Dissertations / Theses on the topic 'Human fetal testis development'

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d'anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l'hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l'environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l'Homme. Dans cette étude, nous avons analysé les effets d'un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l'organe. Nous avons tout d'abord démontré que le MEHP (10-4M) n'altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l'exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l'effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l'exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d'action des phtalates. Différentes études, notamment dans le foie, démontrent l'implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l'expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l'implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l'effet sur les cellules germinales mâles ai pu être mis en évidence dans l'ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras.

Dysregulation of placental and fetal epigenetics can affect gene expression patterns, including the parent-of-origin dependent expression in imprinted genes. While defects of imprinted genes have been implicated in some adverse pregnancy outcomes, little is currently known about the role of epigenetics in regulating normal or pathological human pregnancy and development. The objective of this thesis is to provide fundamental DNA methylation profiles of human fetal and placental development so as to offer insights into the etiology of human disease and adverse pregnancy outcomes. Taking advantage of the unbalanced parental genomic constitutions in triploidies, 45 novel imprinted genes were identified by comparing the genome-wide DNA methylation profiles between 10 diandries and 10 digynies. A comparison of DNA methylation profiles between placentas of different gestations and other somatic tissues showed tissue-specific and gestational age-specific DNA methylation changes in many imprinted genes. To gain insight into the genomic pattern of tissue-specific methylation, DNA methylation profile was evaluated in 5 somatic tissues (brain, kidney, lung, muscle and skin) from eight normal second-trimester fetuses. Tissue-specific differentially methylated regions (tDMRs) were identified in 195 loci, suggesting that tissue-specific methylation is established early in the second trimester. Importantly, only 17% of the identified fetal tDMRs were found to maintain this same tissue-specific methylation in adult tissues, implicating an extensive epigenetic reprogramming between fetus and adult. Besides intra-individual differences, there is also substantial DNA methylation variation between individuals. While many sites show a continuous pattern of DNA methylation variation between different placentas, WNT2, TUSC3 and EPHB4 were identified to have epipolymorphisms at their promoter region. The methylation status at the TUSC3 promoter showed an association with preeclampsia, suggesting a role of DNA methylation change in adverse pregnancy outcomes. A further investigation of DNA methylation profiles in 26 placentas from preeclampsia, IUGR and control subjects showed 34 loci were hypomethylated in the early-onset preeclamptic placentas, with TIMP3 having a potential of being a biomarker for the disorder. These results provided comprehensive DNA methylation profiles for both normal and abnormal fetal and placental tissues, which contribute to the biological and clinical aspects of the pathogenesis of fetal and placental disorders.

γδ T cells are unconventional T cells that that can recognize infected and transformed cells via their γẟ TCR, thus promoting different immune responses. In addition, several studies showed that γδ T cells are important in the protection against different pathogens in early life, such as human cytomegalovirus (CMV). The diversity of the γδ TCR repertoire is mainly generated in the complementarity determining region 3 (CDR3) where V(D)J recombination takes place. One of the main players in the junctional diversity is the terminal-deoxynucleotidyl-transferase (TdT) enzyme responsible for the random template-independent nucleotide addition at the junction of the joining gene segments.In the mouse model it is established that during development, especially before birth, innate γδ T cell subsets are generated in waves and their generation depends on the type of hematopoietic stem and precursor cells (HSPC). These γδ T cells express a semi-invariant γδ TCR and can acquire a functional program already in the thymus. In human, in contrast, the idea of γδ T cells as innate-like lymphocytes is questioned by recent works showing that the γδ TCR repertoire of human pediatric thymuses and of term-delivery cord blood is highly diverse. Here, by analyzing in detail human fetal and post-natal thymi, we observed striking differences between fetal and post-natal γδ thymocytes at the γδ TCR repertoire and functional level. In contrast to post-natal γδ thymocytes, fetal γδ thymocytes were functionally programmed, expressed low levels of TdT and were highly enriched for invariant/public CMV-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). The rearrangements of these invariant sequences were driven by short-homology repeats at the end of the involved gene segments, as it was observed in the mouse. In addition, we investigated the role of HSPC in the generation of this invariant γδ thymocytes by using an in vitro T cell development system and we showed that only fetal HSPC could generate γδ T cells enriched for the same specific features that were found in the ex-vivo fetal γδ thymocytes. Moreover, we showed that the RNA-binding protein Lin28b, highly expressed in fetal γδ T cells, reprogrammed the term delivery HSPC towards the generation of γδ T cells resembling to their fetal counterpart.In conclusion, we show that the human fetal thymus generates, in a HSPC- and Lin28b-dependent manner, innate invariant γδ T cells with programmed effector functions that might provide protection to the fetus during congenital infections, such as against CMV.
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished

During gonad development and fetal life, the germ cells (GC) undergo a range of different developmental processes necessary for correct postnatal gametogenesis and the production of the next generation. If these fetal events are disrupted by genetic or environmental factors, there could be severe consequences that may not present until adulthood. This is of particular importance in relation to human testicular GC tumours (TGCT), the most common cancer of young men, as TGCT is thought to arise from fetal GCs that have failed to differentiate normally during development and thus persist into adulthood, eventually becoming tumourigenic. TGCT is one of several related disorders of male reproductive health thought to comprise a Testicular Dysgenesis Syndrome (TDS), in which faulty testis development in fetal life may predispose to the development of cryptorchidism, hypospadias, reduced sperm count and TGCT. Currently there is no accepted animal model for TGCT, but some insight into human TDS has been gained through the use of a rat model using in utero Di (n-Butyl) Phthalate (DBP) exposure to induce cryptorchidism, hypospadias, low sperm count and reduced fertility (but not TGCT). However, a previous study suggested that DBP exposure can disrupt GC differentiation, resulting in significantly reduced GC number prior to birth and postnatal consequences. This thesis has been directed at investigating the normal process of GC development in the fetal rat and how this is altered by DBP exposure; such understanding may give insights into the origins of human TGCT by showing how and when disruption of normal fetal GC differentiation can occur. The first objective was to characterise GC development in both the rat testis and ovary to understand the normal events that occur between embryonic day (e)13.5 and e21.5, as most data in the literature is based on the mouse. Analysis by immunohistochemical, stereological and mRNA expression indentified that during this time period, a GC will undergo a dynamic sequence of changes involving migration, proliferation followed by differentiation (manifested by loss of specific protein markers), whilst undergoing germ-line specific remethylation. Whilst whole gonad development is vastly different between testes and ovaries, GC development was broadly the same with only minor differences up to the point where GCs in the ovary enter meiosis. Having established the normal process of GC development in the fetal rat testis, the effects of in utero DBP exposure was then investigated. DBP exposure reduced GC number at all ages investigated even after only 24 hours of exposure and simultaneously prolonged GC proliferation. As apoptosis was unaltered by DBP exposure, the consistent reduction in GC number was suggested to be due to an initial reduction in GC number that does not recover to control levels. GC differentiation was assessed by the expression and localization of specific protein markers (OCT4, DMRT1 and DAZL). The pattern of expression of OCT4 and DMRT1 was altered by DBP exposure. GCs in DBP exposed animals also showed a delay in disaggregation from within the centre of seminiferous cords. These results suggested that a delay in GC differentiation was occurring with DBP exposure. This delay in GC development persisted into early postnatal life, following cessation of DBP exposure. Thus at postnatal day (D)6, GC specific re-expression of DMRT1, GC migration to the basal lamina and resumption of GC proliferation all showed a delay. DBP also induced an increase in the presence of multinucleated gonocytes. DNA methylation in the fetal rat testis was also investigated as a mechanism that could be disrupted by DBP exposure. DNA methylation of GCs increased during the last week of fetal life by global methylation of the GC genome and the increased expression of DNA methyl transferases. No effect of DBP exposure was detected. Inhibition of methylation by 5-aza-2’deoxycytidine was then investigated as a way to block GC differentiation in fetal rat testes and this resulted in a similar transient delay in GC differentiation but was perinatally lethal to the fetus. Bisulphite sequencing of the OCT4 promoter was also performed but proved inconclusive. Methylation patterns may be being altered by DBP exposure, but such changes could not be identified in this thesis. To complement the in vivo DBP exposure studies, an in vitro testis explant system using e14.5 testes was investigated. These in vitro testis explants showed some GC effects with MBP, the active metabolite of DBP, and also suggested a novel role for Hedgehog signalling in GC survival in the fetal rat testis. The studies in this thesis have characterised several aspects of fetal GC development in the rat and identified which of these are affected by DBP exposure, resulting in a delay in GC development. As DBP exposure delays but does not block GC differentiation, this may explain why TGCT is not induced in the DBP exposure rat model for TDS.

Thesis (Ph. D.)--University of Glasgow, 2003.
Ph. D. thesis submitted to the Faculty of Veterinary Medicine, University of Glasgow, 2003. Includes bibliographical references. Electronic version also available via Glasgow University e-Theses service.

The mechanisms underlying the high incidence of fetal abnormalities including fetal lung immaturity during maternal diabetes are not fully understood. Utilizing streptozotocin-diabetic rats as the model, I have examined the role of fetal hyperglycemia and hyperinsulinemia and other factors on fetal adrenal and lung functions in culture. Insulin and glucose did not alter fetal adrenal and lung cell proliferation and adrenal corticosterone output. On the other hand, a novel protein-bound, low molecular weight non-proteinaceous cytotoxic factor was detected in the serum of diabetic animals. In addition, a novel protein with cytostatic activity was found in fetal lungs, the concentration of which increased during diabetes. Partial amino acid sequence and Western Blot analysis revealed this protein to be similar to histone H2B. An extra-nuclear role is suggested for this protein because it appears to be present in the microsomal fraction of fetal lungs. It is concluded that fetal lung immaturity during diabetes may be contributed by cytotoxic and cytostatic factors contained in the serum and fetal lungs, respectively.

The identification of tumour antigens (TAs) represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. A group of proteins, the cancer testis (CT) antigens are promising targets for such clinical applications. Their encoding genes show expression restricted to the immunologically privileged testes but their expression is also found in cells with a cancerous phenotype. To facilitate and automate the identification of novel CT genes, bioinformatic analytical pipelines based on publicly available microarray and expressed sequence tag (EST) data were developed and implemented as web tools to support wider application. Human germline-associated datasets were generated and the developed screening pipelines were subsequently used to analyse these datasets, leading to the identification of a. novel cohort of meiosis-speci fic genes, the meiCT genes that exhibit t he characteristics of CT genes and may have oncogenic features. In general, frequent germline gene expression found in cancer could reflect a soma-to-germline transformation occurring in human cells in the course of the development of cancer. The expression of germline-specific genes, in particular of meiotic genes, could lead to the production of proteins that cause oncogenic events and thus contribute to tumorigenesis and to the acquisition of tumour characteristics.

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

Ultrasound is a non-invasive diagnostic tool that could provide important information about pulmonary maturity. Quantitative ultrasound techniques that relate lung tissue acoustic properties, i.e. acoustic velocity, attenuation, and scattering, to its physical properties, such as, elastic and structural properties, have been reviewed. B-mode techniques were equipment dependent and produced conflicting results. A-mode techniques are less dependent on the measuring equipment and could provide more accurate information about lung development. A-node techniques were applied to the study of regional differences within the fetal lung. The accuracy of these techniques could be instrumental to the eventual determination of lung maturation. To pursue this possibility, the alveolar regions across the upper, middle, and lower lobes of physiologically mature normal preterm lamb lungs were scanned to determine their acoustic properties within the frequency range of 1-15 MHz. Average speed of sound, attenuation and size of backscatterers were found to be independent of lung regions. Comparison between the lung mean size of backscatterers and mean alveolar sac diameter, histologically measured from the whole lung, showed that these data were not statistically different. This suggested that the collagen rich alveolar sac septal walls were the principal sources of scattering. Histological measurements on the size of alveolar sacs across different regions of the lung were also independent of the lung regions. The results of this study on the fetal lamb lungs suggested that A-mode ultrasound is sensitive to lung developmental changes. The ability of A-mode ultrasound to determine lung maturity appears promising. Further experiments on regional lung development and lung maturation at the pre and post-surfactant synthesis stages of the gestational life may establish the basis for an accurate and risk-free ultrasound assessment of lung maturation that is reliable in a clinical setting

The aim of the experimental work described in this thesis was to investigate the effects of a ubiquitous environmental contaminant, the synthetic platiciser Di-butylphthalate (DBP) on the developing male reproductive tract. Observed changes were compared to those evident in the human males Testicular dysgenesis syndrome (TDS), which is increasing in incidence. To investigate whether DBP treatment induced any TDS-like changes, pregnant rats were gavaged daily with DBP at doses up to 500mg/kg/day from embryonic (e) day e13.5 up to e21.5. Morphological and hormonal parameters (testosterone, inhibin-B) were then assessed in male rats aged e21.5 or in adulthood ± in utero DBP exposure. Concurrently, untreated fetal rat and fetal human testis explants were cultured in vitro ± MBP (Mono-butylphthalate), the DBP metabolite, at levels of up to 1mM, to investigate whether the array of adverse effects seen with the in utero compared ± MBP exposure. The in vitro experiments were restricted to 48h exposure duration, so an additional in vivo treatment regime was established to compare the endopoints induced after just 48h exposure to DBP in utero. Pregnant rats were dosed daily by gavage with 500mg DBP/kg/day on e19.5 and e20.5 only. Morphological and hormonal parameters (testosterone, inhibin-B) were then assessed in male rats aged e21.5 ± 48h. Findings were compared with those from the original in utero studies in which DBP exposure spanned the period e13.5-e20.5. The long-term in utero exposure regime induced an array of changes in the phenotype of the male reproductive tract, evident in e21.5 and adult animals, including testis maldescent (cryptorchidism) and reduced fertility. Changes in testis morphology such as alteration in the distribution of Leydig cells and induction of multinucleated gonocytes at e21.5 were also seen. The production of testosterone in testes at e21.5 was also significantly reduced following the DBP treatment, including a significant reduction in the protein expression levels of the steroidogenic enzyme P450scc. The short-term in utero exposure to DBP regime induced a generally less severe array of changes in the testis at e21.5 male reproductive tract than those seen after 8-day in utero DBP exposure. However, a greater reduction in testis testosterone was seen after short-term exposure than long-term exposure induced, despite less of a reduction in the expression of the steroidogenic protein P450scc. The precise mechanism through which DBP induces its array of developmental abnormalities is still unclear but these studies support the hypothesis that even short term in utero exposure to DBP directly affects the developing testis, probably by acting on Leydig cells and disrupting normal testis endocrinology.

Orientador: Patricia Fernanda Felipe Pinheiro
Resumo: Introdução: A hipótese da “programação fetal” defende que eventos ocorridos durante a vida intrauterina exerçam influência na patogênese de doenças na vida adulta. Fatores ambientais podem programar no indivíduo o surgimento precoce de doenças cardiovasculares e metabólicas. Atualmente, há um aumento no consumo de adoçantes artificiais associados a tratamentos para a perda de peso e no controle do diabetes, sendo a sacarina sódica um dos mais consumidos. Entretanto, durante a gestação e lactação, o uso de sacarina sódica é restrito, por ser permeável a placenta, interagindo com o concepto e, por compor o leite materno. Embora os efeitos do uso de adoçantes sobre o peso corpóreo e o metabolismo sejam bastante conhecidos, não há relatos de pesquisas que relacionam a programação fetal pelo uso de sacarina sódica com o desenvolvimento pós-natal do testículo. Desta forma, o presente estudo visa investigar a influência do uso da sacarina sódica e da glicose na saúde materna e reprodutiva dos descendentes machos. Material e métodos: Ratas Sprague Dawley foram alimentadas durante a prenhez e lactação com dieta padrão para roedores, água filtrada ad libitum e suplementadas com iogurte natural desnatado (Grupo Controle Iogurte, n= 9); iogurte natural desnatado adoçado com solução de glicose (Dinâmica®) a 5% (v/v) (Grupo Glicose, n= 10); iogurte natural desnatado adoçado com solução de sacarina sódica (Dinâmica®) a 0,3% (v/v) (Grupo Sacarina Sódica, n= 10). As dietas líquidas foram prep... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Introduction: The "fetal programming" hypothesis argues that events that occur during intrauterine life have an influence on the pathogenesis of diseases in adulthood. Environmental factors can program in the individual the early onset of cardiovascular and metabolic diseases. Currently, there is an increase in the consumption of artificial sweeteners associated with treatments for weight loss and diabetes control, with sodium saccharin being one of the most consumed. However, during pregnancy and lactation, the use of sodium saccharin is restricted, since it is permeable to the placenta, interacting with the conceptus, and for composing breast milk. Although the effects of the use of sweeteners on the body weight and metabolism are well known, there are no reports of researches which relate fetal programming through the use of sodium saccharin to the postnatal development of the testis. Thus, the present study aims to investigate the influence of the use of sodium saccharin and glucose on the maternal and reproductive health of male offspring. Material and methods: Dams Sprague-Dawley rats were fed during pregnancy and lactation with a standard chow for rodents, filtered water ad libitum and supplemented with low-fat plain yogurt (Yogurt Control Group, n = 9); low-fat plain yogurt sweetened with 5% (v/v) glucose solution (Dinâmica®) (Glucose Group, n = 10); low-fat plain yogurt sweetened with 0.3% (v/v) sodium saccharin solution (Dinâmica®) (Sodium Saccharin Group, n = 10). ... (Complete abstract click electronic access below)
Mestre

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (leaves 66-75).
Eight distinct nuclear shapes, or morphologies, have been discovered in human proto-organs and tumors, including bell-shaped nuclei with stem-like properties. These bell-shaped, or "metakaryotic," nuclei are abundant in fetal tissues and neoplasias, but rare in normal adult somatic tissues. Metakaryotic nuclei employ an unusual process for division in which DNA synthesis, partial genomic condensation, and separation of the two nuclei in a cup-from-cup fashion occur concurrently, as shown by Feulgen densitometry and single-stranded DNA assays by Dr. Elena Gostjeva. This is clearly different from the sequential steps of S-phase DNA synthesis, chromatin condensation, chromosomal separation, and genomic segregation that occur in mitotic eukaryotic cells. In order to discover how a genome apparently devoid of chromosomes might be organized, this thesis focused on recognizable DNA sequences common to all chromosomes: centromeres and telomeres. Fluorescence In Situ Hybridization (FISH) with pan-centromeric and pan-telomeric probes was applied to samples of human tissue. (A collaborating lab used centromeric and telomeric antibodies to confirm results.) An optimized FISH protocol was developed specifically for metakaryotic nuclei and tested in both human cell lines and eukaryotic cells as experimental controls. Staining of metakaryotic nuclei resulted in approximately 23 centromeric regions in each, unlike the expected number of 46 regions seen in eukaryotic nuclei. Many of these staining regions contained paired centromere signals, or doublets. This suggested a genomic organization of homologous chromosomes paired at their centromere regions. If this were the case, one would expect 46 telomeric signals per nuclei, if telomeres were also homologously paired.
(cont.) Unexpectedly, an average of 23 telomeric regions were found in many, if not all, bell-shaped metakaryotic nuclei. This, along with the observation of a condensed double ring around the mouth of the bell-shaped nuclei, suggested the possibility of a genome organized as paired, continuous genomic circles. Studies of telomere joining in metakaryotic nuclei by Dr. Per Olaf Ekstrom have provided further evidence for the paired genomic circle model. The results in this thesis are an original contribution to the field of stem cell physiology, a starting point for further investigation of DNA organization, synthesis, and repair in these metakaryotic cells, and hopefully will lead to a greater understanding of human development, growth, and cancer.
by Amanda Natalie Gruhl.
Ph.D.

Imaging the fetal brain in utero is challenging due to the unpredictable motion of the fetus. Although ultra-fast MRI sequences are able to image a 2D slice in under a second, thus limiting the time in which fetal motion can corrupt images, Cartesian sampling makes these sequences sensitive to signal misregistration and motion-corruption. Corruption of a single 2D slice renders it impossible to reconstruct 3D volumes from these slices without complex slice-to-volume registration. There is a need for motion-robust sequences that can produce high-resolution 3D volumes of the fetal brain. The Siemens Cardiovascular sequence was edited to produce a new radial readout that sampled a 3D spherical volume of k-space with successive diametric spokes. The diameter end points map a spiral trajectory on the surface of a sphere. The trajectory was modified so that multiple sub-volumes of data are sampled during a single acquisition where M is the number of sub-spirals and N is the number of diametric spokes per sub-spiral. This allows reconstruction of individual sub-volumes of data to produce a series of low-resolution navigator images that can be co-registered to provide information on motion during the acquisition. In this way, a segmented sequence suited to self-navigation was developed. Imaging parameters for the 3D radial sequence were optimised based on theoretical calculations and scans performed in adult brains and abdomens. Optimum values for M and N needed to be determined. Increasing M for a constant total number of projections improves the temporal accuracy of motion tracking at the expense of decreased signal to noise ratio in the navigator images. The effects of breathing and rigid body motion on image quality were also compared between 3D radial and equivalent 3D Cartesian acquisitions. Custom reconstruction code was written to separate the incoming scan data according to the sub-spiral trajectories described within the sequence such that individual navigator images could be reconstructed. Successive sub-spiral images were co-registered to the first navigator image to quantify motion during the acquisition. The resulting transformation matrices were then applied to each sub-spiral image after reconstruction and co-registered sub-spiral images combined in image space to generate the final 3D volume. To improve the quality of navigator images, a method is presented to perform navigator image reconstruction at a lower base resolution, thus reducing streaking artifacts and improving the accuracy of image co-registrations. Finally, the methods developed were applied to two fetal scans. The radial sequence was shown to be more motion-robust than an equivalent Cartesian sequence. The minimum number of diametric spokes that provided navigator images that could be accurately co-registered when scanning an adult brain was N=256, which could be acquired in 1.25 s. For abdominal scans, the minimum number of spokes was N=1024, which could be acquired in about 6 s when water excitation is applied. However, the latter could potentially be reduced by reconstructing navigator images at a lower base resolution. Although fetal scans demonstrated poor image contrast, navigator images were able to track motion during the acquisition demonstrating the potential use of this method for self-navigation. In conclusion, a motion-robust radial sequence is presented with potential applications for prospective navigation during fetal MRI.

T cell development is regulated by signals generated in the interactions between developing thymocytes and the thymic stroma. Using fetal thymus organ culture (FTOC) as a model of T cell development, we investigated the ability of two potent signal modulators to influence this process. These studies show that both nicotine and tumor necrosis factor-alpha have the ability to influence T cell receptor (TCR) signaling and the maturational capacity of treated cultures. FTOC treated with low concentrations of nicotine produced more immature T cells and fewer mature T cells. These expanded populations of cells also expressed CD69, CD95 (FAS) and elevated levels of recombinase activating genes (RAG). This phenotype reflects the fact that these cells have received a positive selection signal, are for apoptosis and are likely attempting secondary TCR rearrangements. Nicotine effects were partially blocked by the nicotinic antagonist, d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of T cells entirely, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors and regulate normal thymopoeisis. These observations underscore the linkage between the nervous and the immune systems, not only in terms of shared resources, but also in terms of direct interactions between these two systems. In another study we used FTOC and an associated in vitro Type 1 diabetes mellitus model to reconcile the role of TNF-alpha in thymopoiesis with its role in diabetes. Our data indicate that thymocytes from NOD FTOC express lower levels of TNF receptors and produce more TNF-alpha compared to non-diabetic C57BL/6 (B6) FTOC. Neutralization of endogenous TNF-alpha in NOD FTOC with a soluble TNF receptor (sTNF R1) rescued insulin production in our in vitro diabetes model. NOD FTOC treated with TNF-alpha produced greater numbers of mature T cells and a higher percentage of cells expressing CD95L (Fas ligand). Treatment with sTNF R1 had the opposite effect. TNF-alpha's known ability to attenuate TCR signaling coupled with these observations suggest that its overproduction in these animals may be driving T cells to maturity, altering the process of negative selection and ultimately enhancing the survival of potentially diabetogenic T cells resulting in disease susceptibility in these animals.

Selection for increased encephalization in humans necessitated extensive brain growth after birth. To estimate changes in rates of growth and corresponding shape changes during gestation and infancy, chord and arc distances were obtained from the frontal, parietal, and occipital bones of 44 human fetuses, neonates, and infants (one year old and younger). Rates of growth in chord and arc measurements were calculated and compared using linear regression of log-transformed variables, followed by ANCOVA. Curvature of bone lengths and widths were estimated by chord/arc indices. Fetal rates of cranial growth were significantly slower while the fetal frontal and occipital bones were significantly more curved than those of infants. Fetal rates of cranial growth decrease during the first six postnatal months, in conjunction with rapid changes in shape, except for parietal superior-inferior height where bossing of the bone is similar in fetuses and neonates.

The dynamics of β-cell mass are at the focus of an extensive international effort to develop β-cell replacement therapies for type 1 diabetes. During normal fetal development endocrine cells emerge from a pool of PDX1+/SOX9+ multipotent progenitors that transiently express the proendocrine gene NGN3. These cells become hormone-positive and are seen to bud from the ductal structures and aggregate into islet clusters. Congenital hyperinsulinism in its diffuse form (CHI-D) is characterised by an increase in hormone-positive cells associated with ducts and diffuse patterns of insulin expression. CHI-D arises from mutations inactivating the KATP channel and is diagnosed following persistent episodes of hypoglycaemia caused by an inappropriate secretion of insulin. Whilst existing knowledge has focused on the β-cell, we have explored the histology of CHI-D across multiple pancreatic cell lineages. The starting hypothesis considered CHI-D as an over-exuberance of endocrine differentiation with a progenitor population underlying this process. We suggest CHI-D is not simply an excessive proliferation of pre-existing β-cells. Expression of many transcription factors involved in endocrine differentiation were unchanged in CHI-D, NKX2.2 was increased and persisted in δ-cells. The incidence of nucleomegaly was also confirmed in CHI-D samples, predominantly in the β- and δ-cell lineages. Whilst increases in endocrine cell proliferation were subtle, the ductal and acinar cell lineages had significantly elevated proliferation correlating with changes in cell cycle regulation. The expression of NGN3 was profiled in a range of human fetal samples to determine whether a competence window for endocrine differentiation exists during development. Peak expression was observed between 10-17 wpc whilst protein and transcript expression were both reduced by birth and postnatally. Combined with the data in CHI-D and postnatal controls, it is likely that endocrine commitment ceases in human towards the end of gestation and that further increases in β-cell mass rely on proliferation or NGN3-independent pathways. These data provide new clues for the pathological mechanisms of CHI-D and the establishment and maintenance of the β-cell mass in the human pancreas. We have shown an altered potential for cell proliferation in CHI-D in previously unappreciated ways and provide a rationale for studying molecular components of the β-cell to help unlock β-cell proliferation as a therapeutic option in diabetes.

Purpose: To review the roles of the different vascular beds nourishing the inner retina [retinal ganglion cells (RGCs)] during normal development of the human eye, using our own tissue specimens to support our conclusions. Methods: An extensive search of the appropriate literature included PubMed, Google scholar, and numerous available textbooks. In addition, choroidal and retinal NADPH-diaphorase stained whole mount preparations were investigated. Results: The first critical interaction between vascular bed and RGC formation occurs in the sixth to eighth month of gestation leading to a massive reduction of RGCs mainly in the peripheral retina. The first 3 years of age are characterized by an intense growth of the eyeball to near adult size. In the adult eye, the influence of the choroid on inner retinal nutrition was determined by examining the peripheral retinal watershed zones in more detail. Conclusion: This delicately balanced situation of RGC nutrition is described in the different regions of the eye, and a new graphic presentation is introduced to combine morphological measurements and clinical visual field data.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4) catalyzes the formation of hydroxylysine in collagens and other proteins with collagenous domains. The resulting hydroxylysine residues participate in the formation of collagen crosslinks, and serve as attachment sites for carbohydrate units. They have been regarded as non-essential, since the absence of lysyl hydroxylase 1 activity is not lethal, although it leads to the kyphoscoliotic type of Ehlers-Danlos syndrome, and since recombinant collagens I and III lacking any hydroxylysine form native-type fibrils in vitro. A novel human lysyl hydroxylase isoenzyme, lysyl hydroxylase 3, was identified, cloned and characterized here. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to catalyze hydroxylation of lysine residues in vitro. No differences were found in the catalytic properties between the recombinant lysyl hydroxylases 3 and 1. The human lysyl hydroxylase 3 gene was shown to be 11.6 kb in size and to contain 19 exons. The introns contain 15 full-length or partial Alu retroposons, which are known to be involved in most human gene rearrangements that occur by homologous recombination. The three recombinant human lysyl hydroxylase isoenzymes were isolated here for the first time as homogenous proteins. Limited proteolysis data suggested that the lysyl hydroxylase polypeptides might consist of at least three distinct domains, A-C. The N-terminal domain A was found to play no role in lysyl hydroxylase activity as a recombinant B-C polypeptide was a fully active hydroxylase. This work also confirmed that lysyl hydroxylase 3 has collagen glucosyltransferase activity as well as trace amounts of collagen galactosyltransferase activity. However, the levels of these activities were so low that their biological significance remains to be determined. In the last part of this work, lysyl hydroxylase 3 knock-out mice were produced and analyzed. The homozygous null embryos were found to die at a very early stage of development due to lack of type IV collagen in the basement membranes. The data demonstrated that hydroxylysine formed by lysyl hydroxylase 3 is essential for early mouse development and that lysyl hydroxylase 1 or 2 cannot compensate for the lack of its function.

Movement is a prerequisite for normal fetal development and growth. Intrauterine movement restrictions cause a broad spectrum of disorders in which the unifying feature is a reduction or lack of fetal movement, giving rise to the term fetal akinesia deformations sequence (FADS [OMIM 208150]). FADS corresponds to a clinically and genetically heterogeneous constellation of properties and is characterized by multiple joint contractures, facial abnormalities, and lung hypoplasia as a result of the decreased in utero movement of the fetuses. Affected babies are often prematurely and stillborn, and those born alive typically die within minutes or hours after birth. The genetic causes for this fatal disorder are ill-defined as a genetic diagnosis is rarely executed, but mutations in three genes, namely RAPSN, DOK7, and MUSK, as well as in the subunits of the muscular nicotinic acetylcholine receptor (AChR) have been described. These mutations are thought to affect neuromuscular junctions, although this has not been proven experimentally.The nucleoporin NUP88 is a constituent of the nuclear pore complex (NPC), the gate for all trafficking between the nucleus and the cytoplasm. NUP88 resides on both the cytoplasmic and the nuclear side of NPCs, and it is found in two distinct subcomplexes. It associates with NUP214 and NUP62 on the cytoplasmic face, while on the nuclear side NUP88 binds NUP98 and the intermediate filament protein lamin A. The NUP88-NUP214-NUP62 complex plays an essential role in the nuclear export of a subset of proteins and pre-ribosomes, which is mediated by the nuclear export receptor CRM1. NUP88 in particular somewhat participates in the nuclear export of NF-κB proteins in a CRM1-dependent manner. Moreover, NUP88 is frequently overexpressed in a variety of human cancers, and its role in cancer appears linked to the deregulation of the anaphase-promoting complex. Here, we report the first Mendelian disorders caused by mutations in NUP88 and with that the first lethal developmental human disease due to mutations in a nuclear pore component. We demonstrate that biallelic mutations in NUP88 are likely to cause fetal akinesia of the Pena-Shokeir subtype. We confirm in zebrafish that loss of NUP88 impairs movement and the mutations identified in the affected individuals resemble a loss-of-function phenotype. We show that loss of NUP88 affects expression and localization of rapsyn, the protein encoded by RAPSN, in human and mouse cell lines, and patient samples. Consistent with altered rapsyn, AChR clustering and neuromuscular junction formation in zebrafish are abnormal. We therefore propose that defective NUP88 function cause FADS by affecting neuromuscular junction formation.Keywords: Nuclear pore complex, NUP88, Fetal Akinesia Deformation Sequence, rapsyn, acetylcholine receptor clustering, synaptic transmission, fetal development, inherited developmental disorder.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Puerto Rico has the highest prevalence of type 2 diabetes, low birth-weight, and the second highest prevalence of preterm-birth in all the U.S. and its non-incorporated territories. These conditions are related. Birth-weight at both ends of the spectrum and preterm-birth are associated with an increased risk for developing type 2 diabetes and immune-inflammatory dysregulations. Maternal psychosocial stressors during pregnancy have also been recognized as potential risk factors for type 2 diabetes, and have been consistently associated with preterm-birth and low birth-weight across populations. Current evidence points toward epigenetic fetal metabolic-programming as the mechanism that underlies the increased risk for the previously mentioned morbidities. However, the particular psychosocial stressors that may contribute to the high prevalence of low birth-weight and preterm-birth in the population of Puerto Rico have not been well studied.

The present study assesses the relationships between particular psychosocial stressors, socioeconomic status, food insecurity, and birth outcomes. The results of this study show that low-risk pregnancy women were more likely to have babies with a higher ponderal index if they were exposed to stressors during gestation months 5, 6, and 7, or if exposed to "relationship stress" at any time during pregnancy. Women exposed to "financial difficulties" at any time during pregnancy were more likely to deliver babies at an earlier gestational age. Differences in birth outcomes between the exposed and non-exposed women were independent of maternal anthropometric measurements, maternal age at birth, number of previous births, and sex of the baby. Significant differences in birth outcomes were found between categories of father's self-identified and identified by others ethnicity, but sample size within categories was small. Although mothers with children at home had higher levels of food insecurity, and the level of food insecurity was correlated with higher levels of stress, no birth outcome measure was associated with food insecurity.

Some results are atypical in comparison with other populations, and therefore these findings may contribute to the understanding of population differences in the relationship between maternal stress during pregnancy and birth outcomes. The relatively small sample size and strict exclusion criteria of this study may limit the generalizability of the findings. Epidemiological similarities between Puerto Rico and other populations, and the possibility of a higher ponderal index increasing the risk for type 2 diabetes in the population of Puerto Rico need to be examined in future research.

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents
Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes

Les malformations du cortex cérébral (MDC) représentent une cause importante de handicap et d'épilepsie pharmaco-résistante. Le séquençage à haut débit a permis une amélioration considérable de l'identification des bases moléculaires des MDC non syndromiques. Toutefois, certaines formes, notamment les MDC complexes, demeurent inexpliquées. Mon projet de thèse a pour objectif de progresser dans la compréhension des MDC complexes en utilisant deux modèles : les microlissencéphalies (MLIS) et le syndrome d'Aicardi (AIC), une forme syndromique particulière associant des malformations de l'oeil et du cerveau uniquement rapporté chez les filles. L'étude par séquençage d'exome en trios de 16 familles MLIS m'a permis d'identifier et de caractériser un nouveau gène, WDR81, impliqué dans le cycle cellulaire. Par la même stratégie, j'ai pu identifier un variant homozygote pathogène dans TLE1, un partenaire majeur de FOXG1 dans la balance prolifération/différenciation de progéniteurs neuronaux, dans une famille consanguine de microcéphalie postnatale dont le phénotype est proche du syndrome FOXG1. En parallèle, mes travaux ont permis de préciser les spectres phénotypiques associés à RTTN, EPG5, COL4A1, COL4A2, TBR1, KIF5C, KIF2A et FOXG1. La deuxième partie de mon projet avait pour objet l'identification des bases moléculaires du syndrome d'Aicardi à partir d'une cohorte internationale de 19 patientes. Après avoir exclu un biais d'inactivation du chromosome X et la présence de microremaniements chromosomiques, j'ai réalisé un séquençage d'exome en trio. Aucun variant récurrent n'a été retrouvé dans les séquences codantes. Dans un second temps, j'ai testé une approche combinant les données du séquençage de génome et l'analyse du transcriptome (RNA-Seq) sur fibroblastes, me permettant d'identifier des transcrits dérégulés qui étaient impliqués dans le développement du cerveau et de l'oeil. J'ai comparé les résultats de cette analyse avec ceux de l'analyse du génome dans le but d'identifier des variants dans ces gènes candidats. En conclusion, mon travail de thèse a permis d'améliorer la connaissance des bases moléculaires des MDC complexes et d'ouvrir des perspectives de nouveaux mécanismes tels que ceux engageant les gènes WDR81 et EPG5, et le rôle des endosomes et de l'autophagie dans les MDC, et aussi TLE1 comme nouvelle cause de microcéphalies postnatales. Mes travaux ont également permis de générer une collection de données de séquençage haut débit (WES, WGS et RNA-Seq) qui seront mises en commun dans le cadre d'un consortium international afin de développer des nouvelles stratégies d'analyse en particulier pour les séquences non codantes. Cette approche permettra également d'ouvrir la voie vers la compréhension des mécanismes cellulaires impliqués dans la formation du cerveau et de l' œil
Malformations of cortical development (MCD) are a major cause of intellectual disability and drug-resistant epilepsy. Next Generation Sequencing (NGS) has considerably improved the identification of the molecular basis of non-syndromic MCD. However, certain forms, including complex MCD, remain unexplained. My PhD project aimed to improve the understanding of complex MCD using two disorders: Microlissencephaly (MLIS) and Aicardi Syndrome (AIC), the latter associating brain and eye malformations and only reported in girls. Trio Whole Exome Sequencing (WES) performed in 16 MLIS families allowed me to identify and functionally characterize a new MLIS gene, WDR81, in which mutations lead to cell cycle alteration. Moreover, using the same strategy, I was able to identify a pathogenic homozygous variant in TLE1 in a patient from consanguineous family with a postnatal microcephaly, suggestive of a FOXG1-like presentation. Interestingly, TLE1 is a major partner of FOXG1, a gene involved in maintaining the balance between progenitor proliferation and differentiation. In parallel, my work allowed me to redefine the phenotypic spectrum associated with RTTN, EPG5, COL4A1 and COL4A2, TBR1, KIF5C, KIF2A and FOXG1. The second part of my PhD program was aimed at identifying the genetic basis of AIC in an international cohort of 19 patients. After excluding a skewed X chromosome inactivation and the presence of chromosomal rearrangements, I performed WES in trios. The analysis of the data from WES did not allow me to identify any recurrent variants. I therefore tested a new approach combining Whole Genome Sequencing (WGS) and RNA-Sequencing (RNA-Seq) on fibroblast cells. I identified a number of deregulated transcripts implicated in brain and eye development. I compared the results of this analysis with the WGS analysis in order to find variants in these candidate genes. In conclusion, these studies have improved the knowledge of the molecular basis of complex MCD, such as TLE1 in postnatal microcephaly, and revealed the pathogenic mechanisms such as WDR81 in cell cycle progression and EPG5 in endosomes and autophagy. My work has also generated a collection of NGS data (WES, WGS and RNA-Seq) that will be shared in an international consortium to develop new analytical strategies, in particular for the non-coding DNA regions. This novel strategy provides opportunities to improve understanding of the cellular mechanisms involved in brain and eye development

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
Ao longo das últimas cinco décadas tem vindo a registar-se um decréscimo da taxa de fecundidade humana. Embora existam vários factores que contribuam para tal (por exemplo, alterações nos comportamentos sociais), são cada vez mais comuns problemas fisiológicos masculinos, como a diminuição da concentração e qualidade espermática, que devem ser também considerados importantes. Adicionalmente, doenças como hipospádias, criptorquidismo e cancro testicular têm também vindo a aumentar de incidência. Devido ao curto período de tempo em que se registam estas grandes diferenças, parece pouco provável que haja apenas influência genética a actuar. Como em tantos outros problemas recentes, a combinação das variações do estilo de vida e da exposição ambiental a moléculas perigosas pode prejudicar o desenvolvimento do sistema reprodutor masculino, diminuindo assim a fertilidade. Foi proposta uma origem comum na vida fetal para as doenças acima referidas, em que uma função deficiente das células somáticas do testículo fetal resultaria numa ruptura da organização e desenvolvimento normal, traduzindo-se em problemas reprodutivos masculinos. Esta teoria dá pelo nome de Síndrome da Disgénese Testicular (Testicular Dysgenesis Syndrome, TDS) e mostra o quão importante é o desenvolvimento fetal das estruturas reprodutivas masculinas para uma saúde e função reprodutiva normal posterior. Uma das doenças incluídas nesta TDS é o cancro testicular com origem na linha germinal (testicular germ cell cancer, TGCC), ao qual é atribuído a maioria das mortes relacionadas com problemas de saúde em homens entre os 15 e os 35 anos e cuja incidência duplicou nos últimos 40 anos. Durante o desenvolvimento fetal no Homem, as células germinais masculinas indiferenciadas mantêm a sua proliferação em níveis baixos após a perda de pluripotência e passagem para um estado diferenciado. Esta perda de pluripotência parece conter a hipótese de como a TGCC surge nos humanos, visto que se o processo for perturbado por factores externos, as células podem entrar num estado intermediário em que mantêm a taxa de proliferação alta, adquirindo características cancerígenas. Um tipo de molécula ambiental ao qual os fetos estão expostos, e que já demonstrou afectar negativamente o sistema reprodutor masculino noutras espécies, são os ftalatos, componentes industriais cuja exposição em roedores levou ao aparecimento de condições semelhantes às da TDS. No entanto, não há informações sobre o seu efeito nos humanos. Outros químicos ambientais cujo efeito no desenvolvimento reprodutivo pode ser negativo são os anti-inflamatórios não esteróides, tais como o paracetamol e a indometacina. Embora o mecanismo exacto ainda não seja conhecido, ambos actuam através da inibição da produção ou acção de prostaglandinas, que são lípidos libertados pelas células quando ocorre uma inflamação, embora a indometacina seja considerada um supressor mais inequívoco do que o paracetamol. A administração de paracetamol e outros analgésicos demostrou ter efeitos negativos na saúde reprodutiva em roedores. Esta tese centrou-se no estudo do efeito da exposição a químicos ambientais como o ftalato di(n-butyl) (di(n-butyl) phthalate, DBP), paracetamol e indometacina no desenvolvimento e diferenciação das células germinais fetais, baseando-se em estudos preliminares e não publicados desenvolvidos pelo grupo de investigação em que me inseri. Para estudar o efeito do DBP, foram recolhidos testículos de fetos humanos (n = 8), com idades compreendidas entre as 14 e 20 semanas de gestação, para serem xeno-enxertados debaixo da pele dorsal de ratos imunodeficientes adultos (machos castrados) em porções pequenas. Sete dias após esta operação, os quais permitem o estabelecimento de irrigação sanguínea nos enxertos, os hospedeiros recebem doses diárias de controlo, DBP ou MBP (ftalato monobutyl, o metabolito activo do DBP) dissolvidos em óleo (500mg/kg/dia), durante 21 dias. Os hospedeiros ainda são sujeitos a três injecções semanais de gonadotropina coriónica humana (20 IU), para replicar as condições normais de gravidez e manter a produção de testosterona. Após o período de tratamento, os enxertos são recolhidos, fixados, cortados em secções e sujeitos a uma imunofluorescência tripla. No total, analisaram-se pelo menos dois enxertos controlos e dois expostos a DBP/MBP para cada feto, resultando, no mínimo, em 32 amostras. O protocolo de imunofluorescência permite visualizar células germinais com antigénios para OCT3/4 (células germinais indiferenciadas), MAGE-A4 (células germinais diferenciadas) e Ki67 (células em proliferação). As imagens foram obtidas por microscopia confocal e as células incluídas num túbulo seminífero definido, que demonstrassem fluorescência para estes antigénios, foram assumidas como pertencendo à linha germinal e contadas manualmente. Os valores registados para os enxertos-controlo e expostos a DBP, do mesmo feto, foram condensados até se obter um valor médio respectivo. A média dos enxertos controlo e dos expostos ao tratamento, de cada um dos fetos, foi utilizada então para análise estatística, usando-se um Teste t para amostras emparelhadas (significância: P<0.05). Os resultados mostraram que os enxertos expostos ao DBP(MBP) registam uma diminuição significativa de mais de 10% na percentagem de células germinais positivas para OCT3/4 (indiferenciadas). Por outro lado, a percentagem de células germinais positivas para MAGE-A4 (diferenciada) em enxertos tratados com DBP(MBP) foi significativamente maior do que os controlos. Porém, a proliferação das células germinais indiferenciadas e diferenciadas não registou alterações significativas em comparação com as amostras controlo. As células germinais reduziram a expressão de OCT3/4 para aumentarem a expressão de MAGE-A4 quando se diferenciaram, o que coincidiu com a redução, e eventualmente a perda, da capacidade proliferativa, demonstrando que o modelo dos xeno-enxertos consegue recapitular o desenvolvimento fetal normal das células germinais masculinas nos humanos. Estes resultados parecem dever-se a uma apoptose selectiva das células indiferenciadas. Se a apoptose fosse geral, a proporção de células germinais indiferenciadas e diferenciadas não mudava após o tratamento com o DBP(MBP), o que não está de acordo com os resultados obtidos, já que há um aumento de células germinais diferenciadas. Devido à falta de dados sobre o efeito do DBP nas gónadas masculinas humanadas, esta hipótese estaria de acordo com dados anteriores de estudos in vitro e in vivo, em humanos e roedores respectivamente, em que a exposição a DBP reduz o número de células germinais fetais positivas para OCT3/4. O segundo projecto desenvolvido e abordado nesta tese foi analisar os efeitos da exposição ao paracetamol e indometacina no número de células germinais nos testículos fetais de ratos. Fêmeas prenhas de ratos Wistar foram tratadas diariamente com paracetamol (350mg/kg/dia), indometacina (1mg/kg/dia ou 0.8mg/kg/dia) ou tratamento controlo. O tratamento decorreu entre o dia embrionário (e) 15.5 até ao e20.5, sendo os testículos dos descendestes recolhidos ao e21.5. As amostras foram fixadas, seccionadas e processadas através de um protocolo imunohistoquímico para o antigénio VASA (marcador típico de células germinais). O número de células germinais foi obtido por estereologia, que é um método uniforme e sistemático que permite analisar a composição celular de um tecido tridimensional, neste caso o testículo, através da contagem de núcleos de células germinais em secções bidimensionais desse mesmo tecido. Para as amostras fetais e21.5, foram contadas todas as células que expressassem VASA e que estivessem contidas num túbulo seminífero definido. A comparação entre amostras controlo e as expostas a paracetamol/indometacina foi analisada estatisticamente através de uma análise de variância ANOVA (significância: p<0.05). Os resultados mostraram uma redução significativa do número de células germinais em amostras expostas à indometacina, quando comparadas com as de controlo. Embora as gónadas masculinas expostas ao paracetamol não tenham apresentado uma diferença significativa, existe uma tendência para a redução do número de células germinais. O desenvolvimento normal das células germinais no rato passa por perderem totalmente a capacidade proliferativa quando se diferenciam, num processo síncrono e uniforme, ao contrário dos humanos. Não se sabe se o decréscimo do número de células pode ser atribuído a apoptose devido à falta de dados nesta área, mas a indometacina pode induzir um aumento da taxa de diferenciação das células germinais fetais, levando à paragem da divisão celular numa idade anormalmente precoce. Outro ensaio experimental foi desenvolvido para investigar o efeito da exposição in utero à indometacina no número de células germinais em testículos de ratos obtidos no dia 25 após o nascimento (postnatal day, pnd25). O protocolo para investigar este efeito foi em tudo idêntico ao seguido para os testículos e21.5, excepto que não houve amostras expostas ao parecetamol e os testículos foram recolhidos após o nascimento (embora o período de exposição à indometacina tenha sido de e15.5 a e18.5). Visto que a gónada pnd25 já produz espermatozóides, as células germinais foram divididas em categorias que correspondem à progressão temporal da espermatogénese: espermatogónia, espermatócito I e espermatócito II. O número de células germinais de cada categoria, mais o número total de células germinais, foi contado também através de esterologia e a comparação estatística entre amostras controlo e tratadas com indometacina foi feita com um Teste t de Student (significância: p<0.05). Os resultados mostraram que o número de espermatogónias e o número total de células germinais aumentaram significativamente em amostras tratadas com indometacina, enquanto os valores para espermatócitos I e II não sofreram alterações significativas. Isto sugere que a exposição in utero à indometacina possa induzir apoptose nas células germinais e aquelas que resistem conseguem recuperar, embora faseadamente, os seus números normais após o nascimento e cessação do período de tratamento. Além disso, ambos os ensaios em ratos mostram que, pelo menos em mamíferos, o desenvolvimento fetal normal das células germinais, e posterior fertilidade dos indivíduos, é dependente de prostaglandinas. Este conjunto de estudos mostrou que a exposição fetal a químicos ambientais comuns, tais como os ftalatos e inibidores de prostaglandinas, pode afectar o desenvolvimento fetal das células germinais nos humanos e no rato, obtendo-se dados sobre este tema pela primeira vez.
Lifestyle and environmental exposure to hazardous molecules have been indicated as a cause for the decrease in human fertility. An increased incidence of hypospadias, cryptorchidism and testicular germ cell cancer has been reported and it has been hypothesized that these may form a Testicular Dysgenesis Syndrome (TDS) with a common origin in fetal life. Although testicular germ cell cancer (TGCC) occurs in young men, it appears that it arises from a failure of fetal germ cells to lose their pluripotency and which then transform into carcinoma-in-situ (CIS) cells, which are germ cells (GC) with pluripotency and proliferative features that probably lead to TGCC in young adulthood. It has been hypothesized that exposure in utero to some environmental factors could play a role in disturbing the testicular endocrine function in the fetus, possibly leading to TGCC. Di(n-butyl) phthalate (DBP), a common environmental chemical, has been shown to affect fetal testis development and endocrine function in the rat. Nonsteroidal anti-inflammatory drugs (paracetamol, indomethacin), which are believed to work through inhibition of prostaglandins, and other inflammation modulators can also affect fetal testis endocrine function in animal models. This study therefore aimed to assess the effect of DBP, paracetamol and indomethacin exposure on fetal GC development and differentiation. To study DBP effects, second-trimester human testis pieces from eight fetuses were xenografted under the backskin of nude castrated male mice, which were then treated with vehicle (control) or DBP for 21 days. Immunofluorescence for OCT3/4 (undifferentiated GC marker), MAGE-A4 (differentiated GC marker) and Ki67 (proliferative cell marker) was used to analyse GC differentiation and proliferation. Exposure to DBP led to a reduction in proportion of undifferentiated GC, although proliferative features were maintained, possibly due to selective apoptosis. To analyse the effect of indomethacin on GC numbers, e21.5 and pnd25 rat testes were evaluated in males exposed in utero to vehicle (control) or indomethacin (1mg/kg or 0.8mg/kg) treatment. Immunohistochemistry for VASA (GC marker) and stereology was used to assess GC number in e21.5 testes and spermatogenic temporal progression in pnd25 testes. Indomethacin exposed e21.5 rat testes showed a significant decrease in GC number, which is in agreement with preliminary results. Pnd25 rat testes from males exposed in utero to indomethacin showed an increase in spermatogonial numbers and total GC number, although the numbers for early and pachytene spermatocytes were not different. This shows that indomethacin affects germ cell development during the exposure period and that there might be a staggered recovery after birth toward normal GC number. Overall, these results show for the first time that exposure to common environmental chemicals affect human and rat male fetal GC development.

Tese de doutoramento em Biologia (Biologia Celular) apresentada à Fac. de Ciências e Tecnologia de Coimbra
Disciplina afim: Imunologia Por volta da sétima semana de gestação, o timo humano é colonizado por progenitores hematopoiéticos provenientes do fígado fetal, os quais se diferenciam predominantemente em linfócitos T no seio do microambiente tímico. No Capítulo 2, é identificada e caracterizada uma população de células estromais que se encontram localizadas na região subcapsular do timo humano, e suportam a diferenciação de precursores tímicos em timócitos imaturos, levando a crer que possam ter um papel importante nos estadios mais precoces do desenvolvimento de células T. As células hematopoiéticas do fígado fetal que colonizam o timo, constituem precursores multipotentes com a capacidade de se diferenciarem em tipos celulares distinctos. No Capítulo 3, nós propusémo-nos investigar se o cometimento à linhagem T teria lugar no seio do fígado fetal, anteriormente à colonização do timo. Foi demonstrado que a capacidade de os precursores de fígado fetal se diferenciarem em linfócitos T, é exclusiva dos progenitores mais imaturos. Pelo contrário, tanto os precursores mais imaturos como os mais diferenciados possuem capacidade de diferenciação em células NK, o que implica o fígado fetal como um possível local de desenvolvimento de células NK. Apesar da medula óssea ser o orgão mais importante na formação de linfócitos B, uma série de observações sugere que a diferenciação B possa ter lugar no fígado fetal. No Capítulo 4, encontram-se descritos dois modelos experimentais de suporte estromal que induzem e suportam a diferenciação de progenitores de fígado fetal em células B, através da aquisição progressiva de marcadores associados a esta linhagem. A via de diferenciação de células B, tal como outros processos biológicos, é muito provavelmente regulada por um conjunto de factores de transcrição. Neste contexto, as proteínas E12 and E47 foram recentemente identificadas como factores cruciais para a diferenciação B no ratinho, e são reguladas negativamente por proteínas Id. No Capítulo 5, é demonstrado que a expressão forçada da proteína Id3 em precursores de fígado fetal através de transferência genética mediada por retrovírus, inibe marcadamente o desenvolvimento de linfócitos B a partir de células estaminais de fígado fetal quando estas são cultivadas nos modelos estromais descritos no Capítulo 4.

Structural brain changes occur in a complex manner throughout life, and understanding healthy brain development is crucial for the study of brain abnormalities in various conditions. Diffusion tensor imaging (DTI) is an advanced magnetic resonance imaging technique that provides information about tissue microstructure not accessible via conventional imaging methods. In this dissertation, DTI is used to assess typical brain development, brain abnormalities in fetal alcohol spectrum disorder (FASD), and relationships between cognition and brain structure in both populations. Cross-sectional and longitudinal DTI studies were used to measure brain maturation from childhood to adulthood. Significant, nonlinear changes of diffusion parameters were noted across the brain, with regional variation in the timing and magnitude of development. Most regions experienced rapid maturation during childhood and adolescence, reached a developmental peak during adulthood, and then, during senescence, underwent a reversal of structural changes that occurred more gradually than the initial development. The genu and splenium of the corpus callosum had the earliest development, while frontal-temporal connections and the corticospinal tracts showed the most prolonged maturation trajectories. DTI was also used to examine brain abnormalities in children with FASD, an acquired brain disorder associated with numerous cognitive, behavioural, and emotional difficulties. DTI revealed widespread differences in children with FASD when compared to healthy controls, suggesting extensive structural brain damage. Finally, significant relationships between cognitive abilities and brain structure were observed in both populations. Brain lateralization of a frontal-temporal pathway correlated with two specific cognitive abilities in typically-developing children. Additionally, a significant relationship between brain structure and mathematical ability was observed in the left parietal lobe of children with FASD. Preliminary results demonstrating reading-brain structure correlations in both healthy and FASD groups are also presented. In conclusion, DTI has shown significant age-related changes in the typically-developing human brain, abnormalities in children with FASD, and correlations between brain structure and cognition in both populations. Normative DTI studies such as the ones presented here are important to establish healthy milestones of brain development and degradation, which may then be used to understand abnormalities in a variety of conditions, including FASD.

Christine May Briton-Jones.
"May 2002."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (p. 149-171).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

碩士
國立交通大學
生物資訊研究所
95
Heart development is a complex process involving many genes which control cell behavior in the embryo and determine its pattern, its form, and much of its behavior. Microarray experiments can generate an enormous amount of data at one time, so we use this technology to obtain gene expression profiles in heart embryonic development. But it is usually very difficult to obtain human heart fetus sample because of the issues of ethical, legal, and social consideration. In order to help us get more understanding of human heart development, we can use the mouse model system that is most often used. Therefore, we must establish a mapping system to make a cross bridge between these two species on developmental stages. To date, the vast majority of researches have focused their study on one species. Specially, we utilize orthologous genes and incorporate the dynamic time warping algorithm in order to map the time points that human and mouse gene expression profiles having highly correlated pattern. Firstly, we apply the algorithm to select the best time-warped orthologous genes having similar pattern. Then, these genes are clustered into groups. Each group has its unique mapping pattern and different biological meaning. The following task is to find relationship and pattern in distinct groups of genes, and to get close understanding into molecular process and gene function, mechanisms of embryogenesis of the heart, and comparative genomics. Ultimately, our aim is to achieve new insights into the heart developmental biology.

by Chung Hoi Sing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 92-106).
Abstract also in Chinese.
ABSTRACT --- p.i
摘要 --- p.iii
ACKNOWLEDGMENT --- p.v
Chapter 1. --- Introduction
Chapter 1.1 --- Angiogenesis in general --- p.1
Chapter 1.1.1 --- The concept of angiogenesis --- p.1
Chapter 1.1.2 --- The process of angiogenesis --- p.1
Chapter 1.2 --- Measurement of angiogenesis --- p.3
Chapter 1.2.1 --- In vivo assays --- p.3
Chapter 1.2.2 --- In vitro assays --- p.5
Chapter 1.3 --- Angiogenic factors --- p.6
Chapter 1.4 --- Angiogenesis in the female reproductive system --- p.7
Chapter 1.5 --- Evidence of hormonally-regulated angiogenesis in endocrine tissues --- p.10
Chapter 1.5.1 --- Ovary --- p.10
Chapter 1.5.2 --- Thyroid --- p.11
Chapter 1.6 --- Angiogenesis in the testis --- p.12
Chapter 1.6.1 --- Structure of testicular vasculature --- p.12
Chapter 1.6.2 --- Angiogenic factors in the testis --- p.13
Chapter 1.6.3 --- Vascular effects of hCG/LH in the testis --- p.17
Chapter 1.6.4 --- Postnatal development of testicular vasculature --- p.17
Chapter 1.7 --- Aims of the present study --- p.19
Chapter 2. --- Materials and methods
Chapter 2.1 --- Animals --- p.20
Chapter 2.2 --- Experimental design --- p.20
Chapter 2.2.1 --- Testicular angiogenesis in adult rats - hormonal stimulation by hCG --- p.20
Chapter 2.2.1.1 --- Changes with time after hCG treatment --- p.20
Chapter 2.2.1.2 --- Effect of Leydig cell depletion --- p.22
Chapter 2.2.1.3 --- Effect of Leydig cell suppression by subcutaneous testosterone-filled silastic implants --- p.22
Chapter 2.2.1.4 --- Effect of testicular macrophage activation --- p.24
Chapter 2.2.1.5 --- Effect of testicular macrophage depletion --- p.26
Chapter 2.2.2 --- Developmental changes in testicular angiogenesis --- p.29
Chapter 2.3 --- Perfusion of testes with fixative or Indian Ink --- p.29
Chapter 2.4 --- Processing of the testes for histological sections --- p.30
Chapter 2.5 --- Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) --- p.31
Chapter 2.6 --- Immunohistochemical staining for vascular endothelial growth factor --- p.32
Chapter 2.7 --- Quantification of PCNA-positive endothelial cells --- p.33
Chapter 2.8 --- Quantification of blood vessel density --- p.34
Chapter 2.9 --- Estimation of intertubular area in testis section --- p.35
Chapter 2.10 --- Preparation of liposome-entrapped dichloromethylene diphosphonate (Cl2MDP-lp) --- p.38
Chapter 2.11 --- Radioimmunoassay of serum tsetosterone --- p.38
Chapter 2.12 --- Statistical analyses --- p.40
Chapter 3. --- Results
Chapter 3.1 --- hCG-induced increase in endothelial cell proliferation in adult rat testes --- p.41
Chapter 3.1.1 --- Testicular histology --- p.41
Chapter 3.1.2 --- Changes in the number of PCNA-positive endothelial cells --- p.41
Chapter 3.1.3 --- Changes in blood vessel density --- p.44
Chapter 3.1.4 --- Changes in testis weight and serum testosterone concentration --- p.44
Chapter 3.2 --- Effect of Leydig cell depletion by ethane dimethane sulphonate (EDS) on hCG-induced endothelial cell proliferation in adult rat testes --- p.48
Chapter 3.2.1 --- Testicular histology --- p.48
Chapter 3.2.2 --- Changes in the number of PCNA-positive endothelial cells --- p.48
Chapter 3.2.3 --- Changes in serum testosterone concentration and testis weight --- p.52
Chapter 3.3 --- Effect ofLeydig cell suppression by testosterone-filled subcutaneous silastic implants on hCG-induced endothelial cell proliferation in adult rat testes --- p.54
Chapter 3.3.1 --- "Changes in serum testosterone concentration, testis weight, and testicular intertubular area" --- p.54
Chapter 3.3.2 --- Changes in the number of PCNA-positive endothelial cells --- p.58
Chapter 3.3.3 --- Changes in the level of vascular endothelial growth factor (VEGF) immunoreactivity in the testis --- p.60
Chapter 3.4 --- Effect of testicular macrophage activation by polystyrene latex beads on hCG-induced endothelial cell proliferation in adult rat testes --- p.60
Chapter 3.4.1 --- Testicular histology --- p.60
Chapter 3.4.2 --- Changes in the number of PCNA-positive endothelial cells --- p.63
Chapter 3.4.3 --- Changes in testis weight and serum testosterone concentration --- p.65
Chapter 3.5 --- Effect of testicular macrophage depletion by liposome-entrapped C12MDP treatment on hCG-induced endothelial cell proliferation in adult rat testes --- p.67
Chapter 3.5.1 --- Testicular histology --- p.68
Chapter 3.5.2 --- Changes in the number of PCNA-positive endothelial cells --- p.68
Chapter 3.5.3 --- Changes in testis weight and serum testosterone --- p.72
Chapter 3.6 --- Endothelial cell proliferation in rat testes during postnatal development --- p.74
Chapter 3.6.1 --- Changes in the number of PCNA-positive endothelial cells --- p.74
Chapter 3.6.2 --- Changes in blood vessel density --- p.74
Chapter 3.6.3 --- Changes in testis weight and intertubular area of the testes --- p.77
Chapter 4. --- Discussion
Chapter 4.1 --- hCG-induced endothelial cell proliferation and changes in blood vessel density --- p.79
Chapter 4.2 --- Role of Leydig cells in hCG-induced endothelial cell proliferation in adult rat testes --- p.82
Chapter 4.3 --- Role of testicular macrophages in hCG-induced endothelial cell proliferation in adult rat testes --- p.86
Chapter 4.4 --- Testicular angiogenesis during postnatal development --- p.88
Chapter 5. --- References --- p.92

Ng, Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 179-204).
Abstracts in English and Chinese.
Abstract --- p.I
摘要 --- p.IV
Publications --- p.VII
Acknowledgements --- p.VIII
Table of contents --- p.IX
List of figures --- p.XV
List of tables --- p.XVII
List of abbreviations --- p.XVIII
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- The Pancreas --- p.2
Chapter 1.1.1 --- Anatomy of Pancreas --- p.2
Chapter 1.1.2 --- The Exocrine Pancreas --- p.4
Chapter 1.1.3 --- The Endocrine Pancreas --- p.5
Chapter 1.1.3.1 --- Structure of Islets --- p.5
Chapter 1.1.3.2 --- "Functions of α-, β-, y-, ð-, Σ-and PP-cells in Islets" --- p.7
Chapter 1.1.4 --- Overview of Pancreas Development --- p.9
Chapter 1.1.4.1 --- Organ Morphology --- p.10
Chapter 1.1.4.2 --- Cyto-differentiation --- p.12
Chapter 1.1.4.3 --- Control by Transcriptional Factors --- p.14
Chapter 1.1.5 --- Postnatal Pancreas Development and Regeneration --- p.18
Chapter 1.1.5.1 --- Proliferation of Pre-existing β-cells --- p.19
Chapter 1.1.5.2 --- Neogenesis from Precursor Cells --- p.20
Chapter 1.1.5.3 --- Transdifferentiation of other Cells --- p.20
Chapter 1.2 --- Diabetes Mellitus --- p.22
Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus and Current Treatments --- p.24
Chapter 1.2.1.1 --- Type I Diabetes Mellitus --- p.24
Chapter 1.2.1.2 --- Type II Diabetes Mellitus --- p.25
Chapter 1.2.1.3 --- Gestational Diabetes --- p.27
Chapter 1.2.1.4 --- Secondary Diabetes --- p.28
Chapter 1.3 --- Stem Cell therapy --- p.29
Chapter 1.3.1 --- Stem Cell --- p.29
Chapter 1.3.1.1 --- Mesenchymal Stem Sell --- p.31
Chapter 1.3.1.2 --- Embryonic Stem Cell --- p.35
Chapter 1.3.1.3 --- Induced Pluripotent Stem Cell --- p.36
Chapter 1.3.2 --- Islets Engineering --- p.37
Chapter 1.3.2.1 --- Genetic Modification --- p.37
Chapter 1.3.2.2 --- Directed Differentiation --- p.38
Chapter 1.3.2.3 --- Microenvironment --- p.38
Chapter 1.3.2.4 --- In vivo Regeneration --- p.39
Chapter 1.3.2.5 --- Cell Fusions --- p.40
Chapter 1.3.2.6 --- Combinatory Treatments --- p.40
Chapter 1.4 --- The Vitamin A & Vitamin D System --- p.42
Chapter 1.4.1 --- The Vitamin A --- p.42
Chapter 1.4.2 --- Vitamin A Metabolism --- p.44
Chapter 1.4.3 --- Roles of vitamin A in Pancreatic Development --- p.46
Chapter 1.4.4 --- The Vitamin D --- p.48
Chapter 1.4.5 --- Vitamin D Metabolism --- p.49
Chapter 1.4.6 --- Metabolic Functions of Vitamin D in Islets --- p.51
Chapter 1.4.7 --- Cod Liver Oil --- p.53
Chapter 1.4.8 --- Interactions between Vitamin A and Vitamin D --- p.53
Chapter 1.5 --- The Relations of Liver and Pancreas Development --- p.55
Chapter 1.5.1 --- Endoderm Induction for Hepatic and Pancreatic Differentiation of ESCs --- p.55
Chapter 1.5.2 --- Bipotential Precursor Population within Embryonic Endoderm --- p.56
Chapter 1.5.3 --- Pancreatic Islets Promote Mature Liver Hepatocytes Proliferation --- p.57
Chapter 1.5.4 --- Transdifferentiation --- p.57
Chapter 1.5.5 --- Transplantation in Liver Niche Promotes Maturation of Insulin-Producing Cells --- p.60
Chapter 1.5.6 --- Neuronal Relay from the Liver to Pancreatic --- p.61
Chapter 1.5.7 --- Development of Islets in the Nile Tilapia --- p.62
Chapter 1.6 --- The Insulin-like Growth Factor-I (IGF1) --- p.64
Chapter 1.6.1 --- IGF1 System --- p.64
Chapter 1.6.2 --- IGF 1 Regulation --- p.65
Chapter 1.6.3 --- Roles of IGF 1 in Pancreatic Development and Regeneration --- p.68
Chapter 1.7 --- Aims and Objectives of Study --- p.70
Chapter Chapter 2 --- General Materials and Methods
Chapter 2.1 --- Pancreatic progenitor cells (PPCs) and liver stromal cells (LSCs) isolation and cell culture --- p.72
Chapter 2.1.1 --- Tissue procurement --- p.72
Chapter 2.1.2 --- PPC and LSC culture --- p.72
Chapter 2.1.3 --- "Treatments of vitamin A, vitamin D and IGF 1" --- p.76
Chapter 2.1.4 --- "Cell culture of Caco-2, HepG2 and DU-145" --- p.76
Chapter 2.2 --- Induction of Islet-like Cell Clusters (ICCs) Differentiation --- p.77
Chapter 2.2.1 --- In vitro Directed Differentiation --- p.77
Chapter 2.2.2 --- In vitro LSC Microenvironment --- p.77
Chapter 2.3 --- RNA Expression Detection --- p.79
Chapter 2.3.1 --- RNA isolation --- p.79
Chapter 2.3.2 --- Reverse Transcription --- p.79
Chapter 2.3.3 --- Polymerase Chain Reaction (PCR) --- p.80
Chapter 2.3.4 --- Realtime PCR --- p.81
Chapter 2.4 --- Immunocytochemistry --- p.83
Chapter 2.5 --- Western Blotting --- p.85
Chapter 2.5.1 --- Protein extraction and quantification --- p.85
Chapter 2.5.2 --- Western Blotting --- p.85
Chapter 2.6 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.87
Chapter 2.6.1 --- Detection of cell viability --- p.87
Chapter 2.6.2 --- Detection of cell proliferation --- p.87
Chapter 2.6.3 --- Measurement of Cell death --- p.88
Chapter 2.6.4 --- Measurement of IGF 1 level in condition medium --- p.89
Chapter 2.6.5 --- Measurement of glucose induced insulin secretion --- p.90
Chapter 2.7 --- Regeneration model --- p.92
Chapter 2.7.1 --- Regeneration model in neonatal-STZ rat --- p.92
Chapter 2.7.2 --- Change in IGF1 expression in pancreas and liver --- p.92
Chapter 2.8 --- Statistical Data Analysis --- p.93
Chapter Chapter 3 --- Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas. (Stem Cell Rev. 2011;7:53-63)
Chapter 3.1 --- Abstract --- p.95
Chapter 3.2 --- Introduction --- p.97
Chapter 3.3 --- Materials and Methods --- p.101
Chapter 3.3.1 --- Fetal Tissue Procurement --- p.101
Chapter 3.3.2 --- Culture of Pancreatic Progenitor Cells --- p.101
Chapter 3.3.3 --- RNA Expression Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.102
Chapter 3.3.4 --- Western Blot Analysis --- p.103
Chapter 3.3.5 --- Immunocytochemstry --- p.105
Chapter 3.3.6 --- PPC Proliferation Assays --- p.106
Chapter 3.3.7 --- PPC Cell Death Assays --- p.107
Chapter 3.3.8 --- Statistical Data Analysis --- p.108
Chapter 3.4 --- Results --- p.110
Chapter 3.4.1 --- "Expression and Localization of RAR, VDR and RXR, CYP26 and CYP24 in PPCs" --- p.110
Chapter 3.4.2 --- Incubation of PPC with atRA Enhances PPC Viability due to Increased Proliferation and Anti-apoptosis --- p.111
Chapter 3.4.3 --- Incubation of PPCs with Calcitriol Enhances Viability due to Increased Proliferation --- p.111
Chapter 3.4.4 --- Both atRA and Calcitriol Induce Up-regulation of both the RAR and the VDR but not the RXR --- p.112
Chapter 3.4.5 --- Combination Treatment with atRA and Calcitriol on Cell Viability and NGN3 Expression --- p.112
Chapter 3.5 --- Discussion --- p.114
Chapter Chapter 4 --- Human fetal liver stromal cell co-culture enhances the growth and differentiation of pancreatic progenitor cells into islet-like cell clusters (In submission to Gastroenterology)
Chapter 4.1 --- Abstract --- p.128
Chapter 4.2 --- Introduction --- p.129
Chapter 4.3 --- Materials and Methods --- p.133
Chapter 4.3.1 --- Use of human and animal tissues --- p.133
Chapter 4.3.2 --- "Cell preparation, characterizations and Differentiation" --- p.133
Chapter 4.3.3 --- Examination of PPC growth and ICC differentiation and functions with LSC co-culture --- p.133
Chapter 4.3.3 --- Identification of growth factors and investigation of their effects --- p.134
Chapter 4.3.4 --- Statistical Analysis --- p.135
Chapter 4.4 --- Results --- p.136
Chapter 4.4.1 --- "Isolation, Culture and Characterizations of LSCs" --- p.136
Chapter 4.4.2 --- Establishment of LSC co-culture system --- p.136
Chapter 4.4.3 --- LSC co-culture enhances PPC-derived ICC differentiation --- p.137
Chapter 4.4.4 --- Differential expression of mRNA for cytokines and growth factors between 1st and 2nd trimester LSCs --- p.138
Chapter 4.4.5 --- Characterization of IGF 1 receptors in PPCs and the effects of exogenous IGF1 on PPC growth and ICC differentiation --- p.139
Chapter 4.4.6 --- Neutralizing antibodies against IGF1R inhibit ICC differentiation --- p.140
Chapter 4.5 --- Discussion --- p.142
Chapter 4.6 --- Supplementary Materials and Methods --- p.147
Chapter 4.6.1 --- Cell Preparation and culture --- p.147
Chapter 4.6.2 --- In Vitro ICC differentiation --- p.148
Chapter 4.6.3 --- RNA expression analysis --- p.149
Chapter 4.6.4 --- Immunocytochemistry --- p.149
Chapter 4.6.5 --- PPC viability and cell count assays --- p.150
Chapter 4.6.6 --- IGF1 and insulin ELISA --- p.151
Chapter 4.6.7 --- Western blotting analysis --- p.152
Chapter 4.6.8 --- Neonatal streptozotocin regeneration model --- p.153
Chapter Chapter 5 --- General Discussion and Future Studies
Chapter 5.1 --- General Discussion --- p.165
Chapter 5.1.1 --- Proliferative effects and enhance expression of NGN3 by vitamin A and vitamin D on PPC --- p.166
Chapter 5.1.2 --- Induction of PPC derived ICCs by LSCs --- p.169
Chapter 5.1.3 --- Potential effects of liver stroma derived IGF1 on PPC derived ICCs differentiation --- p.172
Chapter 5.1.4 --- Significance of islet engineering in the management of diabetes --- p.174
Chapter 5.1.5 --- Conclusions --- p.176
Chapter 5.2 --- Future Studies --- p.177
Chapter Chapter 6 --- Reference
Reference --- p.180

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

Philosophiae Doctor - PhD
The use of ontologies in the mapping of gene expression events provides an effective and comparable method to determine the expression profile of an entire genome across a large collection of experiments derived from different expression sources. In this dissertation I describe the development of the developmental human and mouse eVOC ontologies and demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse, identifying transcription factor complexes, and exploring the mouse orthologs of human cancer/testis genes.Model organisms represent an important resource for understanding the fundamental aspects of mammalian biology. Mapping of biological phenomena between model organisms is complex and if it is to be meaningful, a simplified representation can be a powerful means for comparison. The implementation of the ontologies has been illustrated here in two ways.Firstly, the ontologies have been used to illustrate methods to determine clusters of genes showing tissue-restricted expression in humans. The identification of tissue restricted genes within an organism serves as an indication of the finetuning in the regulation of gene expression in a given tissue. Secondly, due to the differences in human and mouse gene expression on a temporal and spatial level, the ontologies were used to identify mouse orthologs of human cancer/testis genes showing cancer/testis characteristics. With the use of model systems such as mouse in the development of gene-targeted drugs in the treatment of disease, it is important to establish that the expression characteristics and profiles of a drug target in the model system is representative of the characteristics of the target in the system for which it is intended.

博士
國立陽明大學
環境與職業衛生研究所
102
Nonylphenol (NP) is an environmental hormone with proven estrogenic effects. Although its adverse effects on animals are well documented, the effects of NP exposure on humans remain unclear, and those on the human fetus are completely unknown. Additionally, human amniotic fluid-derived mesenchymal stem cells (AFMSCs) containing heterogeneous population of stem cells from various fetal organs have the capacity to differentiate into multiple lineages. AFMSCs may provide a promising cells source to identify the effects of NP on human embryo. The aim of this study was to establish a pregnant cohort to explore the association between maternal NP exposure level and birth outcomes. Furthermore, AFMSCs were treated with NP in 3 concentration levels by different time periods to assess possible effects on characteristics of AFMSCs and their Oct4, Nanog, and Sox2 expressions. A pregnant cohort was followed-up. Maternal urine samples were collected at the first, second, and third pregnancy trimester. The umbilical cord blood at delivery and amniotic fluid for those undergoing amniocentesis were collected as well. NP levels were determined for every specimen. Fetal development were determined by ultrasonic scan and birth outcomes were assessed by a pediatrician. AFMSCs were isolated and cultured by a two-stage culture protocol and then treated with NP (10, 50, 100μM) for 24, 48, and 72 hours, respectively. The effect of NP on the proliferation of AFMSCs was determined by the trypan blue dye exclusion assay. The total number of viable cells was calculated in the microscopy. Reverse transcription and quantitative PCR (polymerase chain reaction) were used to assess the Oct4, Nanog, and Sox2 expressions of AFMSCs. A total number of 201 pregnant women consented to participate. But complete data were available from 162 singletons for data analyzed. After adjusting for the urinary creatinine concentration, NP concentrations during the three trimesters were 4.27, 4.21, and 4.10 μg/g cre. respectively. The NP concentrations were 8.22 ng/ml and 5.91 ng/ml in amniotic fluid and cord blood respectively. No statistically significant correlations between urinary NP concentrations and gestational ages or maternal body weights were observed in a mixed-effects model using a generalised estimating equation. Maternal NP concentrations in each trimester were not associated with birth sex, preterm status, or low birth weight. Data analysed further stratified women by the median urinary NP concentrations in the first, second, and third trimester. Pregnant women with above-median concentration during the second trimester gave birth to the neonatal body with shorten length in the multivariable regression model (β=-0.47 cm, p value=0.04). Additionally, maternal weight gain was also low for women in the group with NP above-median concentration during the second trimester (β=-1.55 kg, p value=0.02). High NP level in the second trimester had a significant association with neonatal body weight especially in the primiparas (β=-182.49 g, p value=0.02). The odds ratios (ORs) of low infant birth weight, comparing pregnant women with different NP levels, was increased by decreasing the cutoff percentile for birth weight in the logistic regression model (ORs=1.18 for the 50th percentile, 2.12 for the 25th percentile, and 7.81 for the 10th percentile). When treating AFMSCs with NP, the growth rate of AFMSCs was dose- and time-dependently decreased. The higher level of NP as well as the longer of NP exposure, the stronger Oct4, Nanog, and Sox2 gene expressions were found (Oct4: 1.5 fold, Nanog: 2.6 fold, Sox: 3.2 fold). These results indicated that NP might influence the process of cellular differentiation or organgenesis during fetal development. This study demonstrates that maternal high NP exposure is associated with small for gestational age (SGA), decreased fetal body length at birth, and low maternal weight gain. Additionally, NP might influence the process of cellular differentiation or organgenesis during fetal development. The effects of this endocrine-disrupting substance on pregnant women and fetuses should be a concern during gestation.

Hintergrund: Der medizinische Fortschritt der letzten Jahrzehnte verbessert das Überleben insbesondere extrem kleiner Frühgeborener. Bei diesen stellt die adäquate Oxygenierung über die unreife Lunge den kritischsten Prozess in der klinischen Betreuung dar, an dessen Ende häufig eine Beeinträchtigung der postnatalen Lungenentwicklung und -reifung steht. Klinisch als Bronchopulmonale Dysplasie (BPD) imponierend, stellt diese Erkrankung die häufigste Folge der extremen Frühgeburtlichkeit dar und ist im weiteren Verlauf mit einer bedeutenden Langzeitmortalität und gesundheitsökonomischen Belastung verbunden. Außer der Vermeidung der Frühgeburtlichkeit existiert keine Therapie für BPD. Exogene Mesenchymale Stromazellen (MSC) erwiesen sich jedoch in Tiermodellen der BPD als therapeutisch ausgesprochen wirksam und stellen somit einen vielversprechenden Therapieansatz dar. Dennoch ist wenig über die Mechanismen der exogenen MSC-Wirkung in der frühgeborenen Lunge bekannt; ein Verständnis dieser ist jedoch unabdingbar für eine sichere und effektive Translation von MSC-basierten Therapien in die klinische Anwendung. Hypothese: Lungenresidente MSC sind an der normalen Lungenentwicklung beteiligt, werden durch Bedingungen, welche die zu frühe Geburt simulieren, beeinträchtigt, und tragen so zur Pathogenese der BPD bei. Exogene MSC unterstützen die lungenresidenten MSC in ihrer normalen Funktion und/oder schützen sie vor Schaden. Methoden und Resultate: Um mesenchymale Zellen aus human-fetalem Lungengewebe (FLMSC) zu isolieren, wurde eine Methode zur enzymatischen Gewebedissoziation mit anschließender selektiver Dichtegradientenzentrifugation entwickelt. Der überwiegende Mehrheit der isolierten Lungenmesenchymzellen wurde als MSC identifiziert. Damit ist mit der hier vorliegenden Arbeit erstmals die vollständige Beschreibung von humanen, fetalen Lungen-MSC gelungen. Nabelschnur (UC)MSC wurden durch enzymatischen Verdau der Wharton-Sulze gewonnen. Kultur der FLMSC in einer hypoxischen, den intrauterinen Bedingungen ähnlichen Atmosphäre resultierte in einem das Lungenwachstum stimulierenden Cytokin- und Genexpressionsmuster. Zudem produzierten die FLMSC für Lungenwachstum und -reifung unabdingbare Extrazellulärmatrixproteine. Unter Exposition gegenüber hyperoxischen Kulturbedingungen – welche die zu frühe Geburt mit anschließender Behandlung auf einer Neugeborenenintensivstation simulierten – begannen FLMSC einen Transdifferenzierungsprozess und sezernierten proinflammatorische und antiangiogene Signalmoleküle. Zudem wurde eine Reduktion der Produktion von für die Lungenentwicklung unabdingbaren Matrixproteinen beobachtet. FLMSC sendeten zudem “Danger-Signale” an andere Zellen, sobald sie Hyperoxie ausgesetzt wurden. Exogene UCMSC sezernierten in vitro große Mengen an Proteinen, welche Lungenzellen vor Schaden schützen und Lungenwachstum und -differenzierung unterstützen. Diskussion und Schlussfolgerung: Das Mesenchym der humanen fetalen Lunge am Ende der kanalikulären Entwicklungsphase besteht zum Großteil aus MSC, und nicht Fibroblasten. Das impliziert eine mesenchymale Stamm- und Progenitorzellhierarchie in der fetalen Lunge sowie bislang unbeschriebene zelluläre mesenchymale Transdifferenzierungsprozesse im weiteren intrauterinen Entwicklungsverlauf. In vitro wurde Evidenz für eine Beteiligung der endogenen Lungen-MSC an der normalen Lungenentwicklung generiert; eine Beteiligung an der Koordination von epithelialem und endothelialem Lungenwachstum und -reifung durch die endogenen MSC kann auf Grund der vorliegenden Daten angenommen werden. Nach Exposition gegenüber Hyperoxie entwickelten FLMSC einen die BPD unterstützenden Phänotyp. Exogene UCMSC besitzen das Potential, die in diesem Zustand fehlenden Faktoren bereitzustellen. Endogene pulmonale MSC sind daher potentielles Ziel und potentielle Effektorzellpopulation einer MSC-basierten Therapie für BPD. Dennoch sind weitere in vivo Experimente mit Tiermodellen der extremen Frühgeburtlichkeit unabdingbar, um die Rolle der endogenen MSC in der normalen, und insbesondere gestörten Lungenentwicklung zu verstehen und folgend potente, und vor allem sichere zellbasierte Therapeutika für unsere wohl verletzlichste Patientenpopulation – Frühgeborene – bereitzustellen.
Background: Despite great achievements in neonatal and perinatal medicine over the past decades, the immature lung remains the most critical organ to care for after premature birth. As a consequence, impairment of of postnatal lung development – bronchopulmonary dysplasia or BPD – remains the most common complication of extreme prematurity and a major healthcare burden. There is no therapy for BPD, except prevention of premature birth. Recently, exogenous mesenchymal stromal cells (MSC) have been shown to prevent and rescue impaired lung development in animal models. Understanding the mechanisms behind the beneficial action of these cells is crucial for a successful, safe, and effective clinical translation of these promising MSC-based cell therapies in neonates. Hypothesis: Endogenous lung-resident MSC contribute to normal lung development and become impaired in conditions resembling premature birth, thus playing a part in the pathogenesis of BPD. Exogenous MSC act by supporting and/or preserving the endogenous mesenchymal cell’s function. Methods and Results: Using lung tissue from aborted fetuses, a novel enzyme/density gradient technique was employed to obtain endogenous human fetal lung mesenchymal cells (FLMSC). The vast majority of the so-isolated cells fulfilled all criteria of MSC, making the herein presented work the first complete description of MSC from human fetal lung tissue. Human umbilical cord-derived (UC)MSC were isolated by enzymatic digestion of the Wharton’s jelly. When cultured in hypoxic atmospheres resembling intrauterine conditions, resident FLMSC exerted a gene expression- and cytokine profile supporting epithelial and endothelial lung development, and secreted extracellular matrix components crucial for normal lung growth. After exposure to hyperoxia – thus mimicking premature birth and subsequent treatment on a neonatal intensive care unit – FLMSC showed signs of transdifferentiation, acquired a pro-inflammatory / anti-angiogeneitic secretory profile, diminished production of crucial extracellular matrix components and send out danger signals to other cells. Conversely, UCMSC secreted various paracrine factors protecting lung cells, and proteins contributing to lung growth and alveolarization. Discussion and Conclusions: The human fetal lung’s mesenchyme at the late canalicular stage of development mainly consists of MSC rather than fibroblasts, thus implying a complex mesenchymal stem-/progenitor cell hierarchy and previously undescribed cellular transdifferentiation processes of human endogenous lung mesenchymal progenitors during late pregnancy. Evidence for a contribution of FLMSC to normal lung development was generated in vitro, suggesting a co-ordination of endothelial and epithelial cell fate by human endogenous lung MSC. When challenged with hyperoxia, FLMSC cells acquire a phenotype contributing to the pathogenesis of the BPD. Conversely, UCMSC harbor the potential to provide the factors that these damaged resident MSC lack to produce. The endogenous MSC may therefore represent a potential target of cell-based therapies of BPD. However, in vivo data obtained from premature animals is inevitable to gain further insights into the contribution of endogenous lung MSC to normal and disrupted lung development and to clinically translate potent and safe MSC-based therapeutics for our most vulnerable patient population - premature infants.

Dissertação de Mestrado em Estudos sobre a Europa apresentada à Universidade Aberta
Com o objetivo de verificar a existência de relação entre a diminuição da mortalidade fetal tardia e o aumento da mortalidade natal e natal precoce, foi consultada bibliografia relevante sobre o assunto e bases de dados disponíveis, em Portugal e na União Europeia. Apesar da limitação de informação, principalmente a nível dos dados europeus, verificaram-se vários indicadores relevantes, como reprodução medicamente assistida, doenças na grávida, como diabetes e hipertensão arterial, fatores de risco como obesidade e tabagismo, bem como no feto, o baixo peso à nascença e a gemelaridade. Foi também considerado o Índice de Desenvolvimento Humano como fator relevante na evolução. Apurados os números e em função dos resultados foi efetuada uma comparação entre Portugal e a União Europeia durante a década de 2004-2013. Esta comparação foi efetuada utilizando gráficos elaborados a partir das tabelas apuradas e permitiu concluir que até à data não existe evidência de tal relação, mas que a mesma não é de excluir, pelo que mais investigação sobre o tema deverá ser efetuada. Para tal deverão ser considerados alguns novos indicadores para futuros estudos.
In order to verify the existence of a relationship between the decrease of late foetal mortality and the increase of neonatal and early neonatal mortality, relevant literature on the subject and available databases, in Portugal and the European Union, were consulted. Despite limited information on European Union data, there were several relevant indicators, such as assisted reproduction, diseases in pregnant women as diabetes and hypertension, risk factors such as obesity and smoking, as well as in the foetus, low birth weight and gemelarity, The Human Development Index was also considered as relevant factor on this evolution. Upon the results, a comparison between Portugal and the European Union during the decade of 2004-2013 was made. This comparison was established using graphics from elaborated tables and came to the conclusion that up until now there is no evidence of such a relationship, but that it cannot be excluded, so more research on this subject should be made. Some new indicators are also mentioned for future studies.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

by Kwan Kwok Yiu.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1997.
Includes bibliographical references (leaves 145-155).
Acknowledgment --- p.i
Abstract --- p.ii
List of Tables --- p.vii
List of Figures --- p.x
List of Abbreviations --- p.xii
Chapter Chapter 1 --- Literature review
Chapter 1.1 --- Historical background --- p.1
Chapter 1.2 --- Chemistry of trans and cis fatty acids --- p.3
Chapter 1.3 --- Dietary source of trans fatty acids --- p.6
Chapter 1.4 --- Consumption of trans fatty acids among Western countries --- p.9
Chapter 1.5 --- Current health concern for excessive intake of trans fatty acids --- p.10
Chapter 1.6 --- Metabolism of trans fatty acids --- p.13
Chapter 1.6.1 --- Absorption --- p.15
Chapter 1.6.2 --- Oxidation --- p.15
Chapter 1.6.3 --- Incorporation --- p.16
Chapter 1.6.4 --- Selectivity --- p.17
Chapter 1.7 --- Impact of trans fatty acids on essential fatty acid metabolism --- p.19
Chapter 1.8 --- Desaturation and elongation of trans fatty acids --- p.21
Chapter 1.9 --- Trans fatty acids and neonatal growth --- p.23
Chapter Chapter 2 --- Amount of trans fatty acids in Hong Kong fast foods
Chapter 2.1 --- Introduction --- p.25
Chapter 2.2 --- Objective --- p.25
Chapter 2.3 --- Materials and methods --- p.26
Chapter 2.4 --- Results --- p.27
Chapter 2.5 --- Discussion --- p.31
Chapter Chapter 3 --- Cross-cultural study of trans fatty acids in human milk
Chapter 3.1 --- Introduction --- p.35
Chapter 3.2 --- Objective --- p.35
Chapter 3.3 --- Materials and methods --- p.36
Chapter 3.4 --- Results
Chapter 3.4.1 --- Dietary information --- p.38
Chapter 3.4.2 --- Fatty acid composition of Chinese and Canadian human milk --- p.40
Chapter 3.4.3 --- Difference between Chinese and Canadian human milk --- p.40
Chapter 3.4.4 --- Difference between Hong Kong and Chongqing Chinese human milk --- p.43
Chapter 3.4.5 --- The change in milk fat and LCPUFA as lactation progresses --- p.43
Chapter 3.5 --- Discussion
Chapter 3.5.1 --- Trans fatty acids in human milk --- p.46
Chapter 3.5.2 --- Content of LCPUFA in human milk --- p.47
Chapter 3.5.3 --- Content of 18:2n-6 in human milk --- p.48
Chapter 3.5.4 --- Fat content in Hong Kong and Chongqing Chinese human milk --- p.49
Chapter 3.6 --- Conclusion --- p.50
Chapter Chapter 4 --- Trans fatty acids and maternal and neonatal essential fatty acid metabolism
Chapter 4.1 --- Introduction --- p.51
Chapter 4.2 --- Objectives --- p.53
Chapter 4.3 --- Materials and methods --- p.53
Chapter 4.4 --- Results
Chapter 4.4.1 --- Experiment1
Chapter 4.4.1.1 --- Relationship between the trans fatty acids in maternal diet and those in milk --- p.64
Chapter 4.4.1.2 --- Relationship between the trans fatty acids in maternal diet and those in neonatal liver --- p.64
Chapter 4.4.1.3 --- Content of 20:4n-6 in milk and in neonatal liver relative to that in maternal diet --- p.72
Chapter 4.4.2 --- Experiment2
Chapter 4.4.2.1 --- Amount of trans fatty acids in rat milk --- p.75
Chapter 4.4.2.2 --- Trans fatty acids in rat liver phospholipids --- p.75
Chapter 4.4.2.3 --- Linoleic acid (18:2n-6) content in rat and its relation to maternal diets --- p.86
Chapter 4.4.2.4 --- Content of 20:4n-6 in rat milk --- p.86
Chapter 4.4.2.5 --- Content of20:4n-6 in rat liver --- p.89
Chapter 4.4.2.6 --- Suppression of the synthesis of 20:4t isomers in maternal and neonatal liver --- p.89
Chapter 4.5 --- Discussion
Chapter 4.5.1 --- Relationship between fatty acid composition of diet and that of milk --- p.93
Chapter 4.5.2 --- 20:4n-6 in rat milk --- p.95
Chapter 4.5.3 --- Transfer of trans fatty acids from maternal diet to neonatal liver phospholipids --- p.98
Chapter 4.5.4 --- The inhibitory effect of trans fatty acids on synthesis of 20:4n-6 in neonatal liver --- p.99
Chapter 4.5.5 --- Effect of 18:2n-6 supplement on 20:4n-6 level of neonatal liver --- p.101
Chapter 4.5.6 --- Suppression of 18:2n-6 supplement on synthesis of 20:4t isomers --- p.101
Chapter 4.6 --- Conclusion --- p.104
Chapter Chapter 5 --- Accumulation and turnover of trans fatty acids
Chapter 5.1 --- Introduction --- p.105
Chapter 5.2 --- Objective --- p.105
Chapter 5.3 --- Materials and methods --- p.106
Chapter 5.4 --- Results
Chapter 5.4.1 --- Accumulation of trans fatty acids in liver and adipose tissue --- p.108
Chapter 5.4.2 --- Selectivity of individual 18:2 trans isomersin liver and adipose tissue --- p.112
Chapter 5.4.3 --- Turnover of trans fatty acids --- p.112
Chapter 5.4.4 --- Accumulation and turnover of 18:lt in brain --- p.115
Chapter 5.5 --- Discussion
Chapter 5.5.1 --- Accumulation of trans fatty acids in liver and adipose tissue --- p.120
Chapter 5.5.2 --- Turnover of trans fatty acids --- p.122
Chapter 5.5.3 --- Accumulation and turnover of trans fatty acidsin brain --- p.124
Chapter 5.6 --- Conclusion --- p.125
Chapter Chapter 6 --- In vivo Oxidation of trans fatty acids in rat
Chapter 6.1 --- Introduction --- p.126
Chapter 6.2 --- Objective --- p.127
Chapter 6.3 --- Materials and methods --- p.127
Chapter 6.4 --- Results --- p.129
Chapter 6.4.1 --- Apparent oxidation of saturated fatty acids --- p.136
Chapter 6.4.2 --- Apparent oxidation of 18:lt relative to 18:ln-9 --- p.136
Chapter 6.4.3 --- Oxidation of 18:2t isomers relative to 18:2n-6 --- p.137
Chapter 6.4.4 --- Effect of 18:2n-6 supplement in PHCO diet on oxidation per se --- p.137
Chapter 6.5 --- Discussion --- p.138
Chapter 6.5.1 --- Oxidation of 18:lt and 18:2t isomers --- p.139
Chapter 6.5.2 --- Effect of 18:2n-6 supplement on oxidation per se --- p.140
Chapter 6.6 --- Conclusion --- p.141
General conclusion --- p.142
References --- p.145

Une diète faible en sodium donnée à des rates lors de la dernière semaine de gestation induit une diminution de l’expansion volumique, du diamètre des artères utérines et du poids des placentas comparativement à des rates témoins. Ces perturbations suggèrent une diminution de la perfusion placentaire affectant l’apport foetal en nutriments. Les ratons naissent avec une restriction de croissance intra-utérine (RCIU). Chez le foetus, le substrat énergétique cardiaque principal est le glucose via la glycolyse. À la naissance, la source principale d’énergie est l’utilisation des acides gras par la β-oxydation. Nous émettons l’hypothèse que dans ce modèle de RCIU, le coeur foetal répond à la diminution d’apport nutritionnel due à une atteinte maternelle en adaptant son métabolisme énergétique cardiaque à la baisse. Les rates gestantes (témoins et recevant la diète faible en sodium) sont sacrifiées au jour 22 de gestation (sur 23). Les coeurs foetaux sont prélevés afin de caractériser les protéines dites « limitantes » in vitro des voies de la glycolyse et de la β-oxydation. Les expressions protéiques de GLUT1, GLUT4, HK1, HK2, CPT2, CPT1β, cytochrome c, PFK1, PKM1/2, mesurées par immunobuvardage de type Western, sont similaires entre les coeurs des foetus RCIU et témoins, mâles et femelles. L’expression protéique de CPT1α est diminuée dans les coeurs des femelles RCIU seulement. Il n’existe aucune différence significative entre les différents groupes quant à l’activité enzymatique de PKM1/2. Nos résultats dressent un profil métabolique général suggérant que le sexe du foetus peut avoir un effet sur la réponse cardiaque foetale à une atteinte du volume sanguin maternel causée par la diète restreinte en sodium. Ce profil métabolique semble démontrer une atteinte du catabolisme des lipides. Afin de bien caractériser cette réponse du mécanisme énergétique, l’activité enzymatique des autres enzymes principales de la glycolyse (HK1, HK2, PFK1), le flux intra-mitochondrial d’acyl CoA à travers les CPTs ainsi que la quantité totale d’acétyl CoA devront être quantifiés.
A low sodium diet was given to pregnant rats during the last week of gestation. This diet diminished the maternal expansion of blood volume, the uterine arteries diameter, and placental weight, when compared to their controls. Together, these results suggest a lower placenta perfusion and a decreased output of nutrients to the fetus. The offspring of these pregnant rats were born with an intra-uterine growth retardation (IUGR). The fetal heart utilizes glucose through glycolysis as the major cardiac energy substrate. At birth, the principal source of energy switches to the oxidation of fatty acids, through β-oxydation. We hypothesized that within our IUGR model, the fetal heart could respond to a diminished nutritional intake due to the maternal input when a decreased cardiac energy metabolism was present. The pregnant rats of both groups (controls and on the low sodium diet) were sacrificed on day 22 of a 23 day gestation. The fetal hearts were then analyzed looking for signs of the limiting proteins glycolysis and β-oxidation. The GLUT1, GLUT4, HK1, HK2, CPT2, CPT1β, cytochrome c, PFK1, PKM1/2 proteins obtained through a Western immunoblot method were similar between the hearts of the IUGR and their control fetuses, whether they were male or female. The protein expression of CPT1α was lower only in female IUGR fetal hearts. There was no significant difference between the groups with respect to the enzymatic activity of PKM1/2. Our results suggest that the metabolic profile changes with regards to the fetus gender and could affect the fetal cardiac metabolism, due to a lower maternal blood flow caused by a sodium controlled diet, by diminishing its lipid metabolism and sparing glucose metabolism. To characterize the energy metabolism, the enzymatic activity of the other principal limiting enzymes glycolysis (HK1, HK2, PFK1), the intra-mitochondrial flux of acyl CoA through the CPTs and the total quantity of acetyl CoA must also be analyzed.