Von Hippel-Lindau (VHL) syndrome is an autosomal dominant predisposition to cancer in neurological tissues, the kidneys, adrenal glands, pancreas, and liver, including neurological hemangioblastoma (HB), pheochromocytoma (PCC), pancreatic neuroendocrine tumors (PNET), pancreatic and renal cysts, and clear cell renal cell carcinoma (ccRCC). The disease process follows Knudson's two-hit model, requiring spontaneous loss or mutation of a normal VHL tumor suppressor allele to induce expression of the disease. VHL syndrome principally involves dysregulation of oxygen sensing pathways including the Hypoxia Inducible Factor (HIF)-Vascular Endothelial Growth Factor-A (VEGF-A) and HIF-Erythropoietin (EPO) pathways. RNA sequencing (RNA-Seq) data from our previously published experiments revealed a potentially novel VEGF-A splice variant with excision of the VEGF Receptor-1 (VEGFR-1)/Flt-1 binding domain, rendering this isoform resistant to native down-regulation. Additionally, phenotypic changes were observed in adult VHL mutant mice, specifically very red appearing extremities with prominently visible vasculature. In order to determine the etiology of this phenotype, we observed red blood cell count, Epo gene expression levels, and arterialization of the blood vessels in these experimental mice as compared to littermate controls. Current research into the VEGF-A isoform is ongoing in the lab, and preliminary evidence for the etiology of the apparent chronic erythema phenotype is inconclusive due to lack of experimental replicates due to COVID-19 quarantine orders. / Master of Science / Von Hippel-Lindau (VHL) syndrome is characterized by cancer development primarily in the brain and spinal cord, kidneys, adrenal glands, pancreas, and liver. VHL syndrome involves mutations which render the VHL gene dysfunctional. Since the VHL gene's normal role is one of preventing cancer development, sensing oxygen levels, and impacting blood vessel development, it follows that the loss of this gene results in tumor development with a rich blood vessel network. One of the downstream effectors of this process is a signaling molecule called Vascular Endothelial Growth Factor-A (VEGF-A). Our lab found a unique variant of VEGF-A, which may be overactive in the body in the setting of VHL disease. Additionally, we noted that our VHL mutant mice turned very red, and we sought to identify the biological cause of this phenomenon. In order to determine the cause of this redness, we studied red blood cell counts and their regulatory gene (Erythropoietin, EPO), as well as potential blood vessel abnormalities using high-power microscopy.

Abstract Advances over the past few decades in ante- and neonatal care have led to the survival of a growing number of premature infants of extremely low gestational age. However, the occurrence of serious diseases, particularly those affecting the most immature infants, remains high. Bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, is one such disease. Our current understanding of the molecular pathogenesis of BPD is incomplete; consequently, there are few preventive and therapeutic options for BPD. Moreover, it is challenging to predict the risk of BPD. Previous studies of BPD in twins revealed that the heritability of BPD is quite high. However, the individual genes that predispose premature infants to BPD are largely unknown. The aim of this study was to identify and study genes associated with BPD in order to investigate its pathogenesis. An additional aim was to add to knowledge of the risk of BPD in newborn premature infants, with an emphasis on twins. A candidate gene study found no consistent association between common polymorphisms of vascular endothelial growth factor receptor 2 and BPD. A second candidate gene study noted an association between the gene encoding Kit ligand and BPD. A genome-wide association study found a suggestive association between a locus close to the gene encoding C-reactive protein (CRP) and BPD, and in subsequent analyses, plasma levels of CRP during the first week of life predicted BPD. Finally, a nationwide register study found that the risk of BPD was lower in twins than in singletons. The results of this study add to what is known of the genetics and pathogenesis of BPD. They also provide new data on the risk of BPD, which may be used to improve early identification of infants for whom the risk of developing BPD is high. / Tiivistelmä Ennenaikaisen syntymän ja keskoslasten hoidon kehittymisen myötä yhä useammat huomattavan epäkypsinä syntyneet lapset jäävät henkiin. Samalla erityisesti juuri näitä lapsia uhkaavien sairauksien esiintyvyys on pysynyt korkeana. Bronkopulmonaalinen dysplasia (BPD, keskosen krooninen keuhkosairaus) on yksi näistä sairauksista. BPD:n molekyylitasoinen tautimekanismi on vielä osin tuntematon, eikä BPD:tä tehokkaasti estävää tai siitä parantavaa hoitoa ole. Myös BPD riskin arvioiminen vastasyntyneen keskoslapsen kohdalla on vaikeaa. BPD on huomattavan perinnöllinen tauti. BPD:lle altistavista geeneistä on kuitenkin vasta vähän tietoa. Tämän tutkimuksen tavoitteena oli lisätä tietoa BPD:n tautimekanismista tutkimalla BPD:lle altistavia geenejä. Lisäksi tutkimuksessa tarkasteltiin BPD:n esiintyvyyttä ja syntymää edeltäviä riskitekijöitä erityisesti kaksosten osalta. Ehdokasgeenitutkimuksessa verisuonten endoteelikasvutekijää koodaava geeni ei assosioitunut toistuvasti BPD:hen. Kit ligandia koodaava geeni sen sijaan assosioitui. Koko genomin assosiaatiotutkimuksessa C-reaktiivista proteiinia (CRP) koodaavan geenin lähistöltä löydettiin BPD:hen mahdollisesti assosioituva alue. Lisäksi ensimmäisen viikon CRP-arvojen osoitettiin ennakoivan myöhemmin kehittyvää BPD:tä. BPD-riskin todettiin olevan matalampi kaksi- kuin yksisikiöisistä raskauksista syntyneillä lapsilla. Tutkimuksen tulokset lisäävät tietoa BPD:n perinnöllisyydestä ja sitä kautta BPD:n tautimekanismista. Tutkimus toi myös uutta tietoa BPD:n riskitekijöistä parantaen vastasyntyneen keskoslapsen BPD-riskin arviota.

<p>Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state.</p><p>We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy. </p>

<p>UVOD: Hipertenzivna oboljenja u trudnoći predstavljaju heterogenu grupu bolesti koja se javljaju kod 3-8% trudnica op&scaron;te 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 jo&scaron; uvek nedovoljno razja&scaron;njena 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, &scaron;to se nepovoljno održava i na majku i na plod. U cilju razja&scaron;njenja patofiziolo&scaron;kih 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 &scaron;to 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, free&szlig;hCG, 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 &ndash; 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 fiziolo&scaron;kim ishodom trudnoće u terminu, bez maternalnih i fetalnih komplikacija. Prilikom regrutovanja trudnica (između 11+0 i 13+6 nedelja gestacije) za uče&scaron;će u istraživanju, uzeti su anamnestički podaci o faktorima rizika za pojavu hipertenzivnih oboljenja u trudnoći, i u okviru kliničkog i aku&scaron;erskog 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 aku&scaron;erskog 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&amp;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&lt;0,05. REZULTATI: Vrednosti serumskih koncentracija sFlt-1 se statistički značajno razlikuju u sve tri grupe ispitanica i značajno su vi&scaron;e u grupama sa hipertenzivnim oboljenjima u odnosu na zdravu grupu ispitanica, p&lt;0,001. Serumske koncentracije VEGF-A su značajno niže u grupi trudnica sa preeklampsijom u odnosu na zdrave trudnice kontrolne grupe (p&lt;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&lt;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&gt;0,05. Nivoi free &szlig;hCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno vi&scaron;e 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&gt;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&lt;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&lt;0,001. Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na indeks telesne mase majke tako da su vi&scaron;e 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&lt;0,001. Serumske koncentracije sFlt-1 u prvom trimestru trudnoće su značajno povezane sa parametrima inflamacije (hsCRP), vrednostima dijastolnog krvnog pritiska i nivoima free &szlig;hCG. 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 vi&scaron;e 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 &szlig;hCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno vi&scaron;e 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 vi&scaron;e 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.</p> / <p>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, free&szlig;hCG, 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&#39;s voluntary participation in the study. Serum levels of sFlt1, VEGF-A and PlGF were determined by quantitative ELISA (R &amp; 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 &lt;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 &lt;0.001. Serum levels of VEGF-A are significantly lower in the preeclampsia group compared to healthy control group (p &lt;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 &lt;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&gt; 0.05. Levels of free &szlig;hCG 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&gt; 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 &lt;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 &lt;0.001. Serum concentrations of sFlt-1 and PlGF are significantly different in relation to the mother&#39;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 &lt;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 &szlig;hCG. 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 &szlig;hCG 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.</p>

INTRODUÇÃO: O pênfigo foliáceo (PF) caracteriza-se pela síntese de autoanticorpos contra a desmogleína 1 (Dsg1) com acantólise na epiderme superior. As lesões na face e tronco podem evoluir para eritrodermia (PFE), cuja patogênese é pouco conhecida. Estudos prévios sugerem a participação do fator de crescimento endotelial vascular (VEGF) e da endoglina (Eng). OBJETIVOS: Avaliar dados demográficos, expressão tecidual e níveis séricos do VEGF e Eng, bem como o perfil dos imunocomplexos no PFE. PACIENTES, MATERIAIS E MÉTODOS: Foram selecionados pacientes de PFE (n=31) e indivíduos controles com pênfigo vulgar (PV; n=10), psoríase (PSO; n=10) e saudáveis (CS; n=14) com amostras de soro e pele armazenadas. Os níveis séricos do VEGF, receptor solúvel do VEGF (sVEGFR-1), anti-Dsg1 e -Dsg3 foram avaliados por meio da técnica de ELISA, e a presença do VEGF, Eng, e imunocomplexos in situ foi determinada por imuno-histoquímica (IH) em plataforma automatizada (n=19). Após digitalização das lâminas, a reatividade aos anticorpos supracitados foi classificada manualmente em: 0 (ausente), 1 (discreta-moderada) e 2 (intensa), e analisada por software. RESULTADOS: PFE ocorreu no início da doença em 25/31 pacientes (80,6%), com média de idade de 42,7 anos e predomínio feminino (23/31; 74,2%). Os pacientes foram hospitalizados em média por 41,2 dias; infecção bacteriana foi a principal complicação (30/31; 96,8%), com bacteremia em 10/31 (32,3%) causada por Staphylococcus aureus em 7/10 (70%) dos pacientes. Infecção pelo vírus do herpes simples (HSV) em 11/31 (35,5%) doentes determinou internação prolongada. Valores de imunofluorescência indireta, e anti-Dsg1 e VEGF séricos (ELISA) foram superiores no PFE vs. PF não-eritrodérmico (PFNE) (p < 0,05). Níveis do sVEGFR-1 foram semelhantes no PFE e PFNE, e correlacionaram-se fracamente com a anti-Dsg1 no PFNE (p=0,034). A avaliação manual da IH com anti-VEGF no PFE foi estatisticamente diferente do PFNE (p=0,042) e CS (p=0,004), e similar ao PV (p=0,667) e PSO (p=0,667). Já no PFNE, a expressão do VEGF foi estatisticamente diferente do PV (p=0,049) e PSO (p=0,049) e semelhante ao CS (p=0,247). A contagem automatizada dos vasos marcados com anti-Eng no PFE foi similar ao PFNE (p=0,700) e PSO (p=0,133), e diferente do PV (p=0,0009) e CS (p=0,0009). A avaliação de 6 espécimes de PFE mostrou depósitos de imunocomplexos: intercelulares intraepidérmicos com IgG e C3 (n=6), IgA (n=5) e IgM (n=1); nas células inflamatórias com IgG e C3 (n=6), IgM e IgA (n=1); nos vasos com IgG, C3 e IgA (n=6), e IgM (n=5); e nos anexos com IgG e C3 (n=6), IgA (n=3) e IgM (n=1). CONCLUSÕES: O PFE predomina no início da doença e em mulheres, com maior risco de infecção. O aumento do VEGF sérico e tecidual sugere uma resposta reparadora ao dano tecidual causado pelos níveis elevados de autoanticorpos no PFE. A menor expressão de Eng no PFE indica uma desregulação da angiogênese na eritrodermia. De forma pioneira, a IH automatizada demonstrou a presença de imunocomplexos intraepidérmicos e nas estruturas dérmicas / BACKGROUND: Pemphigus foliaceus (PF) is characterized by the production of autoantibodies against desmoglein 1 (Dsg1), triggering superficial acantholysis. Lesions on the face and trunk may evolve to erythroderma (PFE). The pathogenesis of PFE is not fully understood. Previous studies suggest the role of vascular endothelial growth factor (VEGF) and endoglin (Eng). OBJECTIVES: To evaluate demographic data, VEGF and Eng expression, and immune complexes deposition in patients with PFE. METHODS: This study included patients with PFE (n=31) and controls with pemphigus vulgaris (PV; n=10), psoriasis (PSO; n=10), and health individuals (CS; n=14) that had serum and skin samples stored. Serum levels of VEGF, soluble VEGF receptor (sVEGFR-1), anti-Dsg1 and Dsg3 were measured by ELISA, and the in situ expression of VEGF, Eng, and immune complexes was evaluated utilizing an automated immunohistochemistry (IH) platform (n=19). After digitalizing the slides, the reactivity was manually classified as 0 (negative), 1 (mild-to-moderate) and 2 (intense), and also analyzed by software. RESULTS: PFE occurred at the onset of the disease in 25/31 (80.6%) patients, with a mean age of 42.7 years and a female predominance (23/31; 74.2%). Patients were hospitalized with an average length of stay of 41.2 days. Bacterial infection was the main complication (30/31; 96.8%), with bacteremia in 10/31 (32.3%) due to Staphylococcus aureus in 7/10 (70%) patients. Herpes simplex virus infection in 11/31 (35.5%) PFE patients caused prolonged hospitalization. Indirect immunofluorescence titers and serum anti-Dsg1 and VEGF (ELISA) were increased in PFE vs. non-erythrodermic PF (PFNE) (p < 0.05). Serum levels of sVEGFR-1 were similar in PFE and PFNE, and weakly correlated with anti-Dsg1 in PFNE (p=0.0342). Manual analysis of anti-VEGF positivity in PFE was statistically different from PFNE (p=0.042) and CS (p=0.004), and similar to PSO (p=0.667) and PV (p=0.667). VEGF expression in PFNE was statistically different from PSO (p=0.049) and PV (p=0.049) and similar to CS (p=0.247). The automated positive vessel count with anti-Eng was similar between PFE and PFNE (p=0.700) and PSO (p=0.133), but different from PV (p=0.0009). Immune complex deposits were evaluated in 6 specimens obtained during PFE and exhibited: intraepidermal intercellular deposits with IgG and C3 (n=6), IgA (n=5) and IgM (n=1); reactivity to inflammatory cells with IgG e C3 (n=6), IgM and IgA (n=1); vascular deposits with IgG, C3 and IgA (n=6), and IgM (n=5); and adnexal positivity with IgG and C3 (n=6), IgA (n=3) and IgM (n=1). CONCLUSIONS: Erythroderma predominates at the onset of PF, especially in women, with higher infectious risk. Increased expression of serum and in situ VEGF suggests that healing processes are triggered in response to the tissue damage caused by high levels of circulating autoantibodies in PFE. The reduced expression of Eng in PFE demonstrates a dysregulated angiogenesis during erythroderma. To the best of our knowledge, this is the first study that showed intraepidermal and dermal deposits of multiple immune complexes utilizing automated IH analysis

INTRODUÇÃO: Recidivas locais e loco-regionais são as principais causas de falha do tratamento em pacientes portadores de carcinomas epidermóides de boca e orofaringe. A cirurgia de resgate é geralmente a melhor opção terapêutica para esses pacientes. Esse estudo tem por objetivo avaliar a importância prognóstica da expressão das proteínas EGFR, MMP-2, MMP-9 e VEGF em pacientes com recidiva local submetidos à cirurgia de resgate. CASUÍSTICA E MÉTODOS: Os prontuários de 111 pacientes portadores de recorrência local de carcinomas epidermóides de boca e orofaringe foram analisados de forma retrospectiva. A localização do tumor primário foi o lábio em 10 casos (9%), a cavidade oral em 68 (61%) e a orofaringe em 33 (30%). O tratamento prévio foi cirurgia em 33 casos (30%), radioterapia associada ou não à quimioterapia baseada em cisplatina em 46 (41%) e cirurgia com radioterapia adjuvante em 32 (29%). A expressão das proteínas EGFR, MMP-2, MMP-9 and VEGF foi avaliada com a técnica do Tissue Microarray. RESULTADOS: O intervalo livre de doença variou de 0,89 a 140,9 meses, com uma mediana de 6,87 meses. As recidivas foram diagnosticadas em intervalo de tempo inferior a 1 ano em 69 pacientes (62,2%) e após 1 ano em 42 (37,8%). Os pacientes com intervalo livre de doença inferior a 1 ano apresentaram pior resultado de sobrevida (p=0,01). O estádio clínico da recidiva (rEC) foi I ou II em 31 casos (27,9%) e III ou IV em 80 (72,1%). Pacientes com doença em estádio clínico mais avançado (rEC III ou IV) apresentaram piores taxas de sobrevida específica por câncer (p=0,04). Hiper-expressão do EGFR foi associada a pior resultado do tratamento. Os casos com EGFR positivo obtiveram sobrevida específica por câncer em 3 anos de 27,2%, enquanto pacientes com EGFR negativo alcançaram 64,3% de sobrevida em 3 anos (p=0,001). A expressão das proteínas MMP-2, MMP-9 e VEGF não se mostrou significativa para o prognóstico (p=0,83, p=0,15 e p=0,86, respectivamente). Na análise multivariada, apenas o intervalo livre de doença e a expressão do EGFR foram associadas à maior risco de morte. CONCLUSÕES: Recidivas locais de carcinomas epidermóides de boca e orofaringe são associadas a mau prognóstico. Intervalo livre de doença superior a 1 ano e ausência de expressão do EGFR foram os principais fatores associados a melhores resultados de sobrevida específica por câncer em pacientes submetidos à cirurgia de resgate. / INTRODUCTION: Local and regional relapses are the main sites of treatment failure in patients with oral and oropharyngeal squamous cell carcinoma. In these instances, salvage surgery is the most widely used treatment approach. The aim of this study is to analyze the prognostic effect of the expression of EGFR, MMP-2, MMP-9 and VEGF in patients with recurrent cancer sumitted to salvage surgery. METHODS: The charts of 111 patients with local recurrence of oral or oropharyngeal squamous cell carcinomas were retrospectively analyzed. The tumor sites were: the lip in 11 cases (9%), the oral cavity in 68 (61%) and the oropharynx in 33 (30%). The previous treatment was: Surgery in 33 patients (30%), radiotherapy with or without cisplatin based chemotherapy in 46 (41%) and surgery with adjuvant radiotherapy in 32 (29%). EGFR, MMP-2, MMP-9 and VEGF expressions were analyzed with tissue microarray immunohistochemical technique. RESULTS: The disease-free interval ranged from 0.89 to 140.9 months with a median of 6.87 months. The patients were categorized into two groups: Those with recurrence in less than 1 year (69 patients - 62.2%) and those with recurrence after 1 year (42 - 37.8%). The group with the shorter disease-free interval presented a worse prognosis (p=0.01). The clinical stage of recurrence (rCS) was I/II in 31 cases (27.9%) and III/IV in 80 cases (72.1%). Patients with more advanced diseases (rCS III/IV) had worse rates of cancer specific survival (CSS) than patients with rCS I/II (p=0.04). An over-expression of EGFR was associated with worse treatment results. Positive EGFR cases had a 3 year CSS of 27.2%, while EGFR negative patients had 64.3% (p=0.001). The MMP-2 and MMP-9 over-expression were also associated with a worse prognosis but without statistical significance (p=0.83 and p=0.15). VEGF expression did not show prognostic significance in this group of patients. In a multivariate analysis only the disease-free interval and over-expression of EGFR were associated with a higher risk of death. CONCLUSION: Local recurrence in oral and oropharyngeal squamous cell carcinomas usually indicates an unfavorable prognosis. A disease-free interval greater than 1 year and a negative EGFR expression are the main prognostic factors which indicate a better cancer specific survival rate in patients submitted to salvage surgery.

INTRODUÇÃO: Recidivas locais e loco-regionais são as principais causas de falha do tratamento em pacientes portadores de carcinomas epidermóides de boca e orofaringe. A cirurgia de resgate é geralmente a melhor opção terapêutica para esses pacientes. Esse estudo tem por objetivo avaliar a importância prognóstica da expressão das proteínas EGFR, MMP-2, MMP-9 e VEGF em pacientes com recidiva local submetidos à cirurgia de resgate. CASUÍSTICA E MÉTODOS: Os prontuários de 111 pacientes portadores de recorrência local de carcinomas epidermóides de boca e orofaringe foram analisados de forma retrospectiva. A localização do tumor primário foi o lábio em 10 casos (9%), a cavidade oral em 68 (61%) e a orofaringe em 33 (30%). O tratamento prévio foi cirurgia em 33 casos (30%), radioterapia associada ou não à quimioterapia baseada em cisplatina em 46 (41%) e cirurgia com radioterapia adjuvante em 32 (29%). A expressão das proteínas EGFR, MMP-2, MMP-9 and VEGF foi avaliada com a técnica do Tissue Microarray. RESULTADOS: O intervalo livre de doença variou de 0,89 a 140,9 meses, com uma mediana de 6,87 meses. As recidivas foram diagnosticadas em intervalo de tempo inferior a 1 ano em 69 pacientes (62,2%) e após 1 ano em 42 (37,8%). Os pacientes com intervalo livre de doença inferior a 1 ano apresentaram pior resultado de sobrevida (p=0,01). O estádio clínico da recidiva (rEC) foi I ou II em 31 casos (27,9%) e III ou IV em 80 (72,1%). Pacientes com doença em estádio clínico mais avançado (rEC III ou IV) apresentaram piores taxas de sobrevida específica por câncer (p=0,04). Hiper-expressão do EGFR foi associada a pior resultado do tratamento. Os casos com EGFR positivo obtiveram sobrevida específica por câncer em 3 anos de 27,2%, enquanto pacientes com EGFR negativo alcançaram 64,3% de sobrevida em 3 anos (p=0,001). A expressão das proteínas MMP-2, MMP-9 e VEGF não se mostrou significativa para o prognóstico (p=0,83, p=0,15 e p=0,86, respectivamente). Na análise multivariada, apenas o intervalo livre de doença e a expressão do EGFR foram associadas à maior risco de morte. CONCLUSÕES: Recidivas locais de carcinomas epidermóides de boca e orofaringe são associadas a mau prognóstico. Intervalo livre de doença superior a 1 ano e ausência de expressão do EGFR foram os principais fatores associados a melhores resultados de sobrevida específica por câncer em pacientes submetidos à cirurgia de resgate. / INTRODUCTION: Local and regional relapses are the main sites of treatment failure in patients with oral and oropharyngeal squamous cell carcinoma. In these instances, salvage surgery is the most widely used treatment approach. The aim of this study is to analyze the prognostic effect of the expression of EGFR, MMP-2, MMP-9 and VEGF in patients with recurrent cancer sumitted to salvage surgery. METHODS: The charts of 111 patients with local recurrence of oral or oropharyngeal squamous cell carcinomas were retrospectively analyzed. The tumor sites were: the lip in 11 cases (9%), the oral cavity in 68 (61%) and the oropharynx in 33 (30%). The previous treatment was: Surgery in 33 patients (30%), radiotherapy with or without cisplatin based chemotherapy in 46 (41%) and surgery with adjuvant radiotherapy in 32 (29%). EGFR, MMP-2, MMP-9 and VEGF expressions were analyzed with tissue microarray immunohistochemical technique. RESULTS: The disease-free interval ranged from 0.89 to 140.9 months with a median of 6.87 months. The patients were categorized into two groups: Those with recurrence in less than 1 year (69 patients - 62.2%) and those with recurrence after 1 year (42 - 37.8%). The group with the shorter disease-free interval presented a worse prognosis (p=0.01). The clinical stage of recurrence (rCS) was I/II in 31 cases (27.9%) and III/IV in 80 cases (72.1%). Patients with more advanced diseases (rCS III/IV) had worse rates of cancer specific survival (CSS) than patients with rCS I/II (p=0.04). An over-expression of EGFR was associated with worse treatment results. Positive EGFR cases had a 3 year CSS of 27.2%, while EGFR negative patients had 64.3% (p=0.001). The MMP-2 and MMP-9 over-expression were also associated with a worse prognosis but without statistical significance (p=0.83 and p=0.15). VEGF expression did not show prognostic significance in this group of patients. In a multivariate analysis only the disease-free interval and over-expression of EGFR were associated with a higher risk of death. CONCLUSION: Local recurrence in oral and oropharyngeal squamous cell carcinomas usually indicates an unfavorable prognosis. A disease-free interval greater than 1 year and a negative EGFR expression are the main prognostic factors which indicate a better cancer specific survival rate in patients submitted to salvage surgery.

The citrulline-nitric oxide (NO) cycle, comprised of the enzymes argininosuccinate synthase (AS), argininosuccinate lyase (AL) and endothelial nitric oxide synthase (eNOS), is responsible for the regulated production of endothelial NO. Although most studies have focused on eNOS to uncover important regulatory mechanisms, we and others have determined that AS is an essential and regulated step in endothelial NO production. AS is rate limiting for endothelial NO production and is the primary source of arginine, the substrate for eNOS-mediated NO production, despite saturating intracellular levels of arginine and available arginine transport systems. AS is essential for endothelial cell viability and its expression is regulated coordinately with eNOS by TNF and thiazolidenediones with concomitant effects on NO production. Given the importance of AS for endothelial health, we explored three independent regulatory mechanisms. In Chapter One, the functional consequences of altered AS expression due to overexpression, insulin, VEGF and ceramide were studied. We demonstrated that overexpression of AS leads to enhanced NO production and that insulin, VEGF and ceramide coordinately regulate the expression of AS and eNOS. In Chapter Two, the first post-translational modifications of AS in the endothelium were characterized. We determined that AS is an endogenous phosphoprotein in the endothelium, described several levels of biological significance of AS phosphorylation, identified 7 sites of AS phosphorylation and began to uncover the direct impact of phosphorylation on AS function. Finally, in Chapter Three, endothelial AS subcellular localization was defined and important protein interactions were identified including caveolin-1 and HSP90. The work presented in this dissertation demonstrates that multiple mechanisms regulate the function of AS, often coordinately with eNOS, and have a direct impact on nitric oxide production. Our findings suggest that the global understanding of the citrulline-NO cycle as a metabolic unit will unravel new paradigms that will re-define our understanding of the regulation of vascular function by NO.

Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state. We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy.

New biomarkers are needed to improve knowledge about pathophysiology, in order to provide earlier correct diagnosis and to follow disease progression of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). The aim of this thesis was to find new biomarkers for these diseases. First, increased serum levels and unchanged levels in postmortal spinal cord of vascular endothelial growth factor (VEGF) were demonstrated. VEGF was not detected in cerebrospinal fluid (CSF) in ALS. Second, increased levels of fibroblast growth factor 2 were found in the CSF and serum of ALS patients. Both studies used enzyme-linked immunoassays. Third, a proteomics method for CSF analysis was explored, based on tryptic digestion and subsequent separation and detection of the peptides by on-line liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. ALS-specific patterns were observed. Four out of five samples were correctly assigned, but no single protein biomarker could be identified. Fourth, [11C](L)-deprenyl-D2 (DED) positron emission tomography (PET) demonstrated increased retention in the pons and white matter in ALS. DED binds to monoamino oxidase B, which in the brain is primarily located in astrocytes. Thus evidence was provided that astrocytosis may be detected in vivo in ALS. Fifth, normal [11C]-PIB binding in five nondemented patients with PD was reported, in contrast to previous findings of increased retention in Alzheimer's disease reflecting amyloid aggregation. Finally, the combined use of fluorodeoxyglucose and L-[β 11C]-DOPA PET for the differential diagnosis of parkinsonian syndromes was evaluated. PET provided support for the clinical diagnosis in 62 out of 75 patients, and served to exclude suspected diagnoses in another five patients.