects who develop preeclampsia compared with those who do not. Although a growing number of studies in the high-risk population have shown the role of these biomarkers in diagnosing preeclampsia, superimposed preeclampsia, which can be a challenging diagnosis, remains partially understudied and the literature regarding this subject continues to be relatively scarce as well as controversial. By this study, we aimed to evaluate the performance of serial measurements of maternal circulating sFlt-1 and PlGF levels for the prediction of superimposed preeclampsia in chronic hypertensive subjects and to compare it to the prediction of preeclampsia in normotensive control subjects. For this purpose, we evaluated a two-armed prospective cohort of women with normotensive and chronic hypertensive pregnancies and assessed the serum levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio at gestational ages of 20, 26, 32 and 36 weeks, having preeclampsia as the primary outcome to be predicted. A total of 97 women were followed-up, 37 in the normotensive group and 60 in the chronic hypertensive group. Among them, 4 (10.8%) women developed preeclampsia and 14 (23.3%) developed superimposed preeclampsia. For predicting preeclampsia, PlGF at 20 gestational weeks presented an AUC=0.83 (CI 95% = 0.68 - 0.99, P=0.035) and the sFlt-1/PlGF ratio at 26 gestational weeks presented an AUC=0.92 (CI95% = 0.81 - 1.00, P=0.007). The percent change of the PlGF levels between 26 and 32 gestational weeks presented an AUC=0.96 (CI 95% = 0.89 - 1.00, P=0.003). For predicting superimposed preeclampsia, the sFlt-1/PlGF ratio at 32 gestational weeks presented an AUC=0.69 (CI 95% = 0.53 - 0.85, P=0.039). Between 20 and 26 gestational weeks, the percent change of PlGF and the sFlt-1/PlGF ratio presented, respectively, an AUC=0.74 (CI 95% = 0.58 - 0.90, P=0.018) and an AUC=0.71 (CI 95% = 0.52 - 0.91, P=0.034). By our results, we concluded that, although the PlGF level and the sFlt-1/PlGF ratio present good performances in the prediction of preeclampsia, caution is required when using them for the prediction of superimposed preeclampsia. Sequential assessments slightly improve the test performances for predicting superimposed preeclampsia at earlier gestational ages

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第5666号
医博第1553号
新制||医||582(附属図書館)
UT51-94-J98
京都大学大学院医学研究科外科系専攻
(主査)教授 西川 伸一, 教授 藤田 潤, 教授 森 崇英
学位規則第4条第1項該当

Placenta growth factor (PGF) expression is downregulated in preeclampsia (PE), a leading cause of maternal morbidity and mortality. The pathophysiology of PE is thought to be manifested by a poorly perfused placenta hampered by hypoxic stress. Two stress-mediate angiogenic responses include post-transcriptional regulation of mRNA stability, and regulation of PML sequestering protein. We investigate whether these mechanisms occur in hypoxic stressed trophoblast and preeclamptic placenta. Methods: To determine transcript stability, PGF mRNA was measured in normal vs. stressed conditions, and the PGF 3'UTR was analyzed for consensus 3'AREs. To characterize stability regulation, the PGF 3'UTR was cloned into a reporter construct. To investigate the association between PGF mRNA and RNA binding proteins, an RNA-Immunoprecipitation assay was performed on trophoblast. PML study: Normal (n=6) and preeclamptic (n=6) placentae were assessed for PML expression. Immunoblot, qRT-PCR and immunohistochemistry techniques were used to determine PML gene expression and localization in placental tissue and primary cells. Results: Two consensus ARE motifs were detected within the human PGF 3'UTR at the 42nd nucleotide and the 91st nucleotide downstream from the PGF coding region. Identical and spatially conserved ARE motifs were found in bovine, rat and mouse PGF 3'UTR. Actinomycin D transcription inhibitor ii studies that were used to measure RNA decay rates in hypoxic and normal conditions, demonstrated a transcriptional response of PGF mRNA to hypoxic stress. Additionally, the PGF 3'UTR did not alter gene expression significantly relative to a site-directed mutant after 24 hours hypoxia. However, PGF 3'UTR decreased reporter activity relative to parental control, suggesting that it could function to regulate stability, but not in hypoxic stress. Western blots and immunohistochemical analyses showed the presence of three potential ARE binding proteins in trophoblast. RNA-Immunoprecipitation assays suggest that PGF mRNA may interact weakly with HuR protein. Upregulation of RNABP expression by insulin was investigated for its effects to upregulate RNABP, and was found to transcriptionally downregulate PGF mRNA. Tumor necrosis factor alpha (TNFa) treatment initiated a short-term upregulation of PGF mRNA. PML Study Results: PML protein was immunolocalized within nuclei of villus mesenchyme, but largely absent in trophoblast nuclei. A trend for increased PML reactivity in placenta of preeclamptic patients was observed. Immunoblot analyses of nuclear extracts confirmed relative increases (~3 fold) of PML expression in preeclamptic placentae (p< 0.05). Conversely, less PML mRNA (~2 fold) was detected in preeclamptic versus normal placental samples. In vitro, PML expression could be increased by hypoxia in cultured endothelial cells but not trophoblast. Conclusion: These results suggest that a post-transcriptional mechanism directed through the 3'UTR does not regulate PGF mRNA expression in stressed trophoblast. PML Study conclusion: Increased PML protein expression in preeclamptic villi suggests it could contribute to decreased vascularity and placental growth and/or function.

A novel molecular mechanism of autophosphorylation-dependent activation of the ser/thr S6/H4 kinase isolated from human placenta is described. Phosphopeptide mapping of the enzyme was used to determine the rate and extent of site-specific autophosphorylation. These data were correlated to phosphotransferase activity of the protein kinase. The results indicated that a sequential phosphorylation of two sites in the catalytic domain is required for maximum activation. Kinetic analysis determined that site 1 is modified by an intramolecular phosphorylation, and site 2 is modified by an intermolecular phosphorylation. On the basis of these data a model is proposed in which autophosphorylation of the pseudosubstrate domain and on a serine residue in subdomain VIII are both required for maximum activation of the S6/H4 kinase.

PP14 has been shown to suppress the incorporation of [3]H-Thymidine into both mitogenically and allogeneically stimulated lymphocytes in a dose dependent manner. The suppressive activity was shown to be specific, in that PP14 did not affect cellular viability, nor interact with the mitogen phytohaemagglutinin (PHA). Flow cytometric analysis indicated that PP14 had no effect on the expression of the Tac antigen, the transferrin receptor or HLA-DR molecules on the surface of stimulated lymphocytes. Neither did PP14 affect the interaction of interleukin-2 (IL-2) with its cell surface receptor. The suppressive activity was partially reversed by the addition of exogenous IL-2. PP14 inhibited the production of IL-2 from mitogenically stimulated lymphocytes and led to a small, but significant reduction in soluble IL-2 receptor release. Radiolabel binding studies and IL-2 dose response curves indicated that PP14 affected the affinity of the IL-2 receptor on PHA stimulated lymphocytes. This was supported by the observation that PP14 increased the level of cell surface-associated IL-2 on stimulated lymphocytes. There was a small inhibition of gamma interferon levels early in the culture period. PP14 had no effect on the CD4/CD8 ratio following stimulation and was not found to be associated with the cell surface, nor mask cell surface expression of the CD2 antigen. These data suggest that the immunosuppressive activity of PP14 may, in part, be mediated via a modulation of the functional, high affinity IL-2 receptor. It is not known as yet whether such an activity is effective at the level of induction of the receptors or whether the primary control is at another level of the response. PP14 may have implications in the study of implantation and fertility and prove of wider interest in the field of transplantation biology and the control of the immune response.

As Heat Shock Proteins (HSPs) ou proteínas do choque térmico são encontradas em todas as células e são classificadas de acordo com seu peso molecular. Dentre elas encontram-se as de 27, 60, 70, 90 e 110 kDa, sendo as mais estudadas no contexto da reprodução as da família 60 e 70. Essas proteínas são ditas como chaperoninas, em razão do seu importante papel no dobramento e desdobramento de outras proteínas celulares sem alterar sua conformação final, e são expressas frente a qualquer tipo de estresse como calor, vírus, bactéria, hormônios, diferenciação celular, etc, e influenciam nas respostas imune inata e adquirida. A placenta também expressa essas proteínas, uma vez que é um órgão de intenso estresse e diferenciação celular durante toda a gestação. Nesse estudo, busca-se avaliar a expressão ou não dessas proteínas na placenta bovina e para isso foram utilizadas 30 amostras de diferentes animais em estágios distintos de gestação, fixadas em formol tamponado a 10% e processadas pela técnica de imuno-istoquímica. O mesmo numero de amostras foi também processado para a análise de imuno-microscopia eletrônica de transmissão pelas técnicas de \"freeze-substitution\" e marcação por pós-embebição. Na imuno-istoquímica, as HSPs 60 e 70 foram localizadas nos trofoblastos, epitélio materno e células binucleadas. A expressão da HSP 60 foi maior no início declinando no segundo e terceiro terço. Já a expressão da HSP 70 manteve-se praticamente constante, evidenciando a forte expressão dessa proteína durante todo o período. Na análise de imuno-microscopia eletrônica de transmissão, ambas as famílias foram localizadas nas células binucleadas (núcleo, citoplasma e vesículas) e epitélio materno (núcleo e citoplasma) em todos os terços gestacionais. O perfil das proteínas estudadas na placenta bovina foi diferente quando comparada à placenta humana, pois nesta última, a intensidade da expressão para a HSP 70 diminuiu com o decorrer da gestação enquanto para a HSP 60 foi constante durante todo a gestação. Provavelmente essas diferenças podem estar relacionadas ao fato dessas amostras terem sido coletadas de mulheres com gravidez interrompidas e também pelo tipo de placentação distinta. A bovinocultura de corte é de extrema importância para a econômica para o Brasil e se faz necessário o conhecimento de fatores que possam melhorar suas características reprodutivas. Dessa forma os resultados obtidos nesse estudo contribuirão certamente de subsídio para experimentos futuros sobre o papel das Heat Shock Proteins na placenta bovina.
Heat Shock Proteins (HSP) can be found in any kind of cell. These proteins are classified according to their molecular weight and their known families include the HSP 27, 60, 70, 90 and 110 kDa. Among these, HSP 60 and 70 are the ones of interest in reproduction. They were known as chaperonines because of their capacity to fold and unfold other proteins into the cell, without changing their own conformation. They are expressed during several stress conditions likes virus and bacteria infections, hormones, heat, cellular differentiation, etc, and also take part signalizing for innate and acquired immune responses. Heat shock proteins are expressed in several tissues and organs, including the placenta. In this study we have evaluated the expression of these proteins in the bovine placenta, using thirty samples from different animais with distinct gestational periods, fixed in 10% formalin and processed for immunohistochemistry. The same numbers of samples were processed for immunoelectron microscopy using freeze-substitution and post embedding labeling techniques. The immunohistochemistry results show the expression of HSP 60 and 70 in trophoblasts, maternal epithelia and binucleated cells. The HSP 60 expression was higher in the beginning of gestation, becoming lower during the second and third trimester. Heat shock protein 70 expression were practically constant throughout the gestation. The immunoelectron microscopy analysis revealed that both HSP 60 and 70 were located in the cytoplasm and nucleio binucleated cells and maternal epithelia from the beginning to the end of pregnancy. The immunolocalization of HSP 60 and 70 in the bovine placenta were distinct from the ones found in studies on women, probably due to the differences of the placentation type and to the fact that those samples were collected from abnormal or discontinuous pregnancy. Beef production in Brazil is an important economical activity and studies to improve the bovine reproductive characteristics are necessary and must be expended, therefore our results certainly contributes for further studies on HSP function during pregnancy in this species.

Global nutrient restriction or excess can influence umbilical hemodynamics in sheep fetuses (Chapter 2). We hypothesized that a specific component of the diet, namely maternal metabolizable protein (MP), would alter placental function. When MP restriction during late gestation occurs, we hypothesized that there would be a decrease in the sensitivity to bradykinin (BK) of the placental vascular arteries. In experiment 1, ewes received one of three isocaloric dietary treatments during late gestation: MP60: 60% of MP requirements; MP80: 80% of MP requirements; and MP100: 100% of the MP requirements on a dry matter basis from day 100 to 130 of gestation. In experiment 1, fetal and placental mass were not affected by dietary treatment; however, placental function was altered by a maternal diet low in protein. Ewes not meeting MP requirements during late gestation had fetal placental arteries that were more sensitive to BK-induced vasorelaxation; therefore we reject our hypothesis for experiment 1. In order to understand the mechanism of BK-induced vasodilation in the placental arteries, experiment 2 was designed. We hypothesized that MP level would alter the mechanism of BK-induced vasorelaxation in placental arteries. In experiment 2, ewes received one of three isocaloric dietary treatments during late gestation: MP60: 60% of MP requirements; MP100: 100% of the MP requirements; and MP140: 140% of MP requirements from day 100 to 130 of gestation. Maternal protein level during gestation did not impact the mechanism of BK-induced vasodilation; therefore we reject our hypothesis for experiment 2. However, the maternal and fetal placental vessels responded to BK through different iv mechanisms. In maternal placental arteries, pathways involving endothelium-derived hyperpolarizing factors (EDHF) and nitric oxide (NO) were responsible for BK-induced vasodilation, while the prostacyclin (PGI2) pathway did not greatly contribute to BKinduced vasodilation. The fetal placental arteries responded to BK through a mechanism that does not involve EDHF, NO, or PGI2, indicating that BK-induced vasorelaxation of the fetal placental arteries may be mediated through an unclassified EDHF-like pathway. It is important to realize the maternal and fetal placental arteries may respond to BKinduced vasodilation through different pathways when considering possible therapeutics for compromised pregnancies.

HIV protease inhibitors are an important component of Highly Active Antiretroviral Therapy used to treat HIV infected pregnant women. They have a low placental transfer and are highly plasma protein bound. The purpose of this thesis was to characterize the factors limiting placental passage and fetal exposure to lopinavir. These factors include lopinavir plasma protein binding and uptake, cellular binding, and efflux of lopinavir in the placental trophoblast cells. First, we determined the unbound fraction of lopinavir in cord blood and characterized the binding of lopinavir to α1-acid glycoprotein (AAG) and human serum albumin (HSA), and displacement by ritonavir. Serum was obtained from cord blood from placentae obtained after cesarean section of healthy non-HIV infected women (n=4). The unbound fraction of lopinavir in serum obtained from this cord blood was 0.02 ± 0.01. The unbound fraction of lopinavir in separately obtained maternal serum samples (n=4) was 0.009 ± 0.001, which was not significantly different from that observed with cord serum samples. Varying concentrations of lopinavir, AAG, and HSA in buffer solutions were then used to characterize the lopinavir binding. The data were fit to obtain the number of binding sites (N) and equilibrium dissociation constant (KD). Binding of lopinavir to AAG (7-23 µM) was saturable with KD of 5.0 ± 1.1 µM and N of 1.2 ± 0.2. At low HSA concentrations (15-152 µM), lopinavir binding KD was 24.3 ± 8.7 µM and N was 1.1 ± 0.4; however at 758 µM, lopinavir binding was essentially unsaturable. Additionally, lopinavir binding to AAG and HSA was not sensitive to ritonavir within the range of therapeutic concentrations. Next, we examined lopinavir uptake, binding and efflux using the BeWo human trophoblast cell culture model. BeWo cells were treated with 3H-lopinavir in the absence or presence of inhibitors of ATP- Binding Cassette transporters. The radioactivity was then measured in the buffer and the cells after incubating for different time intervals and at two temperatures. Verapamil (100µM) stimulated apparent efflux of 3H lopinavir by two fold, possibly due to ABCC2. In addition, this efflux process was 75% inhibited by reduced temperature (4°C). Ritonavir (10 µM) also stimulated 3H-lopinavir efflux, whereas GF120918 (1 µM) had no effect. Reduced temperature (4°C), verapamil (100 µM) or ritonavir (10 µM) individually did not significantly affect the binding of 3H-lopinavir to cell homogenates. However, slight but significant binding displacement by verapamil at 4°C was observed. 3H lopinavir uptake was not sensitive to verapamil, bromosulfophthalein, taurocholate or to reduced temperature suggesting uptake involves diffusion rather than Organic Anion Transporting Polypeptide transporters. The results suggested that interplay between cellular binding and ABC efflux transporters, in addition to simple diffusion, determines the extent of 3H-lopinavir distribution into BeWo cells.

[Truncated abstract] Glucocorticoids are critical for the maturation of the fetus late in pregnancy. Indeed, clinical administration of glucocorticoids is used to accelerate fetal lung maturation in mothers at risk of pre-term delivery. Increased glucocorticoid exposure, however, can have detrimental effects on fetal and placental growth and increase the risk of disease in later life. Many studies have focused on the effect of an increase in the transplacental passage of glucocorticoids on both fetal growth and subsequent postnatal development. But there is a growing body of evidence to suggest that the impact of glucocorticoids on fetal growth is mediated, in part, via their direct effects on the placenta . . . Overall, these studies quantify the labyrinth zone-specific increases in placental expression of PPARG and VEGF in association with a marked increase in vascularisation observed near term. Furthermore, this study demonstrates for the first time that these increases in gene expression are prevented by maternal dexamethasone treatment which also inhibits growth of the fetal capillary network. Elevated expression of SFRP4 in the regressing basal zone late in gestation and in both placental zones after dexamethasone-induced placental growth restriction is consistent with a role for SFRP4 in glucocorticoid-mediated inhibition of wnt signalling. Collectively, the data presented in this thesis show that glucocorticoid inhibition of fetal growth is mediated in large part via effects on the placenta, specifically through inhibition of signals that promote proliferation and vascularisation.

A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully

Fibrinogen-like protein 2 (FGL2) is a known immunomodulator and prothrombinase, expressed by several subsets of immune cells. This thesis explores its potential role during the establishment of pregnancy, in mice, as well as in trophoblast function and in an immune-mediated subtype of preeclampsia (PE), in humans. We first noticed a marked subfertility in Fgl2 knockout (ko) and Fgl2 overexpressing (tg) colonies, where litters were fewer and smaller. To explain this, we mapped spatiotemporal patterns of FGL2 expression in the female reproductive tract and through the estrous cycle. FGL2 is expressed in the ovarian stroma and theca cell layer, peaking shortly before ovulation. Fgl2 ko and tg mice do not show a defect in natural or induced ovulation. FGL2 is expressed in secretory cells of the oviductal epithelium, and Fgl2 ko mice have reduced fertilization efficiency. Fgl2 tg pups are noticeably small, and we find that a reduced ratio of glycogen cells in the junctional zone of their placenta partly explains this. We next investigated the role of FGL2 in trophoblast function, using BeWo and HTR-8/SVneo cell lines. Inflammatory cytokines increase FGL2 expression in BeWo, and FGL2 overexpression promotes syncytialization. We show that it therefore rescues the deleterious effect of inflammation on syncytium formation. In a large cohort of PE and non-PE human placentas, FGL2 is high in a subtype with immune activation, and low in a canonical, anti-angiogenic subtype. Its expression correlates with incidence of chronic inflammatory histopathological lesions, likely driven by immune rejection gene sets. High FGL2 also associates with a high incidence of fibrin deposition in the placenta. Overall, we conclude that FGL2 is involved in several steps of maternal immune adaptation, both before and after pregnancy. Its absence and excess both contribute to mouse subfertility. In the developing and mature placenta, FGL2 is increased by inflammation in the trophoblast and immune compartment of the mature placenta, as a physiological attempt to re-establish immune equilibrium and protect the ongoing pregnancy.

Movements of calcium (Ca) across the maternal and fetal aspects of the human placenta were investigated using the isolated placental lobule dually perfused in vitro. Tissues uptakes and releases of calcium were measured and the effects on calcium movements by calcium-protein binding in the perfusion fluids, (associated with extracellular pathways and non-uniform perfusion), evaluated. The effects of ouabain, dinitrophenol (DNP), and cooling on calcium movements were measured and compared to movements of Na⁺ and K⁺. These indicated the presence of active transport of calcium but no evidence was obtained for Ca²⁺/Na⁺ exchange. Cyclic adenosine 3', 5' -monophosphate (cAMP) levels in dually perfused tissue were measured following microwave fixation. This technique was used to measure changes in tissue cAMP production following exposure to forskolin, 3-iso-butyl-l-ethyl-xanthine (IBMX), and various fragments of both bovine parathyroid hormone (bPTH) and human parathyroid hormone-related peptide (hPTHrP). Rises in cAMP were produced by exposure to bPTH(1-34) but not hPTHrP(1-34), hPTHrP(67-86) or hPTHrP (107-138). It is concluded that calcium is actively transported across the placenta but there is no major contribution via a Ca²⁺/Na⁺ exchanger. The patterns of calcium uptake as a function of perfusate calcium concentration support the evidence of other workers that extracellular pathways are present in the syncytiotrophoblast. A significant amount of passive movement of calcium may therefore take place across the perfused placenta. The 1 to 34 region of the PTH molecule stimulates the production of cAMP by the trophoblast, but there is no indication that this has any effect on transplacental Ca transport.

This thesis describes investigations into Placental Protein 14 (PP14), a immunomodulator involved in human reproduction. The studies included the development of a purification procedure and an investigation of the activity of the protein. In addition the cDNA coding for the protein was cloned and expressed as a recombinant fusion protein and the molecular structure of the protein was predicted and analysed using computer-assisted modelling. Finally the clinical significance of the protein was studied in a range of patient groups. The purification scheme consisted of ion exchange, hydrophobic interaction and gel filtration chromatography, and the pure protein obtained was analysed by SDS-PAGE and Western blotting. The results demonstrate that the purification procedure is a suitable method to obtain PP14 in large quantity and with high purity. PP14 purified by this method retained its activity and was shown to suppress, in a dose-dependent manner, the uptake of 3H-Thymidine by peripheral blood mononuclear cells stimulated with interleukin-2. Purified PP14 was also shown to suppress the uptake of 3H-Thymidine by the cell line U937, also in a dose-dependent manner. This suppression could be removed by the incubation of the PP14 sample with an immunoabsorbent gel linked to monoclonal antibodies against PP14, demonstrating that PP14 was the molecule responsible for the observed activity. Based on the suppression by PP14 of U937 cell growth a bioassay for PP14 was developed, this assay was used to express the specific activity of PPM in Units/ml. To obtain recombinant PP14, mRNA was purified from a tissue sample and reverse transcription used to prepare cDNA. Specific primers were used to amplify the portion of cDNA coding for PP14 which was then ligated into the plasmids pUC 18 and pGEX-KG. Recombinant PP14 was then expressed as a fusion protein with glutathione-S-transferase. The expression conditions were optimised and the fusion protein was purified using affinity chromatography. The structure of PPM was investigated using computer assisted modelling. PPM is a member of the lipocalin family of proteins which share the feature of binding small hydrophobic molecules. The X-ray coordinates of two lipocalins known to share sequence homology with PP14 were used as a basis to model a predicted structure for PP14. An analysis of the structural motifs of the protein was carried out, and it was established that PP14 shares many of the characteristic features of this family of proteins including the presence of a binding pocket. The model was then used to predict potential ligands for PP14.PP14 was measured by radioimmunoassay in uterine flushings from fertile women, women with unexplained infertility and women suffering from recurrent miscarriages, and in plasma samples from fertile and infertile women. The results from the uterine flushings from fertile women showed that PP14 levels rose during the second half of the menstrual cycle reaching ug/ml levels by the end of the cycle. These physiological concentrations are in the same range as the concentrations at which the immunomodulatory activity of PP14 was observed in vitro. The levels of PP14 measured in uterine flushings were lower in infertile women than in fertile women, indicating that a deficiency in PP14 may be associated with infertility. The levels measured in plasma samples from these two groups of women did not pick up this difference. These results suggests that the measurement of proteins such as PP14 in uterine flushings instead of plasma samples may be a more sensitive indicator of local uterine function. In women suffering from recurrent miscarriage a significant lack of secretion of PP14 was observed around the time of implantation. This may be conected with the failure of implantation in these patients. A correlation was observed between the PP14 levels measured in uterine flushings from recurrent miscarriage patients and the level of endometrial development.

Le placenta humain est richement irrigue et ses fonctions dependent essentiellement de sa vascularisation. Neanmoins, la physiopathologie des cellules vasculaires et perivasculaires de la circulation ftale intraplacentaire a ete peu etudiee. Dans cette etude, nous avons examine la repartition des proteines contractiles et la proportion de leurs differentes isoformes dans les vaisseaux ombilico-placentaires issus de grossesses normales. Nous avons ensuite realise l'isolement et la culture des cellules perivasculaires (cellules endotheliales et pericytes) provenant de microvaisseaux ftaux intraplacentaires. Des differences majeures ont ete observees dans la composition en isoformes des proteines contractiles des vaisseaux preplacentaires et intraplacentaires. Contrairement aux vaisseaux preplacentaires, qui renferment surtout des isoformes musculaires, les vaisseaux villositaires possedent principalement des isoformes non musculaires de l'actine et de la myosine. Les cellules endotheliales des microvaisseaux placentaires que nous avons isolees par digestion enzymatique puis cultivees sur matrice de gelatine, satisfont aux principaux criteres classiques de caracterisation immunocytochimique. Elles incorporent en particulier les lipoproteines acetylees et elles reagissent positivement a l'anticorps anti-von willebrand. Les pericytes ont ete isoles et cultives a partir d'explants microvasculaires, puis caracterises par un anticorps anti- -actine de type musculaire lisse. En culture avec les cellules endotheliales, ils donnent naissance a des nodules mixtes endothelio-pericytaires. Differentes applications physiopathologiques de ces methodes de culture peuvent etre envisagees: l'etude en monocouches de la permeabilite capillaire placentaire, celle de la repartition des isoformes des proteines contractiles dans le cas d'hypertension gravidique ou du retard de croissance intra-uterin et l'analyse en co-culture des phenomenes de vasculogenese

The placenta is thought to play a vital role in the transfer of essential fatty (EFA) and their long-chain polyunsaturated derivatives (LCPUFA) from mother to the fetus. There is a preferential accumulation of these fatty acids from maternal to fetal tissues. However, little was known about the manner in which these nutrients preferentially traversed the placenta. This study investigated part of this placental transport mechanism. The results from these investigations demonstrated that the preferential transport of LCPUFA to the fetal circulation may at least be partially mediated by a preferential uptake system in the placenta involving a 40 kDa, placental membrane fatty acid binding protein (p-FABP_pm). This protein was found exclusively in the maternal facing microvillous membranes. It was characterised as different from previously identified ubiquitous FABP_pm by virtue of having a different pl value, different amino acid composition, no aspartate aminotransferase activity and a higher binding affinity for LCPUFA over non-essential fatty acids. The human choriocarcinoma cell line (BeWo) expressed a protein immunoreactive to anti-p-FABP_pm anti-serum. This anti-serum inhibited the binding of LCPUFA to placental membranes and the uptake of LCPUFA by BeWo cells, to a greater degree than it inhibited the uptake and binding of non essential fatty acids. In addition to p-FABP_pm the existence of multiple types of both cytosolic (L-FABP and H-FABP) and membrane (FAT and FATP) fatty acid-binding proteins was demonstrated in placental cells. These proteins could play important roles in both the uptake of fatty acids by the placenta and in controlling the fate of fatty acids inside placental cells.

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo. In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls. In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups. In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo. In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC. In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.

[Truncated abstract] The placenta forms a barrier regulating the transfer of gases, nutrients and wastes between the mother and the developing conceptus, and also produces hormones affecting both the fetus and the mother. This barrier is formed by the differentiation of the outer layer of the blastocyst- the trophoblast- to facilitate implantation and subsequent invasion of the uterus. The trophoblast consists of an underlying proliferative pool of cytotrophoblasts, which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast that forms the barrier between the mother and fetus. Moreover, the location of the syncytiotrophoblast directly in contact with the maternal circulation suggests an endothelial role for the trophoblast regulating blood flow, thrombosis and immune cell adhesion. Disruption to the function of the human trophoblast may result in preeclampsia, a maternally manifested disorder of pregnancy characterised by hypertension and proteinurea. Blood flow to preeclamptic placentae is reduced and the cytotrophoblast pool is diminished; however the exact cause (or causes) remains elusive. Many potential causes are hypothesised, including endothelial damage, premature remodelling of maternal spiral arteries, increased oxidative stress and impaired trophoblast differentiation and apoptosis. Caspase-14 is an unusual caspase in that it is not involved in apoptosis. Furthermore, it possesses a limited, predominantly epithelial, tissue distribution. In the epidermis, caspase-14 is expressed in the apical differentiating layers. Here it cleaves profilaggrin to stabilise intracellular keratin intermediate filaments, and indirectly provides natural hydration and UV protection to the corneocytes. Thus, caspase-14 is vital to the maintenance of the barrier function of the skin. ... As differentiation-associated genes were elevated in the absence of caspase-14, this implies that caspase-14 suppresses biochemical trophoblast differentiation. The cytoskeletal keratin network was also examined following RNA Interference. The synthesis of cytokeratin 18 was significantly enhanced after caspase-14 suppression during BeWo differentiation, linking caspase-14 with keratin homeostasis. Therefore caspase-14 suppresses trophoblast differentiation, potentially through modulation of the cytoskeletal keratin filament network. The precise mechanism remains to be elucidated, however the identification of pathways regulated by caspase-14 advances our knowledge of trophoblast differentiation and potential causes of disorders of pregnancy. In summary, caspase-14 appears to be involved in the suppression of differentiation in the human trophoblast. As disorders of pregnancy such as preeclampsia often feature disturbed differentiation and a diminished cytotrophoblast pool, a greater understanding of caspase-14 biology in the human placenta could lead potential therapies for various disorders of pregnancy.

Breast Cancer Resistance Protein, BCRP, is highly expressed in many different tumor tissues conferring multi-drug resistance against many chemotherapy drugs. BCRP has also been reported to be present in normal tissues including the human placenta during pregnancy. It is believed that in the placenta, BCRP controls the levels of toxins, drugs and xenobiotics that may cross maternal circulation into fetal circulation. The expression of BCRP was examined in the human placenta and in placental membranes (amnion and chorion leave) and attached decidua. In addition, the effect of cytokines and hypoxia on BCRP expression in placental cells was examined in vitro. BCRP was found to be highly expressed in the placenta throughout pregnancy as well as in the amnion, chorion laeve and attached decidua. Our data suggest that increased cytokine expression and reduced oxygen levels may not have any effect on BCRP mRNA or protein levels in the placental syncytiotrophoblast cells. This may suggest that BCRP is stably expressed in the placenta, even under adverse conditions, and may imply that activity of this transporter protein is essential for normal placental function.

Les syncytines sont des gènes d'enveloppes rétrovirales (env) capturés qui sont essentiels pour l'établissement du placenta chez les mammifères. Il a été proposé que la diversité des syncytines capturées explique pourquoi le placenta est l'organe le plus variable chez les mammifères. Ici nous avons employé deux approches pour étudier le lien entre la capture d'env et l'émergence et la diversité des structures placentaires. D'abord, nous avons étudié la placentation des Hyaenidae, les seuls carnivores à présenter un placenta très invasif hémochorial, comme l'humain. Comme tous les carnivores, les hyènes expriment la syncytin-Car1 précédemment décrite, mais nous avons identifié une nouvelle env, capturée uniquement chez ces dernières, que nous avons nommée Hyena-Env2. Ce nouveau gène est présent au même locus chez toutes les hyènes, ayant été capturé pendant la radiation de la famille. Il est non-fusiogène mais a néanmoins été conservé pendant plus de 10 millions d'années et est exprimé à l'interface materno-fœtale du placenta, ce qui en fait un gène candidat pour expliquer le passage à la placentation hémochoriale qui a eu lieu chez les Hyaenidae. Ensuite, nous avons cherché des gènes syncytine dans le genre non-mammifère Mabuya, des lézards vivipares présentant un type rare de placenta très complexe et proche de celui des mammifères. Nous avons identifié une env qui a été capturée et conservée dans ce genre depuis sa radiation, il y a 25 millions d'années. Ce gène, que nous avons appelé syncytin-Mab1, est capable d'induire la fusion cellule-cellule et est exprimé dans une couche de cellules fusionnées à l'interface materno-fœtale du placenta, deux propriétés canoniques de syncytine. Nous avons aussi identifié le récepteur de syncytin-Mab1, MPZL1, et avons montré que leur interaction induit son activation et sa phosphorylation. L'activation de MPZL1 a été liée à la migration et à l'invasion cellulaire, indiquant que cette interaction env-récepteur pourrait jouer un rôle dans l'invasion placentaire du tissu maternel observée chez les Mabuya. Pour conclure, la caractérisation de ces deux nouvelles env indique que les gènes de type syncytine ont pu jouer un rôle à la fois dans l'émergence du placenta de Mabuya et dans la structure atypique du placenta des hyènes, supportant la notion que la capture d'env est une force évolutive majeure
Syncytins are captured retroviral envelope genes (env) that are essential for the establishment of placental structures in mammals. The syncytins present in different mammalian families are highly diverse, resulting from distinct capture events, and it has been suggested that this might play a role in making the placenta the most diverse structure in mammals. Here we used two different approaches to investigate the links between env capture and emergence and diversity of placental structures. First, we investigated placentation in Hyaenidae, the only carnivorans that present a highly invasive hemochorial placenta, as is also found in humans. Hyenas express the previously identified syncytin-Car1 gene, as do all carnivorans, but we identified a new hyena-specific captured env that we named Hyena-Env2. This new gene is present at the same locus in all hyenas, having been captured during the radiation of this family. It is non-fusiogenic but still conserved over at least 10 million years of evolution and expressed at the materno-fetal interface in the hyena placenta, making it a candidate gene for explaining the endotheliochorial to hemochorial placental transition that occurred in Hyeanidae. Second, we searched for syncytin-like genes in the non-mammalian Mabuya lizards, which are viviparous and present a rare type of highly complex placenta that is very reminiscent of mammalian placentas. We identified an env gene that was captured and conserved in this genus since its radiation 25 million years ago. This gene, that we named syncytin-Mab1, is able to mediate cell-cell fusion in vitro and is expressed in a fused cell layer at the materno-fetal interface of the placenta in vivo, characteristic features of canonical mammalian syncytin genes. We also identified the cellular gene MPZL1 as the cognate receptor of syncytin-Mab1 and showed that their interaction induces activation and phosphorylation of the former. MPZL1 activation has been linked with cell migration and invasion, indicating that this env-receptor interaction could play a role in the placental invasion of maternal tissues observed in Mabuya. In conclusion, the characterization of these two novel env genes indicates that syncytin-like env might have played a role both in the emergence of the Mabuya placenta and the atypical placental structure of hyenas, reinforcing the notion that env capture is a major driving force in evolution

Since retinol is insoluble in aqueous media, transport of vitamin A to epithelial tissues from its storage sites in the liver, is mediated by a specific plasma carrier protein called Retinol-Binding Protein (RBP). There have been controversial views expressed by different workers as to the mechanism of active transfer of retinol through the lipid bilayer of target cell membrane. The supply of Vitamin A and its derivatives from mother to foetus is of vital importance for fetal growth and development. Placenta plays an important role in the supply of these nutrients to the fetus. Since little is known about the manner of conveyance of these retinoids from the maternal circulation to the foetus, a study of the placental transport of vitamin A is important and the current work is aimed to investigate these mechanisms. In order to study the retinol transport mechanisms, as a first step, a method to isolate RBP from both adult blood, as well as from the foetal blood, was developed in this laboratory. For the first time foetal RBP has been isolated from placental blood and its characteristics compared with that of adult RBP. This study found that no significant differences exist between the two RBPs. In addition, the heterogeneity observed in certain RBP preparations was explained by reviewing the methodologies for RBP purification. In the second part of this work, transport of retinol across the placental membrane was studied using BeWo cell monolayers grown on special membrane plates as a model. These results have been discussed and the existing theories on retinol transport have been reviewed. In conclusion, this study has shown that there are no significant differences between foetal RBP and adult RBP. Although the possibility of a simple diffusion mechanism cannot be ruled out this study found that transport of retinol across BeWo cells mainly takes place through a receptor-mediated mechanism.

An essential event during early pregnancy is the invasion of trophoblasts into the maternal decidua, which is necessary for proper implantation and establishment of maternal-fetal interface and ultimately allows for proper nutrient exchange and immunological tolerance of the growing fetus. For this invasion to occur, cells originating from the trophectoderm undergo an epithelial to mesenchymal transition to become invasive extravillous trophoblasts and begin invading the uterine decidual tissue. Through the secretion of matrix metalloproteinases and through interactions with many cytokines and cell-adhesion molecules, this well-orchestrated process of trophoblast invasion results in extensive remodeling of the maternal spiral vasculature by the extravillous trophoblasts. Ultimately, the spiral arteries are transformed from high resistance, low flow vessels to low resistance, high flow vessels to allow for adequate perfusion of the placenta and developing fetus. Preeclampsia is a leading cause of maternal morbidity worldwide and is associated with the onset of hypertension and proteinuria, typically after 20 weeks of gestation. While the hypertension typically resolves following delivery of the fetus and placenta, both the mother and growing child are faced with long-term adverse health effects such as the development of cardiovascular disease and metabolic disorders. Preeclampsia is characterized by widespread maternal inflammation and endothelial dysfunction triggered by the secretion of soluble factors from the placenta into the maternal circulation. It is thought that the onset of these adverse systemic conditions is initiated by poor placental perfusion and pathologically hypoxic conditions in the placenta. In many cases of preeclampsia, there is evidence of shallow trophoblast invasion which results in incomplete spiral artery transformation, ultimately leading to poor placental perfusion. However, the exact mechanisms underlying the inadequate extravillous trophoblast invasion and remodeling are incompletely understood. LIN28 is an RNA binding protein that is highly expressed in embryonic stem cells, fetal tissues and many cancers, and was discovered as a regulator of the maturation of the Let-7 family of miRNAs. However, as an RNA binding protein, LIN28 has been shown to interact with thousands of mRNA transcripts, leading to both increased and decreased protein expression, and control of many cellular processes such as differentiation, proliferation, migration, invasion, and cellular metabolism. In vertebrates, LIN28 exists as two highly homologous paralogs, LIN28A and LIN28B, however LIN28B is slightly larger and contains a nuclear localization signal not found in LIN28A. While they both function to inhibit Let-7 maturation, there is evidence to suggest they also have independent functions. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to preeclampsia. We found that LIN28B mRNA expression is ~1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in human primary cytotrophoblasts, syncytiotrophoblasts, and decidual cells. We also found that LIN28B mRNA and protein levels are significantly reduced in human placentas from preeclamptic pregnancies compared to placentas from normal pregnancies, while LIN28A expression is unchanged. Upon investigation of human first trimester placenta sections, we found that LIN28B is more highly expressed in the invasive extravillous trophoblasts and syncytial sprouts compared to villous trophoblasts. To support this with in vitro evidence, we found that overexpression of LIN28B in human HTR8/SVneo cells resulted in increased proliferation, migration and invasion, while knockdown of LIN28B in JEG3 cells resulted in decreased proliferation. Furthermore, knockdown of LIN28B in JEG3 cells led to decreased expression of SYN-1, ELABELA, and the chromosome 19 miRNA cluster, with accompanying increases in the pro-inflammatory cytokine TNFα and ITGβ4, an integrin enriched on non-invasive trophoblasts. Moreover, culture of JEG3 and BEWO cells in hypoxia resulted in significantly decreased levels of LIN28B mRNA and protein expression, as well as syncytin-1 and ELABELA mRNA levels, while TNFα was increased. These results provide the first evidence that LIN28B is the predominant paralog expressed in human placenta and decreased LIN28B may play a crucial role in PE pathogenesis by reducing trophoblast invasion, syncytialization and by promoting inflammation.

La concentration élevée de l'hPL dans la plasma de la femme enceinte, en fin de grossesse, sa sécrétion placentaire estimée à plus de 1 gramme par 24 heures, son pourcentage appréciabvle de l'ensemble des protgéines placentaires, sa structure composée d'une simple chaîne d'acides aminés, sans participation de résidus glycosylés ou phosphorylés, les inconnues actuelles concernant les mécanismes de régulation de sa production, sont les facteurs qui nous ont déterminé à réaliser sa synthèse in vitro

Astrocitomas constituem o tipo mais comum de tumor cerebral neuroepitelial primário apresentando grande heteogeneidade. De acordo com a Organização Mundial de Saúde, os astrocitomas podem ser histologicamente divididos em graus I- IV. Astrocitomas pilocíticos (grau I) são tumores circunscritos, de crescimento lento e bom prognóstico. Astrocitomas difusos (grau II) apresentam hipercelularidade, crescimento relativamente lento e propensão para invadir o tecido cerebral normaladjacente. Astrocitomas anaplásicos (grau III) apresentam aumento da celularidade, atipia nuclear e figuras mitóticas. Glioblastomas (GBMs - grau IV) representam o mais frequente e maligno tumor cerebral humano com crescimento extremamente agressivo, anaplasia, células altamente proliferativas, com frequente neoangiogênese e necrose. O comportamento altamente invasivo dos GBMs, caracterizado pela infiltração difusa para o parênquima cerebral normal adjacente, inviabiliza a remoção cirúrgica total do tumor. Além disso, as células dos GBMs são relativamente resistentes às terapias disponíveis. Analogamente a outros tipos de câncer, os GBMs demonstram comportamentos semelhantes às de células trofoblásticas, sugerindo vias de sinalização compartilhadas no controle dos processos tumorigênicos e de implantação da placenta. Em ambos os casos, o estabelecimento de um fenótipo invasivo compreende processos celulares que incluem aumento da proliferação, expressão ou repressão de moléculas de adesão celular específicas, produção de enzimas que digerem a matriz extracelular, expressão de produtos de proto-oncogenes, ativação da telomerase, evasão ou edição da resposta imune do hospedeiro e angiogênese. Com base nas características comuns entre células tumorais e trofoblastos, o presente trabalho teve como objetivo a busca in silico de genes expressos em placenta e tecidos tumorais e que podem contribuir para o estabelecimento e manutenção do fenótipo maligno, utilizando os bancos de dados de MPSS e SAGE. Dentre os 12 genes avaliados, CD99 foi o que apresentou o maior valor de expressão média nas amostras de GBM em comparação a amostras de tecido cerebral não neoplásico. Em uma casuística ampliada de astrocitomas , observou-se uma maior expressão relativa de CD99 em todos os graus de malignidade, sendo que os GBMs apresentaram os valores mais elevados. Esses achados foram confirmados em nível proteico por western blot e imunoistoquímica. Além disso, foi realizada a análise de imunolocalização de CD99 em amostras de tumores astrocíticos, com localização restrita a membrana ou citoplasma, em contraste ao tecido cerebral não neoplásico ou astrocitomas pilocíticos não infiltrantes, que não apresentaram marcação nestas estruturas. Ao compararmos três linhagens celulares derivadas de GBM, CD99 apresentou maior expressão na membrana e maior capacidade migratória nas linhagens A172 e U87MG, enquanto que a linhagem T98G apresentou menor expressão da proteína e ausência de capacidade migratória. O silenciamento da expressão de CD99 por siRNA diminuiu significativamente a migração das linhagens celulares A172 e U87MG. Além disto, anticorpo anti-CD99 apresentou maior marcação por em lamelipódios das células U87MG, possivelmente por reorganização do citoesqueleto de actina. Os resultados integrados de expressão gênica e proteica sugerem que a expressão de CD99 em astrocitomas de diferentes graus de malignidade pode contribuir para a capacidade infiltrativa destes tumores, ressaltando a importância desta proteína como um potencial alvo para a redução da capacidade infiltrativa dos astrocitomas nos processsos de migração e invasão
Astrocytomas are the most common type of primary neuroepithelial brain tumour and show great heterogeneity. According to World Health Organization criteria, astrocytomas can be histologically separated into grades I through IV. Pilocytic astrocytomas (grade I) are circumscribed, slow growing tumours with a good prognosis and mainly occur in children or young adults. Low-grade astrocytomas (grade II) show hypercellularity, relatively slow growth, and a propensity to invade surrounding normal brain tissue. Anaplastic astrocytomas (grade III) have increased cellularity, nuclear atypia, and mitotic figures. Glioblastomas (GBMs - grade IV), are the most common malignant and aggressive of all brain malignancies, exhibiting anaplastic, highly proliferative cells, with frequent neoangiogenesis and necrosis. GBM cells can escape complete resectability and are relatively resistant to the available therapies (radiation and chemotherapy). Similar to other cancer types, GBMs demonstrates behaviours that are analogous to trophoblastic cells, suggesting shared pathways to control tumourigenic processes and placental implantation. In both cases, the establishment of an invasive phenotype comprises cellular processes that include increased proliferation, the expression or repression of specific cell adhesion molecules, the production of enzymes that digest the extracellular matrix, the expression of proto-oncogene products, telomerase activation, evasion or edition of the host immune response, and angiogenesis. Based on the shared characteristics of tumour cells and trophoblasts, we searched in silico for genes that are in both placenta and tumour tissues using MPSS and SAGE databases and that could contribute to the establishment and maintenance of a malignant phenotype. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both A172 and U87MG cell lines. Additionally, a higher anti-CD99 antibody staining was observed in lamellipodia of U87MG, probably because of actin citoskeletal reorganization. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

La placentation humaine est de type hémomonochoriale, le sang maternel est directement en contact avec le syncytiotrophoblaste. Les flux sanguins maternels, dans la chambre intervilleuse, exercent des forces mécaniques de cisaillement (shear stress) sur la surface microvillositaire du syncytiotrophoblaste. Les effets physiologiques du shear stress exercé par les flux sanguins sur l’endothélium vasculaire artériel et veineux ont fait l’objet de nombreux travaux scientifiques. En revanche, les effets biologiques du shear stress sur le syncytiotrophoblaste humain n’ont jamais été explorés. L’objectif de ce travail était premièrement d’évaluer les valeurs du shear stress exercé in vivo sur le syncytiotrophoblaste humain au cours des grossesses normales, puis de mettre au point un modèle de culture primaire dynamique afin de reproduire les conditions physiologique et d’étudier in vitro la réponse biologique du syncytiotrophoblaste au shear stress. En dépit d’un débit sanguin maternel intraplacentaire important, estimé entre 400 et 600 mL.min-1, le shear stress moyen exercée par le syncytiotrophoblaste est estimée entre 0.5±0.2 et 2.3±1.1 dyn.cm-2. Nos résultats montrent cependant que l’intensité du shear stress est très hétérogène tant à l’échelle de la chambre intervilleuse que de la villosité terminale. Nous avons développé un modèle de culture cellulaire dynamique en condition de flux adapté au syncytiotrophoblaste humain. Ce modèle permet d’appliquer un shear stress égal et constant sur toutes les cellules cultivées et reproductible à chaque culture primaire. Aux gammes de shear stress étudiées (1 dyn.cm-2), nous n’avons pas mis en évidence de diminution de la viabilité cellulaire ni de déclenchement des processus précoces d’apoptose en conditions dynamiques comparativement aux conditions statiques. Deux types de chambre de perfusion permettent d’étudier des réponses cellulaires au shear stress à court et long terme selon des temps d’exposition allant de 5 minutes à 24 heures. Ce modèle expérimental a permis de montrer que le syncytiotrophoblaste humain en culture primaire est mécanosensible. La réponse cellulaire à des niveaux de shear stress de 1 dyn.cm-2 est multiple selon les temps d’exposition et le niveau d’intégration étudié. Après 45 minutes de shear stress les taux d’AMP cyclique intracellulaires sont augmentés ce qui a pour effet d’activer la voie de signalisation intracellulaire PKA-CREB. Cette augmentation d’AMP cyclique est secondaire à la synthèse et la libération de prostaglandine E2 qui, par une boucle de régulation autocrine stimule l’adenylate cyclase. L’augmentation de la synthèse/libération de PGE2 est dépendante de l’augmentation rapide du calcium intracellulaire sous shear stress. L’exposition au shear stress de 24 heures stimule l’expression et la sécrétion du PlGF, un facteur de croissance indispensable à l’angiogenèse placentaire et pour l’adaptation maternelle à la grossesse sur le plan vasculaire. Nos travaux montrent que l’augmentation de l’AMPc intracellulaire et l’activation de la PKA contribuent à la phosphorylation de CREB, facteur de transcription régulant l’expression du PlGF
Human placentation is hemomonochorial, maternal blood circulates in direct contact with the syncytiotrophoblast. In the intervillous space, the maternal blood exerts frictional mechanical forces (shear stress) on the microvillous surface of the syncytiotrophoblast. Flowing blood constantly exerts a shear stress, on the endothelial cells lining blood vessel walls, and the endothelial cells respond to shear stress by changing their morphology, function, and gene expression. The effects of shear stress on the human syncytiotrophoblast and its biological functions have never been studied. The objectives of this study were (1) to determine in silico the physiological values of shear stress exerted on human syncytiotrophoblast during normal pregnancies, (2) to develop a model reproducing in vitro the shear stress on human syncytiotrophoblast and (3) to study in vitro the biological response of human syncytiotrophoblast to shear stress. The 2D numerical simulations showed that the shear stress applied to the syncytiotrophoblast is highly heterogeneous in the intervillous space. In spite of high intraplacental maternal blood flow rates (400-600mL.min-1), the estimated average values of shear stress are relatively low (0.5±0.2 to 2.3±1.1 dyn.cm-2). To study the shear stress-induced cellular responses during exposure times ranging from 5 minutes to 24 hours we have developed two dynamic cell culture models adapted to the human syncytiotrophoblast. We found no evidence of decreased cell viability or early processes of apoptosis in dynamic conditions (1 dyn.cm-2, 24h) compared to static conditions. Shear stress (1 dyn.cm-2) triggers intracellular calcium flux, which increases the synthesis and release of PGE2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 inhibitors, suggesting that the increase in PGE2 production could activate the cAMP/PKA pathway in an autocrine/paracrine fashion. FSS activates the cAMP/PKA pathway leading to upregulation of PlGF in human STB. Shear stress-induced phosphorylation of CREB and upregulation of PlGF were prevented by inhibition of PKA with H89 (3 μM). The syncytiotrophoblast of the human placenta is a mechanosenstive tissue

Introduction: Abnormal fetal growth remains a major problem in pregnancies complicated by diabetes and is associated with increased maternal and offspring mortality and morbidity. Insulin-like growth factors (IGFs) stimulate fetal growth while their effects are inhibited by binding proteins (IGFBPs). IGFBP-1 is a significant IGFBP in maternal and fetal circulation and the only binding protein acutely affected by glucoregulatory hormones; as such, IGFBP-1 is particularly important in pregnancy with diabetes. In plasma of healthy human adults, the fully phosphorylated (pIGFBP-1) isoform is predominant. During pregnancy the phosphorylation status of IGFBP-1 changes; in addition to pIGFBP-1, non-phosphorylated (np) IGFBP-1 and 3 lesser phosphorylated (lp) IGFBP-1 variants with lower affinity for IGF-I are detected in the maternal circulation. The change in the phosphorylation status of IGFBP-1 in pregnancy may provide a physiological mechanism for the increased IGF-I bioavailability at the maternal/fetal interface required for placental and fetal growth.Hypothesis: IGFBP-1 de-phosphorylation occurs at the maternal/fetal interface and this process is catalyzed by placental alkaline phosphatase (PLAP). Fetal overgrowth (macrosomia) in pregnancy with diabetes may be a consequence of elevated IGF-I action at the placenta secondary to increased PLAP activity.Methods and Results: In vitro: Explants of human term placentas from normal pregnancies (n=5), or their conditioned media (CM), were incubated with pIGFBP-1 in the presence or absence of an anti-PLAP function blocking antibody. Addition of pIGFBP-1 to explants resulted in its binding to the tissue and de-phosphorylation, with npIGFBP-1 isoforms appearing in the medium. pIGFBP-1 was not de-phosphorylated when cultures were carried out in the presence of anti-PLAP antibody. In solution phase assays, PLAP failed to de-phosphorylate pIGFBP-1. Thus, placenta de-phosphorylates IGFBP-1 as a result of PLAP activity, and this requires its binding to the tissue.To investigate factors which may affect the activity of PLAP, placental explants (n=3 for each series of experiments) were incubated with pIGFBP-1 in the presence of insulin, IGF-I/-II or under hyperglycemic or hypoxic conditions. Following incubation, the phosphorylation status of IGFBP-1 present in placental-CM was assessed by native electrophoresis and western blot. PLAP-mediated IGFBP-1 de-phosphorylation was not affected in vitro by hyperglycemia, hypoxia, insulin or IGF-I/-II. In vivo: 30 patients with any type of diabetes in pregnancy and 20 controls were recruited. Maternal/cord blood was collected at term and analysed for IGF-I/-II, total IGFBP-1 and the phosphorylation status of IGFBP-1. Placentas were analysed for PLAP expression and activity. The maternal blood levels of both IGFs and total IGFBP-1 were similar in the diabetes and control groups, while cord IGF-II was elevated in diabetes. Unexpectedly, the p/npIGFBP-1 ratio in maternal serum was elevated in patients with diabetes, which may be a result of decreased IGFBP-1 de-phosphorylation. In controls, maternal p/npIGFBP-1 ratio correlated with infant weight, whilst this correlation was not demonstrated in women with diabetes. Placental PLAP expression/ex-vivo activity and total IGFBP-1 levels in maternal serum were unaltered in diabetes and did not relate to fetal growth in either diabetes or control groups. Conclusions: The hypothesis that activated PLAP and therefore enhanced IGFBP-1 de-phosphorylation may increase the effect of IGF-I on placental cell turnover and accelerate fetal growth in diabetes was not supported by the results of this study. Further work is required to reveal mechanisms by which the maternal/placental/fetal IGF-IGFBP-PLAP pathways modulate fetal growth in normal and compromised pregnancy.

Multidrug resistance phosphoglycoprotein (MDR1/P-gp) and breast cancer resistance protein (BCRP) were first isolated in chemoresistant cancer cells and have since been found in a variety of normal tissue, including the placenta. The potential function of MDR1/P-gp and BCRP in the human placenta is to protect the fetus from maternally circulating endogenous steroids and hormones, therapeutic drugs and toxins. The objective of this study was to examine the role of maternal steroids in the regulation of MDR1/P-gp and BCRP in the human placenta. Trophoblast cells were isolated from term placenta tissues and immunohistochemistry, western blot analysis and transport studies were used to determine the effect of maternal steroids on MDR1/P-gp and BCRP regulation. Maternal steroids, present at high concentrations in maternal serum, did not have an effect on BCRP in human syncytiotrophoblast. Estrogen and progesterone did not alter MDR1/P-gp levels in human syncytiotrophoblast, but cortisol significantly decreased MDR1/P-gp levels.

The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term.

Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue.

The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta.

No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.

Les endothelines (et-1, et-2, et-3) exercent des effets vasoactifs et mitogenes via leurs recepteurs et#a et et#b. Un role important de ces peptides dans le developpement de l'unite foeto-placentaire est envisage. Au cours de cette etude en fin de grossesse, nous avons, dans un premier temps, montre que les arnm de la prepro-endotheline-1 (prepro-et-1) et de la prepro-et-3 sont exprimes dans le syncytiotrophoblaste en culture. Dans la musculature lisse des vaisseaux villositaires, cibles des et, nous avons ensuite identifie des elements des voies de signalisation de ces peptides: les proteine gs alpha, gi alpha, gq-g11 alpha et go alpha. Dans ces vaisseaux, et-1, qui induit la contraction, provoque de facon dose-dependante la liberation a partir de la membrane des proteines gs alpha et des proteines gq-g11 alpha tandis que le vip, un agent vasorelaxant des vaisseaux villositaires, stimule la dissociation des proteines gs alpha. Afin de disposer d'un modele d'etude des interactions des et avec la media des vaisseaux villositaires, nous avons developpe la culture de cellules musculaires lisses (pvsmc). Leur phenotype a ete caracterise par leur morphologie et par l'expression d'isoformes de proteines contractiles du muscle lisse. Les pvsmc expriment exclusivement les arnm de la prepro-et-1 tandis que dans la media des vaisseaux villositaires, les transcrits des prepro-et-1 et prepro-et-3 sont identifies. Seuls les recepteurs et#a sont presents dans les pvsmc alors que dans la media des vaisseaux villositaires, nous decrivons l'expression des recepteurs et#a et et#b. Dans ce tissu, en plus du transcrit et#a-r complet, d'autres arnm et et#a-r, obtenus par epissage differentiel, sont observes. Ce processus, qui est maintenu dans les pvsmc, pourrait constituer un mecanisme de regulation de la quantite de recepteurs et#a fonctionnels. L'ensemble de ces resultats confirme que les et sont impliquees dans la regulation autocrine/paracrine du tonus vasculaire du placenta humain et de son developpement au cours de la grossesse

O objetivo do presente estudo foi avaliar a diferença de resultados maternos e neonatais em gestações complicadas por trombofilias hereditárias em pacientes com e sem tromboembolismo venoso. Apesar do aumento de evidências, na literatura, sobre a associação de trombofilias congênitas e resultados obstétricos adversos, há ainda dúvida se pacientes trombofílicas com tromboembolismo venoso apresentam resultados maternos e neonatais piores que as pacientes trombofílicas sem tromboembolismo venoso. O estudo analisou 66 gestantes com trombofilias hereditárias, de forma retrospectiva observacional e comparativa, das quais 33 apresentavam tromboembolismo venoso e 36 o não apresentavam. Os principais desfechos relacionados a resultados maternos e neonatais adversos foram: pré-eclâmpsia grave, descolamento prematuro de placenta, restrição de crescimento fetal, natimortalidade, prematuridade e complicações hemorrágicas maternas. As trombofilias congênitas incluídas no estudo foram o fator V de Leiden (FVL), mutação da protrombina G20210A, mutação C677T do gene da 5,10-metilenotetrahidrofolato redutase (MTHFR), deficiência de proteína S, deficiência de proteína C e deficiência de antitrombina. Ambos os grupos apresentaram características populacionais similares. A ocorrência de complicações maternas e fetais/neonatais foi similar nos dois grupos: pré-eclâmpsia grave (P=0,097), descolamento prematuro de placenta (P=0,478), restrição de crescimento fetal (P=0,868), natimortalidade (P=0,359), prematuridade (P=0,441) e complicações hemorrágicas maternas (P=0,478). Este estudo concluiu que a presença de tromboembolismo venoso em gestantes com trombofilia hereditária apresenta resultados maternos e neonatais semelhantes àquelas com trombofilias hereditárias sem tromboembolismo venoso.
The aim of this study was to evaluate differences in maternal and neonatal outcomes in pregnancies complicated by inherited thrombophilias between patients with and without venous thromboembolism. Despite increasing evidence in the literature indicating an association between inherited thrombophilias and adverse obstetric outcomes, doubts remain whether thrombophilic patients with venous thromboembolism present poorer maternal and neonatal outcomes than thrombophilic patients without venous thromboembolism. In this retrospective, observational and comparative study, 66 pregnant women with inherited thrombophilias, including 33 with venous thromboembolism and 36 without thromboembolism, were investigated. The main end-points analyzed were severe pre-eclampsia, placental abruption, fetal growth restriction, stillbirth, preterm delivery, and maternal hemorrhagic complications. The congenital thrombophilias included in this study were factor V Leiden (FVL), prothrombin G20210A mutation, C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, protein S deficiency, protein C deficiency, and antithrombin deficiency. The two groups were similar in terms of population characteristics. The frequency of maternal and fetal/neonatal complications was similar in the two groups: severe pre-eclampsia (P=0.097), placental abruption (P=0.478), fetal growth restriction (P=0.868), stillbirth (P=0.359), preterm delivery (P=0.441), and maternal hemorrhagic complications (P=0.478). This study concluded that venous thromboembolism in thrombophilic patients does not worsen maternal or neonatal outcomes when compared to thrombophilic patients without venous thromboembolism.

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

Yuen, Ka Chun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 160-185).
Abstracts in English and Chinese.
ABSTRACT --- p.I
摘要 --- p.IV
ACKNOWLEDGEMENTS --- p.VI
LIST OF ABBREVIATIONS --- p.VII
TABLE OF CONTENTS --- p.VIII
LIST OF TABLES --- p.XII
LIST OF FIGURES --- p.XIII
Chapter SECTION I: --- BACKGROUND --- p.1
Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2
Chapter 1.1 --- Overview --- p.2
Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3
Chapter 1.3 --- Gene expression --- p.4
Chapter 1.3.1 --- Angiogenic factors --- p.5
Chapter 1.3.2 --- Growth factors --- p.5
Chapter 1.3.3 --- Proto-oncogenes --- p.6
Chapter 1.3.4 --- Tumour suppressor genes --- p.8
Chapter CHAPTER 2: --- Epigenetics --- p.10
Chapter 2.1 --- Overview --- p.10
Chapter 2.2 --- DNA methylation in mammals --- p.11
Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12
Chapter 2.4 --- Role of DNA methylation --- p.13
Chapter 2.5 --- Aberrant DNA methylation --- p.16
Chapter 2.6 --- DNA methylation in normal cells --- p.17
Chapter 2.6.1 --- X-chromosome inactivation --- p.17
Chapter 2.6.2 --- Genomic imprinting --- p.18
Chapter 2.6.3 --- Cell-type-specific methylation --- p.19
Chapter 2.6.4 --- Placental-specific methylation --- p.20
Chapter 2.7 --- Aim of Thesis --- p.21
Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23
Chapter CHAPTER 3: --- Materials and methods --- p.24
Chapter 3.1 --- Preparation of samples --- p.24
Chapter 3.1.1 --- Collection of placental tissues --- p.24
Chapter 3.1.2 --- Preparation of blood cells --- p.25
Chapter 3.1.3 --- Preparation of cell lines --- p.25
Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26
Chapter 3.2 --- Nucleic acid extraction --- p.26
Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26
Chapter 3.2.2 --- DNA extraction from blood cells --- p.29
Chapter 3.2.3 --- RNA extraction from cell lines --- p.30
Chapter 3.3 --- Methylation analysis --- p.31
Chapter 3.3.1 --- Principles of bisulfite modification --- p.31
Chapter 3.3.2 --- Bisulfite Conversion --- p.32
Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction
Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33
Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34
Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35
Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39
Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39
Chapter 3.4.2 --- Real-time quantitative MSP --- p.42
Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42
Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43
Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43
Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46
Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48
Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49
Chapter 4.1 --- Introduction --- p.49
Chapter 4.2 --- Materials and methods --- p.50
Chapter 4.2.1 --- Sample collection --- p.50
Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50
Chapter 4.2.3 --- Experimental Design --- p.51
Chapter 4.3 --- Results --- p.63
Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63
Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69
Chapter 4.4 --- Discussion --- p.77
Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80
Chapter 5.1 --- Introduction --- p.80
Chapter 5.2 --- Materials and methods --- p.81
Chapter 5.2.1 --- Sample collection --- p.81
Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81
Chapter 5.2.3 --- Experimental design --- p.81
Chapter 5.3 --- Results --- p.86
Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86
Chapter 5.4 --- Discussion --- p.90
Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94
Chapter CHAPTER 6: --- Imprinting checking --- p.95
Chapter 6.1 --- Introduction --- p.95
Chapter 6.2 --- Materials and methods --- p.96
Chapter 6.2.1 --- Sample collection --- p.96
Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97
Chapter 6.2.3 --- Experimental design --- p.97
Chapter 6.3 --- Results --- p.100
Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100
Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101
Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107
Chapter 7.1 --- Introduction --- p.107
Chapter 7.2 --- Materials and methods --- p.108
Chapter 7.2.1 --- Sample preparation and processing --- p.108
Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108
Chapter 7.2.3 --- Experimental design --- p.108
Chapter 7.3 --- Results --- p.111
Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111
Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114
Chapter 7.4 --- Discussion --- p.115
Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118
Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119
Chapter 8.1 --- Introduction --- p.119
Chapter 8.2 --- Materials and methods --- p.120
Chapter 8.2.1 --- Sample collection --- p.120
Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120
Chapter 8.2.3 --- Experimental design --- p.120
Chapter 8.3 --- Results --- p.124
Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124
Chapter 8.4 --- Discussion --- p.136
Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138
Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139
Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139
Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140
Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142
Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143
Chapter 9.5 --- Future perspectives --- p.145
APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147
APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156
APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158
REFERENCES --- p.160

碩士
國立臺灣大學
生化科學研究所
96
Abstract Fusion of cytotrophoblast into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. Previous studies of placental development have shown that the envelope protein of a human endogenous retrovirus W (HERV-W), syncytin 1, may mediate placental cell fusion. Recently, the envelope protein of HERV-FRD, syncytin 2, has been identified and shown to be highly expressed in placenta. This implies that syncytin 2 may be involved in placental cell fusion. Our previous studies have demonstrated that the cytoplasmic domain (CTM) of syncytin 1 inhibits its fusogenic activity. In this study, we first tested whether the CTM of syncytin 2 regulates its fusogenic activity. We found that the CTM of syncytin 2 is required for its fusogenic activity. We further studied the biosynthesis of syncytins and demonstrated that Furin is involved in the proteolytic cleavage of syncytin 1 and syncytin 2 by using furin inhibitor and in the Furin-defective human colon carcinoma LoVo cell line. Finally, we studied the role of the disulfide-forming CXXC and CX6CC motifs in syncytins and found that the disulfide linkage between SU and TM subunits is essential for the fusogenic activity of syncytins because mutation in the CXXC motif abolishes the fusogenic activity of syncytins and causes a dominant-negative effect. Overall, this study provides a better understanding of the biology of syncytin 1 and syncytin 2.