Angiogenesis is thought to play a pivotal role in the cycling endometrium. Coordinated by oestrogen and progesterone, endometrial blood vessel development is primarily mediated by vascular endothelial growth factor-A (VEGF-A), which promotes endothelial cell (EC) proliferation and protects ECs from induced apoptosis. Studying changes at transcript level in human endometrial endothelial cells (HEECs) in response to mitogenic and inhibitory stimuli is one way towards understanding the regulation of physiological endometrial angiogenesis. Endometriosis, the presence of endometrial-like tissue outside the uterine cavity, is a common gynaecological disorder in women of reproductive age, often causing pelvic pain and reduced fertility. Chronic inflammation in the peritoneal environment and defective endometrial protein expression are some of the contributors to the complex pathophysiology of endometriosis. The aim of this work was to study the changes in the transcriptome induced by VEGF-A and partial serum deprivation in primary HEECs, and to investigate biochemical factors associated with subfertility and chronic pelvic pain in endometriosis patients. Exposing primary HEECs to VEGF-A, and serum withdrawal was found to regulate transcripts associated with survival, migration, apoptosis and progression through the cell cycle, when assessed using microarray technology and bioinformatic tools. A subset of 88 transcripts was reciprocally regulated under the two experimental conditions; thus probably important in HEEC biology. Higher endometrial epithelial staining scores of oestrogen receptor-α and reduced staining of progesterone receptors were seen in subfertile endometriosis patients. Lower levels of the receptivity biomarker leukaemia inhibitory factor (LIF) and its receptor, as well as signs of dysregulated αB-crystallin expression and increased peritoneal fluid concentrations of interleukin (IL)-1α and IL-6 were associated with reduced pregnancy rates. Endometriosis patients with chronic pelvic pain had higher levels of vasoactive intestinal peptide (VIP) in eutopic endometria and in endometriotic lesions compared with patients without chronic pain. The presence of chronic pelvic pain was also associated with increased concentrations of VIP and IL-6 in peritoneal fluid. The present results may constitute a basis for further investigation of regulatory pathways in endometrial angiogenesis as well as for studies of endometrial receptivity and pain in women with endometriosis.

Das Ziel der vorliegenden Arbeit war die Identifizierung von Geschlechterunterschieden (GU) in der Expression von miRNAs im späten Stadium der Myokardhypertrophie, sowie der möglichen Rolle von ERbeta bei der Regulierung dieser GU. Unsere früheren Studien identifizierten ERβ als determinierenden Faktor für die beobachteten GU bei Druckbelastung. Unter anderem führte eine Deletion des ERbeta zur Aufhebung der zuvor beobachteten GU auf physiologischer und fibrotischer Ebene, sowie in der Genexpression. In dieser Studie wurden insgesamt 30 miRNAs mit Geschlechter- und/oder Geschlecht*Operation-Interaktionseffekten 9 Wochen nach TAC in WT Mäusen identifiziert. Die gleichen Effekte waren in ERbeta-/- Tieren nicht zu beobachten, teilweise aufgrund einer höheren Expression dieser miRNAs in ERbeta-/- Weibchen als bei den Männchen. Die vorliegende Studie zeigt eine Hemmung vieler miRNAs durch Östrogen (E2) und seine Rezeptoren in weiblichen Kardiomyozyten, welches somit die in vivo-Ergebnisse bestätigt und die protektive Rolle von E2 und ERβ im weiblichen Herzen unterstreicht. Sechs der miRNAs mit GU in WT-, aber nicht in ERbeta-/- Hypertrophie-Modellen wurden als mögliche Fibroseregulatoren identifiziert, da ihnen gemeinsame Inhibitoren des ERK-MAPK-Signalwegs als Zielgene vorhergesagt wurden. Die Expression dieser miRNAs, miR-106a, miR-106b, miR-21, miR-24, miR-27a und miR-27b, war in kardialen Fibroblasten durch E2 geschlechterabhängig reguliert. Zusammengefasst bestätigt diese Arbeit die schützende Rolle von E2 und ERbeta im weiblichen Herzen. E2 und seine Rezeptoren hemmen die Expression vieler miRNAs in weiblichen Kardiomyozyten und kardialen Fibroblasten, sowie in vivo. In männlichen Herzen und kardialen Fibroblasten scheint ERalpha der Hauptakteur zu sein, welcher insbesondere mögliche Fibrose-bezogene miRNAs reguliert. Die verschiedenen Rollen der ERs in weiblichen und männlichen Herzen sind ein bestimmender Faktor der beobachteten GU bei Myokardhypertrophie. / The present study aimed to identify sex-differently expressed miRNAs in a late stage of hypertrophy (9 weeks) and the possible role of ERs in the regulation of these differences. Our previous studies identified ERbeta as an important determinant factor of the observed sex differences in pressure overload, playing different roles in males and females. This report identified a total of 30 miRNAs with sex and/or sex*surgery interaction effect 9 weeks after TAC in WT mice. The same effects were not observed in ERbeta-/- animals partially due to the higher expression of these miRNAs in ERbeta-/- females than in their WT counterparts. This study reveals a repression of a number of miRNAs by estradiol and its receptors alpha and beta in female cardiomyocytes, confirming the in vivo results and accentuating the important protective role of oestrogen and ERbeta in the female heart. Six of the miRNAs with sex differences in WT but not in ERbeta-/- hypertrophy models were found to be possible fibrosis regulators by putatively targeting common ERK/MAPK pathway inhibitors. MiR-106a, miR-106b, miR-21, miR-24, miR-27a and miR-27b were subjected to a different regulation by estradiol in cardiac fibroblasts in a sex-dependent manner. In conclusion, this study reinforces the oestrogen and ERbeta protective roles in the female hearts. Estradiol and ERs repress many miRNAs’ expression in both female cardiomyocytes and cardiac fibroblasts, as well as in vivo. In male hearts and cardiac fibroblasts, ERalpha is apparently the major player, regulating in particular potential fibrosis –related miRNAs. The different roles of ERs in male and female hearts are a determinant factor of the observed sex differences in cardiac hypertrophy.

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptasepolymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positiveCaucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ERpositive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants.

Das Ziel dieser Arbeit war zunächst in der Methodenoptimierung eines bereits grundlegend etablierten Protokolls zur Isolierung und Kultur equiner endometrialer Epithel (EEZ) und Stromazellen (ESZ) (BUSCHATZ 2007) zu sehen. Zudem wurde die Entwicklung weiterer Möglichkeiten des Handlings angestrebt (Passagierung, Kryokonservierung). So sollten den Zellen optimierte Rahmenbedingungen in vitro geboten werden, welche den Verhältnissen im Organismus weitgehend nahe kommen. Im Anschluss daran wurden die Zellen in vitro hinsichtlich ihrer morphologisch-funktionellen Charakteristika untersucht und die Befunde vergleichend zu den Gegebenheiten in situ betrachtet. Besonderes Augenmerk galt dabei der Expression von Progesteron- (PR) und Östrogenrezeptor-α (ERα) und von Inhibin-α. Wären vergleichbare Konstellationen in vitro und in vivo anzutreffen, könnte ein solches Kultursystem als Modell zum Studium interzellulärer Wechselwirkungen oder pathogenetischer Abläufe am Endometrium dienen. Voraussetzung hierfür wäre jedoch eine fortgeschrittene zelluläre Differenzierung, wie sie beispielsweise durch die Expression von Inhibin-α und der Steroidhormonrezeptoren angezeigt wird. Es wurden transzervikale Uterusbioptate und vollständige Uteri euthanasierter Stuten für die Zellaufreinigung sowie die vergleichende histologische Untersuchung gewonnen. Einer mechanischen und enzymatischen Dissoziation des Gewebes folgte die Separation beider Zellarten durch Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. Die Kultur erfolgte in wasserdampfgesättigter Raumluft bei 37 °C in 5 % CO2-Atmosphäre. Als Kulturmedium diente DMEM/Ham´s F-12 unter Zusatz von 2,5 % fötalem Kälberserums und diverser Additive. Es wurde eine morphologische Charakterisierung der Zellen während der Kultur vorgenommen und zudem ERα, PR und Inhibin-α an allen Kulturen und Gewebeproben immunhistologisch bestimmt. Das Ablösen der Zellen zur Passagierung erfolgte mit Trypsin-EDTA (ESZ) bzw. Alfazyme® (EEZ). Entsprechend abgelöste Zellen wurden zudem in DMSO-haltigem Nährmedium kryokonserviert. Die Kultivierung von EEZ und ESZ gelang sowohl bei Verwendung transzervikaler Uterusbioptate als auch bei Uteri euthanasierter Pferde. Zudem konnten alle physiologischerweise bei der Stute auftretenden Zyklusstände (Anöstrus, Interöstrus, Östrus) sowie ein gravider Uterus kultiviert werden. Die Konfluenz wird von EEZ nach 4 bis 16 d und von ESZ innerhalb von 7 bis 18 d erreicht, wobei Zellen aus ursprünglich proliferativen endometrialen Funktionszuständen tendenziell eher konfluent sind als sekretorisch aktive. Während der Kultur kann eine eindeutige morphologische Unterscheidung beider Zellarten voneinander erfolgen. ESZ besitzen eine spindelige, teils sternförmige, insgesamt „fibroblastenartige“ Gestalt. EEZ sind in zwei morphologischen Subtypen anzutreffen. Der monomorphe Zelltyp „A“ stellt kleine, polygonale Zellen mit regulären und regelmäßigen Zellgrenzen dar. Zelltyp „B“ ist größer, pleomorph, ebenfalls polygonal, mehrkernig und besitzt unregelmäßige, schlecht erkennbare Zellgrenzen. Subkultivierungen waren bis zu 20 (ESZ) bzw. 24 mal (EEZ) möglich. Zudem konnten beide Zellarten in flüssigem Stickstoff gelagert (kryokonserviert) und danach erfolgreich kultiviert werden. Weiterhin gelang der Nachweis von Inhibin-α im Uterus des Pferdes. Hierbei wurde eine zyklische Dynamik in der Expression festgestellt (stärkere Expression während der sekretorischen Phase), welche als Hinweis für eine sekretorische Differenzierung der EEZ anzusehen ist. Ebenfalls konnte dieses Protein in kultivierten EEZ und in viel geringerem Maße auch in den ESZ gefunden werden. Darüber hinaus war erstmalig der Nachweis von PR und ERα in beiden Zellarten in vitro möglich. Insgesamt ist die Expression dieser drei für uterines Gewebe essentiellen Rezeptoren/Proteine in kultivierten EEZ und ESZ als Hinweis für eine fortgeschrittene Differenzierung anzusehen, welche mit der vorgestellten Methode der Isolierung und Kultur auch erreicht werden konnte. Im Hinblick auf die Wachstumsgeschwindigkeit in vitro fanden sich zudem Hinweise auf eine Beibehaltung der ursprünglich im Gewebeverband erlangten zellulären Differenzierung, welche sich bei der Expression von ERα, PR bzw. Inhibin-α allerdings nicht nachvollziehen ließ. Abschließend betrachtet, deuten die vorliegenden Ergebnisse somit auf eine partielle Beibehaltung in situ erlangter endometrialer Funktionen und Spezifika hin, welche als Grundlage für weitere Arbeiten an endometrialen Zellkulturen des Pferdes anzusehen sind.

Contexto: As oscilações hormonais ao longo da vida estão associadas às variações de humor em mulheres normais. O estrógeno (E) parece estar associado aos níveis de fator neurotrófico derivado do cérebro (BDNF) em voluntárias saudáveis. No entanto, essa associação não foi investigada em pacientes com transtorno bipolar (TB). Sabe-se que os episódios de humor do TB estão associados a alterações dos níveis de BDNF; entretanto, não está claro o papel dos hormônios femininos. Objetivo: investigar a existência de uma possível associação entre os níveis de BDNF e os níveis de hormônios do eixo hipotálamo-hipófise-gonadal em mulheres com TB, incluindo pacientes durante o período reprodutivo e também na pós-menopausa. Métodos: Mulheres eutímicas (HAM-D e YMRS com escores menores que 8) com transtorno bipolar I(TB I), II (TB II) ou transtorno bipolar sem outra especificação (TB SOE) foram incluídas. As pacientes em idade reprodutiva tinham ciclos menstruais regulares (CMR) e não faziam uso de nenhum tipo de contracepção hormonal (CH); e as na pós-menopausa não estavam em uso de terapia de reposição hormonal (TRH). Condições endócrinas instáveis foram consideradas um fator de exclusão. Todas as pacientes estavam em tratamento farmacológico, associado ou não a intervenções psicossociais. Amostras de sangue foram retiradas para as medidas de BDNF, estrogênio (E), progesterona (P), LH e FSH, sendo coletadas nas fases folicular (FF) e lútea (FL) do ciclo menstrual, e uma única vez nas mulheres na pós-menopausa. Os diagnósticos foram confirmados através de entrevista clínica estruturada para o DSM-IV Transtornos do Eixo I (SCID-I), administrado por investigadores treinados. Resultados: Foram avaliadas 96 pacientes com TB. Destas, 64 não preenchiam critérios de inclusão ou apresentavam fatores de exclusão. Foram estudadas 32 mulheres com idades entre 22 e 69 anos (média = 52,78 anos). Considerando toda a amostra, o BDNF apresentou uma correlação positiva com os níveis de estradiol (r = 0,36, p = 0,043). Nas pacientes em período reprodutivo, na fase lútea(FL), houve uma correlação negativa entre o BDNF e o FSH (r = 0,831, p = 0,040). Um resultado semelhante foi encontrado com os níveis de LH (r = 0,908, p= 0,012) nessa mesma fase do ciclo menstrual. Conclusão: Os resultados encontrados na amostra de mulheres com TB foram semelhantes aos descritos na literatura em indivíduos saudáveis, que também apresentam correlação entre E e BDNF (Begliuomini et al., 2007). Estes achados indicam que o estímulo estrogênico pode ser importante na manutenção de s níveis fisiológicos de BDNF. A partir desses resultados novas vias devem ser incluídas na investigação na fisiopatologia das alterações de humor relacionadas a variações hormonais, bem como ao tratamento do TB. Além disso, ressalta a importância de incluir as variações hormonais femininas na equação diagnóstica e prognóstica do TB. / Background: Background: Hormonal oscilations across lifetime have been associated with mood variations in healthy women. Oestrogen (E) seems to be associated with Brain Derived Neurotrophic Factor (BDNF) levels in healthy volunteers. This assictaion was not studied in women with Bipolar Disorder (BD). Mood episodes of BD are associated with reductions in BDNF levels, although the role of feminine hormones in pathophysiology of BD hás not been completely studied. Objective: To investigate the association between BDNF levels and hormones involved in hypothalamus-pituitary-gonadal axis in women with BD, comparing a group during reproductive years with a menopausal group. In addition, differences across the two phases of menstrual cycle were also evaluated in the group during reproductive years. Methods: Women euthymic (HAM-D and YMRS scoring less than 8) with bipolar I, II or bipolar disorder not otherwise specified were included. Patients of reproductive age had regular menstrual cycles (RMC) and did not use any type of hormonal contraception (CH) and postmenopausal were not using hormone replacement therapy (HRT). Endocrine instable conditions were considered an exclusion factor. All patients were on pharmacotherapy, associed or not with psychosocial interventions. Blood samples withdrawn for measures of BDNF, oestrogen (E), progesterone (P), LH and FSH levels, being collected in the follicular phase (FP) and luteal (FL) of the menstrual cycle, menopausal women were held only one blood sample. Diagnoses were assessed using structured clinical interview for DSM-IV Axis I Disorders (SCID-I), administered by certified investigators. Results: Ninety six patients with BD were evaluated, of these, 64 did not meet inclusion criteria or met exclusion factors. The sample was constituted by 32 women with BD aged 22 to 69 years (mean = 52.78 years). Considering the whole sample, the BDNF was significantly correlated with estradiol levels (r = 0.36, p = 0.043). In patients in reproductive period, in the luteal phase, there was a negative correlation with FSH (r = 0.831, p = 0.040). A similar result was found with levels o LH (r = 0.908, p = 0.012) in the same menstrual cycle phase. Conclusion: The results found in the sample of women with TB were similar to those previously reported in healthy subjects, which also show a correlation between E and BDNF (Begliuomini et al., 2007). These findings indicate that estrogenic stimulation may be important in maintaining physiological BDNF levels. From these results, new avenues should be included in research on the pathophysiology of mood swings related to hormonal changes as well as the treatment of TB. Furthermore, the importance of including female hormonal changes in the equation of TB diagnostic and.

Contexto: As oscilações hormonais ao longo da vida estão associadas às variações de humor em mulheres normais. O estrógeno (E) parece estar associado aos níveis de fator neurotrófico derivado do cérebro (BDNF) em voluntárias saudáveis. No entanto, essa associação não foi investigada em pacientes com transtorno bipolar (TB). Sabe-se que os episódios de humor do TB estão associados a alterações dos níveis de BDNF; entretanto, não está claro o papel dos hormônios femininos. Objetivo: investigar a existência de uma possível associação entre os níveis de BDNF e os níveis de hormônios do eixo hipotálamo-hipófise-gonadal em mulheres com TB, incluindo pacientes durante o período reprodutivo e também na pós-menopausa. Métodos: Mulheres eutímicas (HAM-D e YMRS com escores menores que 8) com transtorno bipolar I(TB I), II (TB II) ou transtorno bipolar sem outra especificação (TB SOE) foram incluídas. As pacientes em idade reprodutiva tinham ciclos menstruais regulares (CMR) e não faziam uso de nenhum tipo de contracepção hormonal (CH); e as na pós-menopausa não estavam em uso de terapia de reposição hormonal (TRH). Condições endócrinas instáveis foram consideradas um fator de exclusão. Todas as pacientes estavam em tratamento farmacológico, associado ou não a intervenções psicossociais. Amostras de sangue foram retiradas para as medidas de BDNF, estrogênio (E), progesterona (P), LH e FSH, sendo coletadas nas fases folicular (FF) e lútea (FL) do ciclo menstrual, e uma única vez nas mulheres na pós-menopausa. Os diagnósticos foram confirmados através de entrevista clínica estruturada para o DSM-IV Transtornos do Eixo I (SCID-I), administrado por investigadores treinados. Resultados: Foram avaliadas 96 pacientes com TB. Destas, 64 não preenchiam critérios de inclusão ou apresentavam fatores de exclusão. Foram estudadas 32 mulheres com idades entre 22 e 69 anos (média = 52,78 anos). Considerando toda a amostra, o BDNF apresentou uma correlação positiva com os níveis de estradiol (r = 0,36, p = 0,043). Nas pacientes em período reprodutivo, na fase lútea(FL), houve uma correlação negativa entre o BDNF e o FSH (r = 0,831, p = 0,040). Um resultado semelhante foi encontrado com os níveis de LH (r = 0,908, p= 0,012) nessa mesma fase do ciclo menstrual. Conclusão: Os resultados encontrados na amostra de mulheres com TB foram semelhantes aos descritos na literatura em indivíduos saudáveis, que também apresentam correlação entre E e BDNF (Begliuomini et al., 2007). Estes achados indicam que o estímulo estrogênico pode ser importante na manutenção de s níveis fisiológicos de BDNF. A partir desses resultados novas vias devem ser incluídas na investigação na fisiopatologia das alterações de humor relacionadas a variações hormonais, bem como ao tratamento do TB. Além disso, ressalta a importância de incluir as variações hormonais femininas na equação diagnóstica e prognóstica do TB. / Background: Background: Hormonal oscilations across lifetime have been associated with mood variations in healthy women. Oestrogen (E) seems to be associated with Brain Derived Neurotrophic Factor (BDNF) levels in healthy volunteers. This assictaion was not studied in women with Bipolar Disorder (BD). Mood episodes of BD are associated with reductions in BDNF levels, although the role of feminine hormones in pathophysiology of BD hás not been completely studied. Objective: To investigate the association between BDNF levels and hormones involved in hypothalamus-pituitary-gonadal axis in women with BD, comparing a group during reproductive years with a menopausal group. In addition, differences across the two phases of menstrual cycle were also evaluated in the group during reproductive years. Methods: Women euthymic (HAM-D and YMRS scoring less than 8) with bipolar I, II or bipolar disorder not otherwise specified were included. Patients of reproductive age had regular menstrual cycles (RMC) and did not use any type of hormonal contraception (CH) and postmenopausal were not using hormone replacement therapy (HRT). Endocrine instable conditions were considered an exclusion factor. All patients were on pharmacotherapy, associed or not with psychosocial interventions. Blood samples withdrawn for measures of BDNF, oestrogen (E), progesterone (P), LH and FSH levels, being collected in the follicular phase (FP) and luteal (FL) of the menstrual cycle, menopausal women were held only one blood sample. Diagnoses were assessed using structured clinical interview for DSM-IV Axis I Disorders (SCID-I), administered by certified investigators. Results: Ninety six patients with BD were evaluated, of these, 64 did not meet inclusion criteria or met exclusion factors. The sample was constituted by 32 women with BD aged 22 to 69 years (mean = 52.78 years). Considering the whole sample, the BDNF was significantly correlated with estradiol levels (r = 0.36, p = 0.043). In patients in reproductive period, in the luteal phase, there was a negative correlation with FSH (r = 0.831, p = 0.040). A similar result was found with levels o LH (r = 0.908, p = 0.012) in the same menstrual cycle phase. Conclusion: The results found in the sample of women with TB were similar to those previously reported in healthy subjects, which also show a correlation between E and BDNF (Begliuomini et al., 2007). These findings indicate that estrogenic stimulation may be important in maintaining physiological BDNF levels. From these results, new avenues should be included in research on the pathophysiology of mood swings related to hormonal changes as well as the treatment of TB. Furthermore, the importance of including female hormonal changes in the equation of TB diagnostic and.

Contexto: As oscilações hormonais ao longo da vida estão associadas às variações de humor em mulheres normais. O estrógeno (E) parece estar associado aos níveis de fator neurotrófico derivado do cérebro (BDNF) em voluntárias saudáveis. No entanto, essa associação não foi investigada em pacientes com transtorno bipolar (TB). Sabe-se que os episódios de humor do TB estão associados a alterações dos níveis de BDNF; entretanto, não está claro o papel dos hormônios femininos. Objetivo: investigar a existência de uma possível associação entre os níveis de BDNF e os níveis de hormônios do eixo hipotálamo-hipófise-gonadal em mulheres com TB, incluindo pacientes durante o período reprodutivo e também na pós-menopausa. Métodos: Mulheres eutímicas (HAM-D e YMRS com escores menores que 8) com transtorno bipolar I(TB I), II (TB II) ou transtorno bipolar sem outra especificação (TB SOE) foram incluídas. As pacientes em idade reprodutiva tinham ciclos menstruais regulares (CMR) e não faziam uso de nenhum tipo de contracepção hormonal (CH); e as na pós-menopausa não estavam em uso de terapia de reposição hormonal (TRH). Condições endócrinas instáveis foram consideradas um fator de exclusão. Todas as pacientes estavam em tratamento farmacológico, associado ou não a intervenções psicossociais. Amostras de sangue foram retiradas para as medidas de BDNF, estrogênio (E), progesterona (P), LH e FSH, sendo coletadas nas fases folicular (FF) e lútea (FL) do ciclo menstrual, e uma única vez nas mulheres na pós-menopausa. Os diagnósticos foram confirmados através de entrevista clínica estruturada para o DSM-IV Transtornos do Eixo I (SCID-I), administrado por investigadores treinados. Resultados: Foram avaliadas 96 pacientes com TB. Destas, 64 não preenchiam critérios de inclusão ou apresentavam fatores de exclusão. Foram estudadas 32 mulheres com idades entre 22 e 69 anos (média = 52,78 anos). Considerando toda a amostra, o BDNF apresentou uma correlação positiva com os níveis de estradiol (r = 0,36, p = 0,043). Nas pacientes em período reprodutivo, na fase lútea(FL), houve uma correlação negativa entre o BDNF e o FSH (r = 0,831, p = 0,040). Um resultado semelhante foi encontrado com os níveis de LH (r = 0,908, p= 0,012) nessa mesma fase do ciclo menstrual. Conclusão: Os resultados encontrados na amostra de mulheres com TB foram semelhantes aos descritos na literatura em indivíduos saudáveis, que também apresentam correlação entre E e BDNF (Begliuomini et al., 2007). Estes achados indicam que o estímulo estrogênico pode ser importante na manutenção de s níveis fisiológicos de BDNF. A partir desses resultados novas vias devem ser incluídas na investigação na fisiopatologia das alterações de humor relacionadas a variações hormonais, bem como ao tratamento do TB. Além disso, ressalta a importância de incluir as variações hormonais femininas na equação diagnóstica e prognóstica do TB. / Background: Background: Hormonal oscilations across lifetime have been associated with mood variations in healthy women. Oestrogen (E) seems to be associated with Brain Derived Neurotrophic Factor (BDNF) levels in healthy volunteers. This assictaion was not studied in women with Bipolar Disorder (BD). Mood episodes of BD are associated with reductions in BDNF levels, although the role of feminine hormones in pathophysiology of BD hás not been completely studied. Objective: To investigate the association between BDNF levels and hormones involved in hypothalamus-pituitary-gonadal axis in women with BD, comparing a group during reproductive years with a menopausal group. In addition, differences across the two phases of menstrual cycle were also evaluated in the group during reproductive years. Methods: Women euthymic (HAM-D and YMRS scoring less than 8) with bipolar I, II or bipolar disorder not otherwise specified were included. Patients of reproductive age had regular menstrual cycles (RMC) and did not use any type of hormonal contraception (CH) and postmenopausal were not using hormone replacement therapy (HRT). Endocrine instable conditions were considered an exclusion factor. All patients were on pharmacotherapy, associed or not with psychosocial interventions. Blood samples withdrawn for measures of BDNF, oestrogen (E), progesterone (P), LH and FSH levels, being collected in the follicular phase (FP) and luteal (FL) of the menstrual cycle, menopausal women were held only one blood sample. Diagnoses were assessed using structured clinical interview for DSM-IV Axis I Disorders (SCID-I), administered by certified investigators. Results: Ninety six patients with BD were evaluated, of these, 64 did not meet inclusion criteria or met exclusion factors. The sample was constituted by 32 women with BD aged 22 to 69 years (mean = 52.78 years). Considering the whole sample, the BDNF was significantly correlated with estradiol levels (r = 0.36, p = 0.043). In patients in reproductive period, in the luteal phase, there was a negative correlation with FSH (r = 0.831, p = 0.040). A similar result was found with levels o LH (r = 0.908, p = 0.012) in the same menstrual cycle phase. Conclusion: The results found in the sample of women with TB were similar to those previously reported in healthy subjects, which also show a correlation between E and BDNF (Begliuomini et al., 2007). These findings indicate that estrogenic stimulation may be important in maintaining physiological BDNF levels. From these results, new avenues should be included in research on the pathophysiology of mood swings related to hormonal changes as well as the treatment of TB. Furthermore, the importance of including female hormonal changes in the equation of TB diagnostic and.

Ziel dieser Studie war die Etablierung einer Kokultur aus equinen endometrialen Epithel- und Stromazellen. Nach der erfolgreichen Umsetzung des Kokulturmodells sollte im weiteren Versuchsablauf durch die Zugabe von 17β-Östradiol (E2) und/oder Progesteron (P4) zum Nährmedium der Einfluss der Hormone auf die Zellen untersucht werden. Neben einer lichtmikroskopischen Auswertung der zytomorphologischen Charakteristika beider Zellarten sollte die Expression der Steroidhormonrezeptoren Östrogenrezeptor α und Progesteronre-zeptor sowie der uterinen Proteine Uteroglobin und CalbindinD9k immunzytologisch überprüft werden. Für die Etablierung der Kokultur wurden Endometriumproben von lebenden (n = 5) sowie frischtoten (n = 4) Stuten gewonnen. Eine jeweils parallel entnommene Gewebeprobe von jedem Tier wurde in Formalin fixiert und diente als Referenzmaterial (in situ). Auf die Zelliso-lierung (mechanisch und enzymatisch) folgte die Separation von Epithel- und Stromazellen (EZ/SZ) mittels Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. An-schließend wurden die EZ auf die Außenseite von Millicell®-Membraneinsätzen aufgebracht. Nach zwei Tagen erfolgte das Einsäen der bis zu diesem Zeitpunkt separat kultivierten SZ auf die Innenseite der Membranen. Als Nährmedium diente ein Gemisch aus DMEM und Ham’s F-12, wobei diesem 2,5 % fötales Kälberserum sowie verschiedene Additive zugesetzt wurden. Ab Kulturtag 4 wurden dem Medium definierte Konzentrationen und Kombinationen von E2 und P4 zugesetzt. Die Kultivierung erfolgte bei einem CO2-Partialdruck von 5 % in 37 °C warmer wasserdampfgesättigter Raumluft. Mit der polarisationsmikroskopisch er-fassbaren Ausbildung durchgehender Zellrasen („scheinbare Konfluenz“) wurden die Kokul-turen in Formalin fixiert und für die Lichtmikroskopie aufgearbeitet. Das Ausgangsgewebe zeigte mehrheitlich eine sekretorische Funktionsmorphologie (n = 6). Einzelne Endometrien befanden sich in einem Übergangsstadium von der Sekretions- zur Proliferationsphase (n = 1), bzw. vice versa (n = 1) oder wiesen eine irregulär proliferative Differenzierung (n = 1) auf. Im Rahmen der Kokultivierung bildeten die EZ innerhalb der Schnittebene vier und die SZ drei verschiedene morphologische Zelltypen aus. Dabei traten rundovale bis polygonale EZ (Typ 1) selten bis gelegentlich, spindelförmige EZ (Typ 2) gelegentlich bis häufig und iso-prismatische (Typ 3) sowie mehrschichtig wachsende EZ (Typ M) jeweils selten auf. Die SZ zeigten innerhalb der Schnittebene selten eine rundovale bis polygonale Zellform (Typ 1), sehr häufig eine spindelförmige Morphologie (Typ 2) und selten ein mehrschichtiges Wachstum (Typ M). Ein Zusammenhang zwischen der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung oder dem Hormonzusatz und der Häufigkeitsverteilung der Zell-typen sowie der Wachstumsgeschwindigkeit der kultivierten Zellen war nicht offensichtlich. Zytokeratin 19 wurde stets von EZ exprimiert, während es auf Seiten der SZ nur sporadisch in maximal 5 % der Zellen im Bereich mehrschichtig wachsender Zellrasen auftrat. Die Stero-idhormonrezeptoren konnten lediglich in einzelnen Kokulturen aus sekretorisch differenzier-tem Ausgangsgewebe detektiert werden. Uteroglobin wurde in vitro mit einer variablen Häufigkeit in den EZ-Typen exprimiert. Während ein übergreifender Zusammenhang zur hormonellen Supplementierung nicht abgeleitet werden konnte, wurde jedoch ersichtlich, dass im Bereich einschichtig wachsender EZ in Ansätzen aus sekretorisch differenzierten Endometrien unter niedrigen Hormondosen (Zusatz von entweder nur E2 oder nur P4) im Median häufiger Uteroglobin exprimiert wurde. Mit zunehmender Hormonkonzentration im Medium nahm der Anteil immunopositiver Zellen (Typen 1, 2 und 3) deutlich ab. Innerhalb der Stromazellpopulation wurde Uteroglobin selten und ausschließlich in Zellen aus sekretorisch differenziertem Ausgangsmaterial nachgewiesen. CalbindinD9k wurde in vitro vornehmlich intrazytoplasmatisch und sehr vereinzelt intranukleär exprimiert. Insgesamt konnte das Protein in vitro stets in wenigen Typ-1-EZ, sehr selten in Typ-2-EZ und in einer geringen bis mäßigen Anzahl von Typ-3- und Typ-M-EZ beobachtet werden. Innerhalb der Stromazellpo-pulation trat CalbindinD9k ausschließlich in einer geringen (Endometrien aus dem Östrus) bis mäßigen (Endometrien aus dem Interöstrus) Anzahl der Typ-2- und wenigen Typ-M-SZ auf. Insgesamt wurden keine deutlichen Einflüsse der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung und/oder der hormonellen Supplementierung in vitro auf die im-munzytologischen Charakteristika der kokultivierten Zellen ersichtlich. Abschließend betrachtet, konnte ein Kokultursystem equiner endometrialer Epithel- und Stromazellen erfolgreich etabliert und charakterisiert werden. Es bietet dabei, trotz der z. T. fehlenden Kongruenz zu den Gegebenheiten in situ, Ansätze für potenzielle Folgearbeiten, insbesondere hinsichtlich der Erfassung interzellulärer Wechselwirkungen sowie bezüglich der Vermittlung und Wirkung hormoneller Einflüsse auf zellulärer Ebene. / The aim of the present study was the establishment of a coculture system of equine endome-trial epithelial and stromal cells. Subsequent to the successful development of the coculture model the culture medium should be supplemented with 17β-estradiol (E2) and/or progester-one (P4) in order to study the influence of the hormones on the cellular level. In addition to the examination of cytomorphological characteristics of both cell types via light microscopy, the expression of the steroid hormone receptors (estrogen receptor α and progesterone receptor) as well as of the uterine proteins Uteroglobin and CalbindinD9k was investigated. For the establishment of the coculture system endometrial samples were obtained from living (n = 5) as well as freshly deceased mares (n = 4). A simultaneously taken tissue specimen of each animal was fixed in formalin and served as in situ reference material. After an initial mechanical and enzymatical isolation the epithelial and stromal cells (EC/SC) were separat-ed via filtration, density gradient centrifugation and differential adhesion. Subsequently, the EC were applied to the outer surface of Millicell® inserts. The SC were cultivated separately for 2 days before they were seeded onto the inner surface of the same insert. The culture medium used was comprised of a DMEM and Ham‘s F-12 basis as well as 2.5 % foetal calf serum and different additives. Starting on day 4 of cultivation the standardised medium was supplemented with different concentrations and combinations of E2 and P4. Throughout the study the cultures were kept in a humidified atmosphere of 37°C and a 5 % partial pressure of carbon dioxide. Once the cocultures formed continuous cell layers, as determined via a polarisation microscope (“apparent confluency”), the membranes were fixed in formalin and routinely processed for light microscopical evaluation. The initial tissue samples predominantly showed a secretory functional morphology (n = 6), while single specimens were obtained during the transition from the secretory to the prolifera-tive phase (n = 1) or vice versa (n = 1). One endometrial sample exhibited an irregular proli-ferative differentiation. In the course of cocultivation the EC formed 4 and the SC 3 different cellular morphologies within the section plane. EC with a round-oval to polygonal cell form (type 1) were rarely to occasionally encountered, while spindle-shaped EC (type 2) were occasionally to frequently seen and EC with a cuboidal morphology (type 3) as well as such cells growing in stratified layers (type M) were only infrequently detected. The SC only rarely showed a round-oval to polygonal cell form (type 1) or areas of a stratified cell growth (type M), whereas spindle-shaped SC (type 2) were observed very often. A correlation of the endometrial functional morphology at the time of cell isolation or the hormonal supplementation and the frequency distribution of the cell types as well as the growth rate of the cultivated cells was not evident. The EC always expressed Cytokeratin 19, while on the side of the SC only up to 5 % of the cells in areas of stratified cell growth exhibited this filament. Solely in individual cocultures from secretory differentiated endometrial tissue the steroid hormone receptors could be de-tected. Uteroglobin was expressed in vitro in EC with a variable frequency. An overall corre-lation of the hormonal supplementation and the Uteroglobin expression could not be derived. However, under low hormone doses (only E2 or only P4 supplement) Uteroglobin was detect-ed in EC in areas of single-layered cell growth more often (median value). With an increase in hormone concentration the amount of immunopositive cells (types 1, 2 and 3) diminished noticeably. In SC the protein could only rarely be seen and exclusively in cells from endome-tria with a secretory functional morphology. In vitro CalbindinD9k was predominantly detected intracytoplasmatically, while single cells showed an additional intranuclear expression. Alto-gether, CalbindinD9k could always be observed in a few type-1-EC, rarely in type-2-EC and with a variable frequency in small to moderate numbers of type-3- and type-M-EC. In SC the protein was exclusively expressed in a small (endometrial samples form the oestrous phase) to moderate (endometrial tissue from the interoestrous phase) number of type-2-SC and a few type-M-SC. Generally, no distinct influence of the endometrial functional morphology at the time of tissue sampling and/or of the hormonal supplementation in vitro on the immuno-cytochemical characteristics of the cocultured cells could be observed. In summary, a coculture system of primary equine endometrial epithelial and stromal cells was successfully established and characterised. Despite of the partly absent congruence to the in situ conditions/prerequisites, the present study offers a basic approach and scaffold for further investigations, particularly regarding the ascertainment of intercellular dependencies or the mediation and effectiveness of hormonal influences on the cellular level.

(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....

Das Ziel dieser Arbeit war zunächst in der Methodenoptimierung eines bereits grundlegend etablierten Protokolls zur Isolierung und Kultur equiner endometrialer Epithel (EEZ) und Stromazellen (ESZ) (BUSCHATZ 2007) zu sehen. Zudem wurde die Entwicklung weiterer Möglichkeiten des Handlings angestrebt (Passagierung, Kryokonservierung). So sollten den Zellen optimierte Rahmenbedingungen in vitro geboten werden, welche den Verhältnissen im Organismus weitgehend nahe kommen. Im Anschluss daran wurden die Zellen in vitro hinsichtlich ihrer morphologisch-funktionellen Charakteristika untersucht und die Befunde vergleichend zu den Gegebenheiten in situ betrachtet. Besonderes Augenmerk galt dabei der Expression von Progesteron- (PR) und Östrogenrezeptor-α (ERα) und von Inhibin-α. Wären vergleichbare Konstellationen in vitro und in vivo anzutreffen, könnte ein solches Kultursystem als Modell zum Studium interzellulärer Wechselwirkungen oder pathogenetischer Abläufe am Endometrium dienen. Voraussetzung hierfür wäre jedoch eine fortgeschrittene zelluläre Differenzierung, wie sie beispielsweise durch die Expression von Inhibin-α und der Steroidhormonrezeptoren angezeigt wird. Es wurden transzervikale Uterusbioptate und vollständige Uteri euthanasierter Stuten für die Zellaufreinigung sowie die vergleichende histologische Untersuchung gewonnen. Einer mechanischen und enzymatischen Dissoziation des Gewebes folgte die Separation beider Zellarten durch Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. Die Kultur erfolgte in wasserdampfgesättigter Raumluft bei 37 °C in 5 % CO2-Atmosphäre. Als Kulturmedium diente DMEM/Ham´s F-12 unter Zusatz von 2,5 % fötalem Kälberserums und diverser Additive. Es wurde eine morphologische Charakterisierung der Zellen während der Kultur vorgenommen und zudem ERα, PR und Inhibin-α an allen Kulturen und Gewebeproben immunhistologisch bestimmt. Das Ablösen der Zellen zur Passagierung erfolgte mit Trypsin-EDTA (ESZ) bzw. Alfazyme® (EEZ). Entsprechend abgelöste Zellen wurden zudem in DMSO-haltigem Nährmedium kryokonserviert. Die Kultivierung von EEZ und ESZ gelang sowohl bei Verwendung transzervikaler Uterusbioptate als auch bei Uteri euthanasierter Pferde. Zudem konnten alle physiologischerweise bei der Stute auftretenden Zyklusstände (Anöstrus, Interöstrus, Östrus) sowie ein gravider Uterus kultiviert werden. Die Konfluenz wird von EEZ nach 4 bis 16 d und von ESZ innerhalb von 7 bis 18 d erreicht, wobei Zellen aus ursprünglich proliferativen endometrialen Funktionszuständen tendenziell eher konfluent sind als sekretorisch aktive. Während der Kultur kann eine eindeutige morphologische Unterscheidung beider Zellarten voneinander erfolgen. ESZ besitzen eine spindelige, teils sternförmige, insgesamt „fibroblastenartige“ Gestalt. EEZ sind in zwei morphologischen Subtypen anzutreffen. Der monomorphe Zelltyp „A“ stellt kleine, polygonale Zellen mit regulären und regelmäßigen Zellgrenzen dar. Zelltyp „B“ ist größer, pleomorph, ebenfalls polygonal, mehrkernig und besitzt unregelmäßige, schlecht erkennbare Zellgrenzen. Subkultivierungen waren bis zu 20 (ESZ) bzw. 24 mal (EEZ) möglich. Zudem konnten beide Zellarten in flüssigem Stickstoff gelagert (kryokonserviert) und danach erfolgreich kultiviert werden. Weiterhin gelang der Nachweis von Inhibin-α im Uterus des Pferdes. Hierbei wurde eine zyklische Dynamik in der Expression festgestellt (stärkere Expression während der sekretorischen Phase), welche als Hinweis für eine sekretorische Differenzierung der EEZ anzusehen ist. Ebenfalls konnte dieses Protein in kultivierten EEZ und in viel geringerem Maße auch in den ESZ gefunden werden. Darüber hinaus war erstmalig der Nachweis von PR und ERα in beiden Zellarten in vitro möglich. Insgesamt ist die Expression dieser drei für uterines Gewebe essentiellen Rezeptoren/Proteine in kultivierten EEZ und ESZ als Hinweis für eine fortgeschrittene Differenzierung anzusehen, welche mit der vorgestellten Methode der Isolierung und Kultur auch erreicht werden konnte. Im Hinblick auf die Wachstumsgeschwindigkeit in vitro fanden sich zudem Hinweise auf eine Beibehaltung der ursprünglich im Gewebeverband erlangten zellulären Differenzierung, welche sich bei der Expression von ERα, PR bzw. Inhibin-α allerdings nicht nachvollziehen ließ. Abschließend betrachtet, deuten die vorliegenden Ergebnisse somit auf eine partielle Beibehaltung in situ erlangter endometrialer Funktionen und Spezifika hin, welche als Grundlage für weitere Arbeiten an endometrialen Zellkulturen des Pferdes anzusehen sind.