Children with Fetal Alcohol Spectrum Disorder have significantly delayed motor skills, and deficiencies in reflex development. The reasons underlying these motor deficits are not fully understood. The purpose of this thesis was to investigate the effect of embryonic exposure to ethanol (EtOH) on motor neuron and muscle fiber morphology and physiology in zebrafish. We observed that EtOH-exposed fish took longer to hatch and exhibited fewer swimming bouts in response to touch. Immunolabelling of motor neurons indicated that EtOH-exposed fish had significantly higher rates of motor neuron axon defects. Examination of muscle fiber morphology revealed that EtOH exposure resulted in significantly smaller muscle fibers. Miniature endplate current (mEPC) recordings from muscle fibers revealed that event amplitudes, rise times, half widths, frequencies and decay times were affected by EtOH exposure. These findings indicate that motor neurons and muscle fibers of zebrafish are affected by embryonic EtOH exposure, which may be related to deficits in locomotion. / Physiology, Cell and Developmental biology

Cardiovascular and metabolic diseases are still the primary cause of death and disability in modern society. Although genetic factors play a fundamental role in the development of these chronic conditions, the remarkable variability in an individual’s susceptibility to develop these pathologies cannot be completely explained by genetics. The early programming of adult diseases theory became established in the 1980’s, and is now supported by a growing body of evidence demonstrating that exposure to suboptimal environmental conditions during crucial periods of time can predispose an individual to the development of chronic conditions (including cardiovascular and metabolic diseases) later in life. Among the multitude of factors that can cause early programming, we have focused on the study of pregnancy complications leading to fetal hypoxia and causing intrauterine growth restriction (IUGR). To this end, we have used an animal model in which pregnant Sprague Dawley rats were exposed to either normal (∼21% O2) or hypoxic (∼11.5% O2) conditions during the last third of pregnancy. We then followed and studied the cardiovascular and metabolic characteristics of the offspring later in life. The studies presented in this thesis demonstrate that hypoxic prenatal insults have long-term consequences on cardiac structure, function and susceptibility to ischemia. We also demonstrated that programmed susceptibility to myocardial ischemia was associated with changes in cardiac energy metabolism and increased levels of myocardial oxidative stress. Moreover, we described the interaction between prenatal hypoxic insults, aging and sex differences in the later development of cardiovascular conditions. Additional studies presented in this thesis demonstrate that offspring born IUGR are more susceptible to develop most components of the metabolic syndrome when exposed to a high-fat (HF) diet. Furthermore, we also demonstrated that the exacerbated deleterious response to a HF diet described in offspring born IUGR can be prevented by postnatal administration of Resveratrol, which is a natural compound with anti-oxidant and anti-aging properties. In conclusion, the results presented in this thesis are an important contribution to the understanding of the long-term cardiovascular and metabolic effects of prenatal hypoxic insults causing IUGR and provide evidence regarding possible mechanisms and treatment alternatives that could be considered.

Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the α1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.

Ultrasound is the primary modality for fetal imaging, but Magnetic Resonance Imaging nowadays has a valuable complementary role as it often reveals findings that alter pregnancy management. Knowledge on some clinically relevant areas of the normal fetal development is still lacking, and this was the aim of this project. We wanted 1) to obtain reference MRI data of normal brain measurements before 24 gestation weeks (GW), 2) to study the development of the hippocampus, 3) to study the development of the ear and 4) to test the ability of MRI for evaluating the lip and palate. For this, we retrospectively analysed a database with 464 in vivo and 21 post mortem fetal MRI examinations. Study I evaluated a series of 70 normal fetuses. A table of normal brain measurements from 17 to 23 GW was built, the first in the literature that includes ages below 20 GW. Study II focused on the evolution of the hippocampus from 18 to 38 GW by evaluating 3 post mortem and 60 in vivo MRI examinations. Our results suggested this area to develop later and more asymmetrically than previously thought. Study III analysed a series of 122 normal MRI in vivo and 16 MRI post mortem. We described the development of the fetal ear in vivo for the first time in the literature, realizing that the value of MRI is limited by the size of the structures evaluated. In study IV, 60 brain-targeted MRI examinations of 55 normal fetuses and 5 fetuses with orofacial clefts were blindly reviewed by two readers, focusing on the lips and palates. Our results suggest a high accuracy of MRI in the evaluation of this area, regardless of fetal age or previous ultrasound findings. This thesis brings new knowledge on the normal development of the fetal brain and cranium.

Mammalian pregnancy is an immunological paradox. The foetus, which expresses both paternal and maternal cell-surface molecules, has to be protected from rejection by the maternal immune system. At the same time, the mother has to have an efficient immune defence and must provide her offspring with antibodies. The first part of this thesis investigates some of the mechanisms involved in the foetal avoidance of maternal rejection reactions. Placental absence of MHC class II expression, as well as a bias for Th2-cytokines at the maternal-foetal interface are suggested to be important for foetal survival. The results showed that placental MHC class II expression cannot be induced in vivo. Transfections of trophoblast cells with MHC class II genes, however, resulted in detectable MHC class II cell-surface expression, indicating that a post-transcriptional block does not exist in these cells. By using IL-4- and IL-10-double deficient mice, it was shown that neither maternal nor foetal expression of these cytokines were crucial for completion of allogeneic pregnancy. In the second part of the thesis, the effect of transmission of immunoglobulin G (IgG) from the mother to the offspring was studied. It was observed that viable maternal Ig-secreting cells occasionally infiltrated the B cell-deficient offspring and remained functional for long periods. In this study "green fluorescent mice" were used as a tool. Furthermore, neonatal ingestion of wild type milk increased the survival of adoptively transferred B-lineage cells in B cell-deficient mice, suggesting that suckling of IgG-containing milk could be used to facilitate B cell-reconstitution in B cell-deficient mice. Finally, results from studies on normal mice showed that absence of maternal IgG-transmission during their neonatal development resulted in elevated serum-IgG production, as well as enhanced immune reactions upon immunisations in adult life. This showed that maternal IgG can have long-term immunoregulatory effects in the offspring.

CYP27A1 and CYP7B1 are widely expressed in various human tissues and are two key enzymes involved in the pathways for conversion of cholesterol to bile acids. Also, CYP27A1 is involved in bioactivation of vitamin D3 and CYP7B1 plays a role in 7alpha-hydroxylation of dehydroepiandrosterone and other steroids. Both enzymes have been reported to be relevant to prostate cell proliferation. The current study examines the hormonal regulation of CYP27A1 and CYP7B1. CYP7B1 was shown to be regulated by estrogens and androgens in human embryonic kidney HEK293 and prostate cancer LNCaP cells. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. CYP7B1 regulation by estrogens may be of importance in fetal development and in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function. The regulation of CYP7B1 by sex hormones also suggests an important role for CYP7B1 in balancing prostate hormone levels in human cells. Results show that CYP27A1 can be regulated by dexamethasone, growth hormone, IGF-1, PMA, estrogens and androgens in liver-derived HepG2 cells. Dexamethasone, growth hormone and IGF-1 stimulated the promoter and endogenous activity of CYP27A1, whereas thyroid hormones and PMA inhibited CYP27A1. The regulatory effects of estrogens and androgens are different depending on the cell types. Thus, the results imply that human CYP27A1 gene is a target for estrogens and androgens, and the expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects. The mechanism for the dexamethasone-induced effect on the human CYP27A1 promoter was examined. A GRE was identified important for GR-mediated regulation of CYP27A1 transcriptional activity.

Enligt Statistiska Centralbyrån [SCB] föddes det år 2008 109 301 barn i Sverige. Utöver dessa föddes det cirka 600 barn som dött i livmodern innan förlossning. Intrauterin fosterdöd innebär att barn föds döda efter graviditetsvecka 22. Tidigare forskning belyser vikten av att barnmorskor har ett individuellt synsätt vid mötet med par vid intrauterin fosterdöd. Forskningen åskådliggör även hur föräldrar upplevt det stöd som givits under förlossningen. Det finns dock relativt lite forskning kring hur barnmorskor stödjer vid intrauterin fosterdöd. Syftet med detta arbete var därför att undersöka hur barnmorskor upplever sitt stöd i samband med förlossning vid intrauterin fosterdöd. Metoden som användes var kvalitativ innehållsanalys med en induktiv ansats. Fem barnmorskor intervjuades på två förlossningsavdelningar i Västra Götaland och Örebro län. I analysprocessen framkom tre huvudkategorier;individanpassa, ge tid och vara ett verktyg. Resultatet visar att barnmorskors stöd under förlossning inriktar sig på att låta paren styra informationsflödet och genom närvaro lyssna in behoven. Det var av vikt att ge paren tid i förlossningsarbetet men även tid i mötet med barnet. För att stödja bör barnmorskan känna sig trygg både i sig själv och i sin profession. En trygghet kring sin egen syn på döden var också av betydelse i stödet. / According to the Swedish bureau of Statistics [SCB] there were 109 301 children born in Sweden during 2008, in addition to these there where also approximately 600 stillborn children. Intrauterine fetal death means that the child has died in the womb after the 22nd week of pregnancy. Previous research highlights the importance of the midwives´ individual approach to parents with a stillborn child. Research also illustrates how parents experienced the support given during delivery. However, there is a limited amount of research done on how midwives support the parents of a stillborn child. The aim of this study was there for to investigate how midwives experience there support during delivery of a stillborn child. The method used was qualitative content analysis with an inductive approach. Five midwives were interviewed at two maternity wards in Västra Götaland and Örebro County. The analysis process revealed three main categories; adjust to the individual, to give time and to be a tool. The result shows that midwife´s support during delivery focuses on allowing couples to control the flow of information and by being present judge the parents´ needs. It is important to give the parents time both during delivery and to spend time with the stillborn child. To be able to support, the midwife need to feel confident both personally and professionally. It was also significant what the midwife´s own view on death was.

Für die Beurteilung der Hirnentwicklung erfolgte eine postmortale MRT- Untersuchung von totgeborenen Kindern im Rahmen einer virtuellen Autopsie. Besondere Berücksichtigung innerhalb dieses Datenmaterials fand die Oberflächenveränderung im Sinne der Entstehung von Gyri und Sulci. Weiterhin wurde geprüft, wie sich die morphologische Entwicklung und damit die Germination und Migration der Nervenzellen in diesem Schwangerschaftszeitraum in der MRT darstellen und beurteilen lässt. Im Ergebnis der Untersuchung zur Germination und Migration war vor allem zwischen der 18. und 25. SSW die zonale Gliederung des Hirnmantels mit “ventricular zone“, “intermediate zone“, “subplate zone“ und “cortical plate“ durch wechselnde Hypo –bzw. Hyperintensitäten in der MRT gut zu beurteilen. Der zeitlich geordnete Ablauf in der Entstehung einzelner Sulci konnte in dieser Arbeit unter anderem auch mittels eines atlasartigen Teils abgebildet werden. Dabei zeigte sich eine häufig etwas frühere Darstellbarkeit einzelner Sulci im postmortalen MRT im Vergleich zu den Studien an pränatalen MRT´s bei Schwangeren. Eine Streubreite des Auftretens der einzelnen Sulci von 2-3 Wochen bei gesunden Feten ist dabei zu berücksichtigen.

While all women hope to deliver a healthy baby, approximately 3-5% babies are affected by birth defects. Birth defects can occur naturally or be induced by teratogens. Alcohol is a known teratogen that causes fetal alcohol spectrum disorder (FASD), the most commonly known cause of neurobehavioural and neurodevelopmental deficits. Individuals affected with FASD are likely to be involved with or require additional assistance from healthcare, education, social services, and justice sectors. Due to this immense burden, effective prevention of FASD can have a major public impact. Prevention of FASD can occur at different levels: primary prevention (preventing alcohol-induced birth defects from occurring in the first place); secondary prevention (preventing alcohol-induced birth defects from developing or progressing); tertiary prevention (improving the outcome of individuals affected with FASD); and quaternary prevention (preventing another child from being affected with FASD). The objective of this thesis was to explore a multilevel birth defect and FASD prevention strategy. Primary prevention by was investigated by maternal multivitamin supplementation to optimize fetal growing conditions, as alcoholics are commonly deficient in nutrients. A meta-analysis of maternal multivitamin supplementation demonstrated a decreased risk for certain congenital anomalies and pediatric cancers. Secondary prevention was investigated by a randomized double-blinded placebo-controlled evaluating the ability of high doses of antioxidants (vitamin C and vitamin E) to mitigate the effects of prenatal alcohol exposure. The study was ceased due to safety concerns regarding high doses of vitamin C and vitamin E in preeclamptic studies. Tertiary prevention was investigated by anonymous meconium screening of babies of Grey-Bruce, Ontario residents delivering at or transferred to St. Joseph’s Health Care in London, Ontario. A 30% prevalence of fatty acid ethyl esters (FAEE) positive meconium was observed at this high-risk unit. Meconium screening is also a means of quaternary prevention since positive screens also identify mothers who were unable to stop consuming alcohol after 13 weeks of pregnancy, and therefore are at risk of delivering another child who is prenatally exposed to alcohol. The identification and engagement of these mothers into treatment programs constitutes primary prevention of FASD in subsequent pregnancies.

Children with fetal alcohol spectrum disorders (FASD) have deficits across many cognitive, behaviour and social domains. However, despite social difficulty being proposed as a main deficit following prenatal alcohol exposure, the nature of their deficient social behaviour is largely unknown. One process that may underlie difficulties in social functioning is poor social cognition, which refers to one’s understanding of the self, others and social world. The primary goal of this dissertation was to determine whether social cognitive deficits represent a core disability underlying the socio-behavioural problems of FASD using a bottom-up approach. The first level of this approach is represented by face processing. Global and independent face feature processing was compared between FASD and normal controls (NC) using experimental and clinical measures. Eye gaze processing was investigated next using experimental and clinical tasks. At the highest level of the bottom-up approach, social perspective taking, including theory of mind and empathy were examined, along with how these abilities related to parent-rated behaviour. Finally, the lowest level consisted of specific aspects of the social neural network. White matter in three limbic pathways was investigated using diffusion tensor imaging (DTI). Results generally supported the bottom-up approach of social cognition in FASD. These children showed impaired processing of face features, when matching mouth shapes and partially occluded identity, compared with NC. The FASD group was slower to process gaze and arrow cues, suggesting impaired attention shifting. Children with FASD also showed impairments in social perspective taking, including understanding false beliefs and empathy, and these impairments were related to parent-rated attention and social problems, and autistic-like traits. Deficits in theory of mind got worse with age in FASD and empathy showed distinct sex-related differences. Although no group differences were observed on DTI indices, groups did show different age-related changes in white matter. In conclusion, deficits at each level of the current bottom-up approach may underlie the social impairments in FASD and may contribute to their broader social behavioural phenotype. The results from this dissertation have potential to inform clinical practice and lead to more effective diagnostic and treatment approaches in FASD.