Made available in DSpace on 2014-06-11T19:23:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-10Bitstream added on 2014-06-13T18:51:22Z : No. of bitstreams: 1 masseno_apb_me_botfmvz.pdf: 725155 bytes, checksum: 45ef73d33fd9fcb5fbee140412fde1ad (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A biópsia uterina é uma ferramenta valiosa utilizada na avaliação da fertilidade da égua. 0 objetivo deste trabalho foi estudar a relação entre a presença de miofibroblastos, a expressão da MMP-2 e a intensidade do processo fibrótico nas endometrites crônicas das éguas. Foram utilizadas 60 biópsias uterinas de éguas provenientes da rotina do Hospital Veterinário da FMVZ - UNESP, Campus de Botucatu. Biópsias endometriais classificadas histologicamente de acordo com Kenney e Doig (1986) e segundo as definições de Ricketts e Alonso (1991) para endometrite cronica infiltrativa e endometrose. A avaliação dos graus de fibrose endometrial foi feita par meio dos métodos histoquímicos Tricrômico de Masson e Picrosirius Red. A expressão da MMP-2 assim como a expressão de actina de músculo liso α dos miofibroblastos foi avaliada utilizando-se tecnica imunoistoquímica. A quantidade de colágeno na fibrose endometrial foi maior nas regiões periglandulares, perivasculares e no estrato esponjoso, predominando o colágeno do tipo I. Tanto no endométrio hígido como nas endometrites crônicas foi observada imuno-reatividade para as enzimas estudadas. A α-SMA e a MMP-2 estão envolvidas nos processos fibróticos endometriais das éguas uma vez que estas enzimas aumentam em expressão e intensidade de reação conforme o grau de endometrite. / Uterine biopsy is a valuable tool for evaluation of mare's fertility. The aim of this work was to study the relation between the presence of myofibroblasts, the expression of MMP-2 and the intensity of fibrotic process in chronic endometritis in mares. Sixty mare's uterine biopsies from the Veterinary Hospital routine of FMVZ-UNESP, Campus of Botucatu, were used. Biopsies of chronic endometrites were classified according to Kenney and Doig (1986) and definitions Ricketts & Alonso (1991) for chronic infiltrative endometrites and endometrosis. Evaluations of endometrial fibrotic levels were done by histochemical methods. Masson and Picrosirius Red Tricromio. The expression of MMP-2 as the expression of myofibroblasts straight α- smooth muscle actin was evaluated using immunohistochemical techniques. The collagen concentration in fibrotic endometrium was higher at periglandular, perivascular and stratum spongiosum regions with collagen type I predominance. Imnunoreactivity of α-SMA and MMP-2 was detected in healthy endometrium as in severe chronic endometritis. The a- SMA and MMP-2 showed variation in expression and reaction was more intense in the normal and chronic affected endometrium and may plays a hole in the endometrial fibrotic process.

Resumo

Orientador: Marco Antonio Alvarenga / Coorientador: João Pessoa Araújo Junior / Banca: Frederico Ozanam Papa / Banca: Fabiana Ferreira de Souza / Banca: Rubens Paes de Arruda / Banca: Eduardo Malschitzky / Resumo: A expressão das interleucinas no útero e das catelicidinas no plasma seminal podem ser responsáveis pela magnitude e persistência da resposta inflamatória uterina. Os objetivos desse estudo foram verificar a presença das catelicidinas no plasma seminal e caracterizar a resposta imune uterina pela expressão de interleucinas inflamatórias. O plasma seminal foi obtido por coleta de sêmen de 15 garanhões e submetido ao SDSPAGE e DOT-blotting. As éguas foram submetidas a um ciclo estral sem desafio uterino (T1), com infusão de plasma seminal (T2) e inseminação artificial (T3). Exame ultrassonográfico, citologia exfoliativa uterina e colheita de células para a expressão do RNAm das interleucinas por qPCR foram realizados durante 3 ciclos consecutivos aleatoriamente, 24 (M1), 48 (M2) e 72 horas (M3) após a indução da ovulação. A expressão do RNAm no endométrio das éguas resistentes e susceptíveis foi verificada para as interleucinas IL-1β, IL-6, IL-8, IL-10 e TNF-α. A concentração total de proteínas no plasma seminal dos garanhões variou de 6,3 a 25,6 mg/mL. O SDS-PAGE revelou a existência de 3 bandas protéicas dentro do intervalo de peso molecular previamente citado na literatura para as catelicidinas equinas. Os garanhões avaliados mostraram quantidades de eCATH1 < 339,6ng, eCATH2 < 141,3ng e eCATH3 < 283,4ng. No momento M2 não foi possível diferenciar as éguas resistentes das susceptíveis pela citologia ou ultrassonografia independente da infusão com plasma seminal, inseminação artificial ou da ovulação, mas quando há presença de espermatozoides no útero a resposta inflamatória é mais intensa. Durante o momento M3, as éguas susceptíveis apresentaram maior volume de fluido uterino acumulado em todos os tratamentos com menor capacidade de limpeza uterina. Foi verificada maior expressão do RNAm das interleucinas pró-inflamatórias nas éguas susceptíveis e a expressão do RNAm da interleucina-10 é... / Abstract: In the equine species, uterine interleukin expression and seminal plasma cathelicidins can modulate the magnitude and persistence of the post-breeding inflammatory reaction. The objectives were to verify the presence of cathelicidins in the stallion seminal plasma and characterize the uterine immune response by pro and anti-inflammatory interleukins. Seminal plasma was obtained by semen collection of 15 stallions and submitted to SDS-PAGE and DOT-blotting tests. Three experiments were designed to study interleukin expression during baseline (T1), after seminal plasma infusion (T2) and artificial insemination (T3). Endometrial cytology and cell collection as well as ultrassonographic evaluations were performed to access interleukin mRNA through qPCR during three randomly assigned consecutive estrous cycles, 24 (M1), 48 (M2) and 72 hours (M3) after an ovulation was induced. Endometrial mRNA expression in mares resistant and susceptible to persistent post-breeding endometritis was identified for the following pro-inflammatory cytokines: IL-1β, IL-6, IL-8, IL-10 e TNF-α. Total seminal plasma protein concentrations ranged from 6.3 to 25.6 mg/mL. SDS-PAGE analysis identified the existence of three proteic bands with molecular weight similar to what was previously reported for equine cathelicidins. Evaluated stallions showed values < 339.6 ng for eCATH1, < 141.3 ng for eCATH2 and < 283.4 for eCATH3. At M2, resistant and susceptible mares could not be separated through cytology or ultrassonography, even though the presence of sperm strongly stimulates uterine inflammatory reaction. At M3, susceptible mares showed higher uterine fluid accumulation and lower uterine clearance. Pró-inflammatory mRNA expression was higher in susceptible mares and interleukin-10 mRNA expression was similar between resistant and susceptible mares / Doutor

Orientador: Agnaldo Lopes da Silva Filho / Coorientador: Andrezza Vilaça Belo / Banca: Paulo Traiman / Banca: Andrea Teixeira de Carvalho / Resumo: A resposta inflamatória é um processo ativo nas neoplasias de colo do útero, podendo atuar tanto na progressão quanto na regressão da lesão. No local da inflamação estão presentes macrófagos e neutrófilos, além de citocinas como o fator de necrose tumoral-alfa (TNF-α) e o interferon-gama (IFN-γ). Este estudo visa avaliar a resposta inflamatória sérica em mulheres com lesões pré-invasoras e invasoras do colo uterino. Foram analisadas amostras de soro obtidas de mulheres sem evidência de doença (n=30 - grupo controle), com lesões pré invasoras do colo uterino (NIC) (n=30) e com carcinoma invasor de colo uterino (CCE) (n=30). O sangue das pacientes foi coletado, centrifugado e armazenado até análise laboratorial. A atividade das enzimas inflamatórias N-acetilglucosaminidase (NAG) e mieloperoxidase (MPO) foram obtidas por ensaio enzimático. Utilizou-se o método imunoenzimático de ELISA para obtenção dos níveis séricos do TNF-α e IFN-γ. As diferenças entre os grupos foram avaliadas pelo teste do qui-quadrado, Kruskal-Wallis ou Mann Whitney. A correlação entre os grupos foi realizada pelo teste Kendall's Tau. As diferenças com valor de p<0,05 foram consideradas significativas. No grupo de pacientes com lesões pré- invasoras do colo uterino, duas mulheres apresentavam NIC I (6,7%), oito pacientes NIC II (26,7%) e 20 casos apresentavam NIC III (66,7%). O estadiamento (FIGO) das pacientes com CCE invasor foi IA2 em três casos (10,0%), IB1 em 19 (63,3%), IB2 em dois (6,7%) e IIA em seis (20,0%) mulheres pertencentes a esse grupo. As atividades das enzimas NAG, MPO e níveis de TNF-α foram maiores no grupo de mulheres com NIC, quando comparado com o grupo com CCE. Os níveis de IFN-γ foram menores no grupo de mulheres com NIC, quando comparado com o grupo com CCE. Não houve uma associação significativa entre o grau do NIC e o estadiamento do CCE invasor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The inflammatory response is an active process in cervical cancer and may act in the progression and/or regression of the lesion. At the site of inflammation cells are present, among the most important macrophages and neutrophils and some cytokines such as TNF- and IFN- . This study aims to evaluate the inflammatory response levels in women with pre-invasive lesions and invasive cervical cancer. Methods: Were analyzed serum samples obtained from women without evidence of disease (n = 30 - control group), with pre-invasive lesions of the cervix (n = 30) and with carcinoma of the cervix (n = 30). The activity of inflammatory enzymes Nacetylglucosaminidase (NAG) and myeloperoxidase (MPO) were obtained by enzymatic assay. We used the ELISA assay method for obtaining serum levels of TNF- and IFN- . The blood of the patients was collected, centrifuged and stored until laboratory analysis. Differences between groups were evaluated by chi-square, Kruskal-Wallis and Mann Whitney. The correlation between the groups was performed using Kendall's Tau. Differences with p <0.05 were considered significant. Results: The women age ranged from 30 to 79 years. In the group of patients with preinvasive lesions of the cervix, two had CIN I (6.7%), eight patients with CIN II (26.7%) and 20 cases had CIN III (66.7%). Staging (FIGO) of patients with SCC was IA2 in three cases (10.0%), IB1 in 19 (63.3%), IB2 in two (6.7%) and IIA in six women (20.0%). The activities of enzymes NAG, and MPO and the levels of TNF- were higher in women with CIN compared with the group with SCC. The levels of IFN- were lower in the group of women with CIN compared with the group with SCC. There was not a significant association between the degree of the CIN and the staging of the SCC of the cervix with inflammation assessed by the levels of inflammatory markers used... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:18:29Z : No. of bitstreams: 1 lages_el_me_botfm.pdf: 285358 bytes, checksum: 43b1317997b0df835998ac263f0477a7 (MD5) / A resposta inflamatória é um processo ativo nas neoplasias de colo do útero, podendo atuar tanto na progressão quanto na regressão da lesão. No local da inflamação estão presentes macrófagos e neutrófilos, além de citocinas como o fator de necrose tumoral-alfa (TNF-α) e o interferon-gama (IFN-γ). Este estudo visa avaliar a resposta inflamatória sérica em mulheres com lesões pré-invasoras e invasoras do colo uterino. Foram analisadas amostras de soro obtidas de mulheres sem evidência de doença (n=30 – grupo controle), com lesões pré invasoras do colo uterino (NIC) (n=30) e com carcinoma invasor de colo uterino (CCE) (n=30). O sangue das pacientes foi coletado, centrifugado e armazenado até análise laboratorial. A atividade das enzimas inflamatórias N-acetilglucosaminidase (NAG) e mieloperoxidase (MPO) foram obtidas por ensaio enzimático. Utilizou-se o método imunoenzimático de ELISA para obtenção dos níveis séricos do TNF-α e IFN-γ. As diferenças entre os grupos foram avaliadas pelo teste do qui-quadrado, Kruskal-Wallis ou Mann Whitney. A correlação entre os grupos foi realizada pelo teste Kendall's Tau. As diferenças com valor de p<0,05 foram consideradas significativas. No grupo de pacientes com lesões pré- invasoras do colo uterino, duas mulheres apresentavam NIC I (6,7%), oito pacientes NIC II (26,7%) e 20 casos apresentavam NIC III (66,7%). O estadiamento (FIGO) das pacientes com CCE invasor foi IA2 em três casos (10,0%), IB1 em 19 (63,3%), IB2 em dois (6,7%) e IIA em seis (20,0%) mulheres pertencentes a esse grupo. As atividades das enzimas NAG, MPO e níveis de TNF-α foram maiores no grupo de mulheres com NIC, quando comparado com o grupo com CCE. Os níveis de IFN-γ foram menores no grupo de mulheres com NIC, quando comparado com o grupo com CCE. Não houve uma associação significativa entre o grau do NIC e o estadiamento do CCE invasor... / Introduction: The inflammatory response is an active process in cervical cancer and may act in the progression and/or regression of the lesion. At the site of inflammation cells are present, among the most important macrophages and neutrophils and some cytokines such as TNF- and IFN- . This study aims to evaluate the inflammatory response levels in women with pre-invasive lesions and invasive cervical cancer. Methods: Were analyzed serum samples obtained from women without evidence of disease (n = 30 - control group), with pre-invasive lesions of the cervix (n = 30) and with carcinoma of the cervix (n = 30). The activity of inflammatory enzymes Nacetylglucosaminidase (NAG) and myeloperoxidase (MPO) were obtained by enzymatic assay. We used the ELISA assay method for obtaining serum levels of TNF- and IFN- . The blood of the patients was collected, centrifuged and stored until laboratory analysis. Differences between groups were evaluated by chi-square, Kruskal-Wallis and Mann Whitney. The correlation between the groups was performed using Kendall's Tau. Differences with p <0.05 were considered significant. Results: The women age ranged from 30 to 79 years. In the group of patients with preinvasive lesions of the cervix, two had CIN I (6.7%), eight patients with CIN II (26.7%) and 20 cases had CIN III (66.7%). Staging (FIGO) of patients with SCC was IA2 in three cases (10.0%), IB1 in 19 (63.3%), IB2 in two (6.7%) and IIA in six women (20.0%). The activities of enzymes NAG, and MPO and the levels of TNF- were higher in women with CIN compared with the group with SCC. The levels of IFN- were lower in the group of women with CIN compared with the group with SCC. There was not a significant association between the degree of the CIN and the staging of the SCC of the cervix with inflammation assessed by the levels of inflammatory markers used... (Complete abstract click electronic access below)

<p><i>In utero </i>heat stress (<b>IUHS</b>) is a major concern for realizing full production potential in the swine industry. Postnatal phenotypes, such as growth performance, post-absorptive metabolism, and stress response, are negatively altered in pig offspring that have been exposed to IUHS. With current trends in global temperatures predicting a continuation of increased temperatures, it is necessary to further investigate mechanisms driving these altered postnatal phenotypes and to find mitigation strategies to combat the negative effects of IUHS. In a first study, postnatal consequences of IUHS in pigs were evaluated and a mitigation strategy was tested. A second study was conducted to investigate the HPA axis response to a stress challenge in IUHS pigs. The first study found decreased average daily gain in IUHS pigs, and that providing a nutrient-dense diet did not rescue this lost productivity due to a decrease in feed intake for this diet. These results show the importance of maintaining beneficial gestation environments to avoid IUHS and the need to continue looking for alternative strategies to mitigate negative effects of IUHS. In the second study, IUHS pigs had a decreased change in cortisol response (<b>Δ CORT</b>) from baseline when subjected to a corticotropin-releasing hormone (<b>CRH</b>) challenge at 10 wk of age, and 15 wk old pigs had a decreased Δ CORT response when subjected to a dexamethasone suppression test and a CRH challenge as well as decreased glucocorticoid receptor expression in both the hypothalamus and anterior pituitary when compared to 10 wk old pigs. These results show changes in HPA axis function as young pigs mature and that particular focus may need to be put on IUHS pigs at a young age when they might be more vulnerable to negative impacts of stress. Overall, these studies show that IUHS causes a variety of negative postnatal effects in offspring and that a better understanding of mechanisms driving these changes along with developing alternative strategies to combat the incidence of these negative postnatal effects remains of paramount importance for the swine industry.</p>

Yes / Background: Carbon (δ13C) and nitrogen (δ15N) isotope ratios of collagen from teeth and bone are used to study human nutrition and health. As bones are constantly remodelling throughout life, isotopic values of bone collagen represent an average of several years. In contrast, human teeth do not remodel and their primary dentine contains only the isotopic data from the time of formation. In contrast to all other bones, human auditory ossicles also appear not to remodel. As they develop in utero and finish formation in the first 2 years of life, their collagen should also represent isotopic values of these two relatively short periods. Aim: By comparing δ13C and δ15N data from ossicles and incremental dentine, this study aims to investigate how two developmental periods of the ossicles, in utero and the first 2 years of life, reflect in collagen obtained from the ossicles. Subject and methods: Ossicle and tooth samples of 12 individuals aged 0.5 ± 0.4 years to 13 ± 1 years from the nineteenth century St. Peter’s burial ground in Blackburn were collected and processed to obtain bulk bone and incremental dentine collagen which was measured for δ13C and δ15N. Results: Averaged δ13C and δ15N of ossicles are lower when compared to every age group except after 3 years of age. Average offset between ossicles and dentine of different groups ranges from 0.4–0.9‰ for δ13C and from 0.3–0.9‰ for δ15N, with highest counterbalance at birth and after the first 5 months after birth. Conclusions: There appears to be a systematic offset between the dentine and ossicle data. It seems that the second phase of development does not influence the isotopic values of collagen significantly and the data we are obtaining from ossicles represents the in utero period. / Research grant from The Society for the Study of Human Biology.

En la actualidad el cáncer es una enfermedad que está afectando al mundo entero. En el Perú según la oficina de Epidemiología del INEN 2007 las neoplasias malignas más frecuentes en mujeres son las de Cuello uterino y de Mama, presentando altas tasas de incidencia; ante esta situación es importante que el alumno como futuro profesional de enfermería, esté preparado para el desarrollo de actividades preventivo promociónales que contribuyan con la disminución del cáncer en las mujeres. Es por ello que el presente trabajo tiene como objetivo: Determinar el nivel de conocimientos sobre los factores de riesgo y la prevención secundaria del cáncer de Cérvix y de mama en los estudiantes de Enfermería de la UNMSM en el año 2009. El propósito está orientado hacia la creación de estrategias de aprendizaje que enfaticen el conocimiento de los alumnos sobre estos temas a fin de afianzar el rol preventivo promocional sobre el cáncer. El presente trabajo es de tipo cuantitativo, nivel aplicativo, método descriptivo y de corte transversal. Se utilizo como técnica, la entrevista y como instrumento de recolección de datos un cuestionario, el cual fue aplicado a una muestra de 182 estudiantes de enfermería, concluyendo que el nivel de conocimientos que tienen los estudiantes de Enfermería de la UNMSM sobre los factores de riesgo y la prevención secundaria del cáncer de Cérvix y de mama en su mayoría es Medio. / -- At present the cancer is a disease that this affecting the entire world. In Peru according to the office of Epidemiology of the INEN the 2007 malignant but frequent neoplasias in women are those of uterine Neck and Breast, presenting/displaying high rates of incidence; before this situation it is important that the student like professional future of infirmary, is preparation for the development of promotional activities preventive that contribute with the diminution of the cancer in the women. It is for that reason that the present work have like objective: To determine the level of knowledge on the factors of risk and the secondary prevention of the cancer of Cervix and breast in the students of Infirmary of the UNMSM in 2009. The oriented intention this towards the creation of learning strategies that emphasize the knowledge of the students on these subjects in order to strengthen the promotional preventive roll on the cancer. The present work is of quantitative type, aplicativo level, descriptive method and of it cross section. I am used like technique, the interview and like instrument of data collection a questionnaire, which was applied to a sample of 182 students of infirmary, concluding that the level of knowledge which they have the students of Infirmary of the UNMSM on the factors of risk and the secondary prevention of the cancer of Cervix and breast in its majority is Average. / Tesis

En la actualidad el cáncer es una enfermedad que está afectando al mundo entero. En el Perú según la oficina de Epidemiología del INEN 2007 las neoplasias malignas más frecuentes en mujeres son las de Cuello uterino y de Mama, presentando altas tasas de incidencia; ante esta situación es importante que el alumno como futuro profesional de enfermería, esté preparado para el desarrollo de actividades preventivo promociónales que contribuyan con la disminución del cáncer en las mujeres. Es por ello que el presente trabajo tiene como objetivo: Determinar el nivel de conocimientos sobre los factores de riesgo y la prevención secundaria del cáncer de Cérvix y de mama en los estudiantes de Enfermería de la UNMSM en el año 2009. El propósito está orientado hacia la creación de estrategias de aprendizaje que enfaticen el conocimiento de los alumnos sobre estos temas a fin de afianzar el rol preventivo promocional sobre el cáncer. El presente trabajo es de tipo cuantitativo, nivel aplicativo, método descriptivo y de corte transversal. Se utilizo como técnica, la entrevista y como instrumento de recolección de datos un cuestionario, el cual fue aplicado a una muestra de 182 estudiantes de enfermería, concluyendo que el nivel de conocimientos que tienen los estudiantes de Enfermería de la UNMSM sobre los factores de riesgo y la prevención secundaria del cáncer de Cérvix y de mama en su mayoría es Medio. / At present the cancer is a disease that this affecting the entire world. In Peru according to the office of Epidemiology of the INEN the 2007 malignant but frequent neoplasias in women are those of uterine Neck and Breast, presenting/displaying high rates of incidence; before this situation it is important that the student like professional future of infirmary, is preparation for the development of promotional activities preventive that contribute with the diminution of the cancer in the women. It is for that reason that the present work have like objective: To determine the level of knowledge on the factors of risk and the secondary prevention of the cancer of Cervix and breast in the students of Infirmary of the UNMSM in 2009. The oriented intention this towards the creation of learning strategies that emphasize the knowledge of the students on these subjects in order to strengthen the promotional preventive roll on the cancer. The present work is of quantitative type, aplicativo level, descriptive method and of it cross section. I am used like technique, the interview and like instrument of data collection a questionnaire, which was applied to a sample of 182 students of infirmary, concluding that the level of knowledge which they have the students of Infirmary of the UNMSM on the factors of risk and the secondary prevention of the cancer of Cervix and breast in its majority is Average.

Einführung: Eine entscheidende Ursache für das Aufkommen der den modernen Menschen charakterisierenden kognitiven Funktionen ist in der beachtlichen Vergrößerung des menschlichen Neocortex innerhalb der letzten 5-7 Millionen Jahre zu finden. Die Identifizierung der dieser Entwicklung zu Grunde liegenden genomischen Veränderungen ist letztlich nicht nur bedeutsam für die Beantwortung der Frage, welche evolutionären Anpassungen den Menschen kennzeichnen, sondern auch für ein besseres Verständnis einer möglicherweise besonderen Anfälligkeit gegenüber neurologischen und psychiatrischen Erkrankungen. Kürzlich konnten 15 menschenspezifische Gene, deren Expression sich vorzugsweise in neuronalen Vorläuferzellen (NPCs) des menschlichen fetalen Neokortexes nachweisen lässt, identifiziert werden (Florio et al., 2018). Drei davon (FAM72B, C und D) sind vor 3,4 – 1 Millionen Jahren im menschlichen Genom durch Genduplikationen entstanden und gehören zur Family of sequence similarity 72 (FAM72). Zielsetzung und Ansätze: Konkret wurde betrachtet, ob FAM72D durch die spezifischen Substitutionen von Aminosäuren eine sich von der Funktion des anzestralen Gens FAM72A unterscheidende Rolle in der neokortikalen Entwicklung einnimmt. Untersucht wurden deshalb die Effekte von FAM72A und D auf die Proliferationskapazität und Genexpression von NPCs nach der ektopen Expression von FAM72A oder D während der embryonalen Entwicklung des Neocortex der Maus. Methoden: Die in utero Elektroporation (IUE) embryonaler Mäusegehirne erfolgte zur Expression eines rot oder grün fluoreszierenden Proteins (RFP oder GFP) entweder gemeinsam mit einem leeren DNA pCAGGS Vektor als Kontrollbedingung oder aber einem pCAGGS-FAM72A oder pCAGGS-FAM72D Plasmid. Die in der zweiten Ergebnissektion (Results II) präsentierten IUE wurden dabei im dorsolateralen Neokortex zum Höhepunkt der Neurogenese am 14. Entwicklungstag (E 14.5) durchgeführt, im Unterschied zu den Experimenten in der dritten Sektion (Results III), die im medialen Neokortex am 18. Entwicklungstag (E 18.5) während der Spätphase der embryonalen Neurogenese realisiert wurden. Die Proliferation der NPCs wurde durch Immunfluoreszenzanalysen zweier Marker (Ki67 und phosphoryliertes Histon 3) bestimmt. Zudem wurde die Häufigkeit wichtiger Subtypen von NPCs ebenfalls durch Immunfluoreszenzanalysen eines Markers für basale intermediäre Vorläuferzellen (bIPs → Tbr2) sowie für basale und apikale radiale Gliazellen (aRGs, bRGs → Sox2) ermittelt. Die Gliogenese wurde durch Olig2 Immunfluoreszenz quantifiziert. Weitere Experimente wurden durchgeführt, um die Fähigkeit der NPCs, den Zellzyklus nach der IUE von FAM72D erneut einzuleiten, zu untersuchen. Zu diesem Zweck wurde schwangeren Mäusen 24 h nach der IUE das Thymidin-Analogon 5-Ethinyl-2'-desoxyuridin (EdU) intraperitoneal injiziert. Damit wurden alle Zellen markiert, die sich zu diesem Zeitpunkt in der S-Phase des Zellzyklus befanden und damit den Zellzyklus nach der IUE fortsetzten. Nach weiteren 24 h (48 h post-IUE) erfolgte die Auswertung: alle Ki67- und EdU-doppelt positiven Zellen wurden als solche betrachtet, die den Zellzyklus nach IUE fortführten (EdU+) und nach weiteren 24 h noch immer proliferierten (Ki67+). Zur Durchführung der Transkriptomanalyse wurden Mäuse am 13. Entwicklungstag mit pCAGGS-GFP und entweder dem leeren DNA-Vektor (pCAGGS, Kontrolle) oder einem die Expression von FAM72A (pCAGGS-FAM72A) oder FAM72D (pCAGGS-FAM72D) ermöglichenden Vektor elektroporiert. Anschließend wurden die elektroporierten dorsolateralen neokortikalen Bereiche am 14. Entwicklungstag mikroskopisch seziert und in einzelne Zellen dissoziiert. Die Isolation der elektroporierten (GFP+) Zellen erfolgte aus den Einzelzellsuspensionen durch Fluoreszenz-aktivierte Zellsortierung (FACS). Im Anschluss wurden die isolierten Zellen für die RNA-Sequenzierung vorbereitet. Die primäre Datenanalyse der Ergebnisse der RNA-Sequenzierung wurde entsprechend etablierter Protokolle durchgeführt (Florio et al., 2015). Ergebnisse: Die Analyse der Immunfluoreszenzquanitfizierungen (Results II und III) ergab keine signifikanten Veränderungen der proliferativen Parameter oder der Häufigkeit der NPCs in der ventrikulären Zone (VZ) oder subventrikulären Zone (SVZ) des sich entwickelnden Mausneokortex nach der ektopen Expression von FAM72A oder FAM72D im Vergleich zur Kontrollbedingung. Die Transkriptomanalyse (Results IV) zeigte jedoch 88 signifikant hoch- und 52 herunterregulierte Gene in Folge der FAM72A sowie 91 signifikant hoch- und 67 herunterregulierte Gene nach der FAM72D Expression im Vergleich zur Kontrolle. Es wurde festgestellt, dass nur zwei dieser differentiell exprimierten Gene in Folge der ektopen Expression sowohl von FAM72A als auch FAM72D hochreguliert wurden und ein Expressionslevel > 1 fpkm aufwiesen: Syde1 und Shisa5. Darüber hinaus wurden sechs Gene mit > 1 fpkm identifiziert, die spezifisch nach der Expression von FAM72D hochreguliert waren: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Darüber hinaus zeigte die Genontologie-Analyse (Gen Ontology) eine signifikante Anreicherung von Angiogenese-assoziierten Genen (z. B. Vegfc) im Datensatz der artifiziell FAM72A exprimierenden Zellen. Interessanterweise konnte beobachtet werden, dass unter den im Vergleich zur Kontrolle differentiell exprimierten Genen mehr Gene mit typischer Expression in NPCs in Folge von FAM72D als FAM72A Expression hochreguliert und mehr NPC typische Gene nach FAM72A Expression herunterreguliert wurden. Im Falle der Gene, deren Expression eher in Neuronen zu finden ist, zeigte sich ein entgegengesetztes Bild (Results IV). Diese Befunde lassen den vorsichtigen Schluss zu, dass FAM72D stärker als FAM72A die Aufrechterhaltung von NPC-Eigenschaften positiv beeinflussen kann. Schlussfolgerungen: In einer früheren Studie erhöhte der Knockdown von Fam72a in NPCs erwachsener Mäuse die Neurogenese (Benayoun et al., 2014). Dies legt in Verbindung mit den vorliegenden Ergebnissen nahe, dass FAM72A und FAM72D nicht hinreichend, möglicherweise jedoch notwendig sind, um die Aufrechterhaltung des Vorläuferzellcharakters von NPCs zu fördern (Results II, III). Aus diesem Grund sollte das in dieser Studie verfolgte Gain of Function Design durch einen Loss of Function Ansatz ergänzt werden. Als Modellsystem bieten sich hierfür insbesondere Hirnorganoide aus Stammzellen des Schimpansen oder Menschen an. Da alle der kürzlich identifizierten menschenspezifischen Gene in den gleichen NPCs exprimiert werden, sollte auch die potenzielle synergistische Wirkung auf die NPC-Proliferation der FAM72 und der zwölf anderen humanspezifischen Gene wie etwa ARHGAP11B analysiert werden. Neben anderen möglichen Mechanismen, die auf Grundlage der Genexpressionsanalyse im Diskussionsteil dieser Arbeit (Results IV und Discussion) erörtert wurden, könnte die Hochregulierung von Slitrk5 in Folge der ektopen Expression des humanspezifischen FAM72D besonders relevant sein. Es ist bekannt, dass Slitrk5 am Recycling des TrKB-Rezeptors beteiligt ist (Song et al., 2015), der wiederum grundlegende Aspekte der Gehirnentwicklung beeinflusst. Ebenfalls konnte bereits gezeigt werden, dass FAM72A die Funktion des TrKB Rezeptors hemmt (Nehar et al., 2009). Somit ist denkbar, dass FAM72D im menschlichen Neokortex die Wiederherstellung der TrKB-Rezeptorfunktion indirekt über Slitrk5 verbessert und dadurch wesentliche Parameter wie das Überleben von Vorläuferzellen und die Neurogenese beim Menschen verlängern oder verstärken könnte. Diese Studie stellt damit die erste funktionelle Charakterisierung der evolutionär hochinteressanten, die FAM72 Gene beinhaltende Region des menschlichen Genoms während der Entwicklung in utero dar. Daraus ergeben sich zahlreiche Ansatzpunkte für zukünftige Untersuchungen, die in ihrer Gesamtheit ein umfassendes Verständnis der Evolution des menschlichen Gehirns ermöglichen werden.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146 / Introduction: The higher cognitive functions that characterize modern humans can be attributed to the cerebral neocortex and its remarkable expansion in size during the last 5 – 7 million years of human evolution. The identification of the underlying genomic changes will be not only of importance to better understand the unique complexity of the human brain, but also its susceptibility to neurological and psychiatric diseases. Recently, 15 human-specific genes preferentially expressed in neural progenitor cells (NPCs) of the human fetal neocortex were identified (Florio et al., 2018). Three of them, FAM72B, C and D belong to the Family of sequence similarity 72 (FAM72) and occurred in the human genome by gene duplication 3.4 – 1 mya. Aims & Approaches: Specifically, it was asked whether FAM72D plays a diverse role compared to the ancestral FAM72A (Results II, III, IV) due to the specific sets of amino acid substitutions it acquired (Results I). Effects of FAM72A and FAM72D on the proliferative capacity and gene expressions of embryonic mouse NPCs were analyzed upon ectopic expression either of FAM72A or FAM72D during embryonic mouse neocortical development. Methods: In utero electroporation (IUE) of embryonic mouse brains was performed to drive the expression of a red or green fluorescent protein (RFP or GFP) either plus empty DNA vector (pCAGGS; control), pCAGGS-FAM72A or pCAGGS-FAM72D plasmids in the dorsolateral neocortex at mid-neurogenesis (embryonic day 13.5, E13.5; Results II) or in the medial neocortex at late-neurogenesis (E15.5; Results III). NPC proliferation was evaluated by immunofluorescence of Ki67 (immunohistochemistry, IHC), a cell proliferation marker, and phosphorylated Histone H3 (PH3), a marker of cell mitosis. Moreover, the abundance of NPCs using immunofluorescence of basal intermediate progenitor (Tbr2) and apical and basal radial glia (Sox2) markers, and the gliogenesis by Olig2 immunofluorescence was analyzed. Additional experiments were carried out to study the capacity of NPCs to reenter the cell cycle upon IUE of FAM72D. To this end, pregnant mice were intraperitoneally injected with the thymidine analog 5-Ethynyl-2´-deoxyuridine (EdU) 24 h post-IUE, to label all cells undergoing S-phase of the cell cycle (i.e., all cells that reentered the cell cycle after IUE) in the developing mouse brains. Embryonic brains were collected 24 h after EdU injection and co-stained with Ki67. Ki67 and EdU double positive cells were considered as cells that reentered the cell cycle. To execute the transcriptome analysis E13.5 mice were electroporated with pCAGGS-GFP either plus an empty DNA vector (pCAGGS, control), a vector driving expression of FAM72A (pCAGGS-FAM72A) or FAM72D (pCAGGS-FAM72D). Subsequently, the electroporated dorsolateral neocortical areas were microdissected at E14.5 and dissociated into single cells. The electroporated (GFP+) cells were isolated from the single cell suspensions by the fluorescence-activated cell sorting (FACS). The isolated cells were processed for RNA sequencing. Data analysis was performed as previously reported (Florio et al., 2015). Results: By immunohistochemistry, no significant changes in any of the proliferative parameters or in the abundance of progenitors in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing mouse neocortex upon ectopic expression of FAM72D compared to FAM72A and control samples were detected (Results II, III). However, the transcriptome analysis (Results IV) showed 88 significantly up- and 52 down-regulated genes upon FAM72A and 91 significantly up- and 67 downregulated genes upon FAM72D expression compared to the control. Only two of these differentially expressed genes were found to be upregulated upon FAM72A and FAM72D with an expression >1 fpkm: Syde1 and Shisa5. Besides, six genes specifically upregulated upon ectopic expression of FAM72D exhibiting fpkm > 1 were identified and characterized using the existing literature: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Beyond that, gene ontology analysis showed significant enrichment of angiogenesis-related genes (e.g., Vegfc) upon FAM72A expression. Interestingly, there were more genes found to be enriched in NPCs that were upregulated compared to control upon FAM72D than FAM72A expression, but more NPC enriched genes downregulated upon FAM72A compared to FAM72D expression. In the case of differentially expressed neuron-enriched genes, the data was were inverse, which slightly supports the idea that FAM72D rather than FAM72A could positively affect the maintenance of NPC characteristics. Conclusions: In a previous study knockdown of Fam72a in adult mouse NPCs increased neurogenesis (Benayoun et al., 2014). This suggests, in conjunction with the present results, that FAM72A and FAM72D are not sufficient, but may be required, to promote NPC maintenance (Results II, III). This is why the gain of function experiments conducted in this study should be complemented by a loss of function approach in the developing mouse neocortex, in chimpanzee or human-derived brain organoids. Because of their expression in the NPCs of the developing human neocortex, it might be productive to analyze the potential synergistic effect on NPC proliferation of the FAM72s and the 12 other human-specific genes such as ARHGAP11B. Among other mechanisms discussed based on the gene expression analysis in this thesis (Results IV and Discussion), the upregulation of Slitrk5 upon ectopic expression of the human-specific FAM72D could be particularly remarkable. Slitrk5 is known to be involved in the recycling of the TrKB receptor (Song et al., 2015), which affects fundamental aspects of brain development. While FAM72A was found to inhibit the TrKB receptor (Nehar et al., 2009), the occurrence of FAM72D could indirectly rescue the TrKB receptor function via Slitrk5 and thereby prolonging or enhancing essential features such as precursor cell survival and neurogenesis in humans. Therefore, this study provides the first functional characterization of the evolutionary highly interesting region in the human genome comprising the FAM72 genes during embryonic neocortical development in vivo and offers numerous starting points for further investigations, that will collectively facilitate a comprehensive understanding of the genomic adaptations underlying the astonishing evolution of the human brain.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146