Thousands of animal experiments have demonstrated that androgenic hormones, such as testosterone, during the prenatal and early postnatal periods, masculinise and defeminise various neural and behavioural characteristics that differ by sex. Can these findings from animal experiments be generalised to human behaviour? Can early androgen exposure shape subsequent gender-related disorders in humans? Chapter 1 (Introduction) provides an overview of the literature. Chapter 2 (Kung et al., 2016a) is the first study to demonstrate that testosterone concentrations in saliva samples collected during the early postnatal testosterone surge at 1 to 3 months of age can negatively predict subsequent expressive vocabulary size (how many words a child can say) during toddlerhood. Notably, males typically have a smaller expressive vocabulary than do females during toddlerhood and a small expressive vocabulary is predictive of subsequent language difficulties, such as dyslexia and stuttering, which are more common in boys. Chapters 3 (Kung et al., 2016b) and 4 (Kung et al., 2016c) evaluate a popular theory of autism, the extreme male brain theory, which argues that heighted androgen exposure during early development causes the male preponderance in autism. To test the hypothesised relationship, Chapters 3 and 4 use different measures and study populations, including testosterone concentrations in amniotic fluid samples obtained prenatally and saliva samples obtained during the early postnatal testosterone surge in typically developing children, as well as examining the adjustment in children exposed to unusually high levels of androgens prenatally due to congenital adrenal hyperplasia (CAH), a rare clinical condition occurring in approximately 1 in 18,000 births. Findings from these two chapters converge to show that any relationship between early androgen exposure and subsequent development of autistic traits is small, non-existent, or unreliable, providing a much-needed clarification of the role of early androgen exposure in the aetiology of autism. Using data from a general population study, Chapter 5 (Kung et al., 2018a) is the first study to show that male-typical play behaviour in early childhood, a trait that has been linked to increased early androgen exposure in previous research, can positively predict adolescent physical aggression, which is typically higher in males than in females. This positive association between play and aggression supports potential influences of early androgen exposure, as well as socio-cognitive influences involved in gender development. Chapter 6 (Kung et al., 2018b) is the first study to compare emotional and behavioural adjustment in children with CAH, their unaffected siblings, and children in the general population. Findings from this chapter suggest that although within the families with a child with CAH there are generally no differences in emotional or behavioural problems between boys or girls with CAH and their unaffected same-sex siblings, both girls with CAH and their unaffected sisters are at risk of developing behavioural problems when compared with girls in the general population. Familial influences and social stigma may contribute to this gender-specific pattern of behavioural adjustment. Finally, Chapter 7 (Discussion) integrates the findings and previous research and provides directions for further research. Chapter References Chapter 2 Kung, K. T. F., Browne, W. V., Constantinescu, M., Noorderhaven, R. M., and Hines, M. (2016). Early Postnatal Testosterone Predicts Sex-Related Differences in Early Expressive Vocabulary. Psychoneuroendocrinology, 68, 111-116. Chapter 3 Kung, K. T. F., Constantinescu, M., Browne W. V., Noorderhaven, R. M., and Hines, M. (2016). No Relationship Between Early Postnatal Testosterone and Autistic Traits in 18 to 30-Month-Old Children. Molecular Autism, 7:15. Chapter 4 Kung, K. T. F., Spencer, D., Pasterski, V., Neufeld, S., Glover, V., O'Connor, T. G., Hindmarsh, P. C., Hughes, I. A., Acerini, C. L., and Hines, M. (2016). No Relationship Between Prenatal Androgen Exposure and Autistic Traits: Convergent Evidence from Studies of Children with Congenital Adrenal Hyperplasia and of Amniotic Testosterone Concentrations in Typically-Developing Children. Journal of Child Psychology and Psychiatry, 57, 1455-1462. Chapter 5 Kung, K. T. F., Li, G., Golding, J., and Hines, M. (2018). Preschool Gender-Typed Play Behavior at Age 3.5 Years Predicts Physical Aggression at Age 13 Years. Archives of Sexual Behavior, 47, 905-914. Chapter 6 Kung, K. T. F., Spencer, D., Pasterski, V., Hindmarsh, P. C., Neufeld, S. A. S., Hughes, I. A., Acerini, C. L., and Hines, M. (2018). Emotional and Behavioral Adjustment in 4- to 11-Year-Old Boys and Girls with Classic Congenital Adrenal Hyperplasia and Unaffected Siblings. Psychoneuroendocrinology. 97, 104-110.

Atherosclerosis, the leading cause of mortality in Western societies, affects large elastic arteries, causing focal deposition of proliferative inflammatory and lipid-laden cells within the artery. Several risk factors have been causally implicated in the ???reaction to injury??? hypothesis first described by Ross in 1969. The ???injury??? sustained by endothelial cells may be either mechanical or chemical. Environmental factors have a role in the production of chemical agents that are injurious to the endothelium. Mechanical stresses such as wall tensile stress are proportional to systemic blood pressure and pulse pressure. Essentially, these systemic pressures are fairly evenly distributed throughout the circulation. However, atherosclerotic lesions characteristically occur at focal sites within the human vasculature; at or near bifurcations, within the ostia of branch arteries and at regions of marked or complex curvature, where local haemodynamic abnormalities occur. The most discussed haemodynamic factor seems to be low or highly oscillating wall shear stress which exists on the outer wall of bifurcations and on the inner aspect of curving vessels. The magnitude of these haemodynamic forces may not be great but the subtleties of their variable spatial distribution may help to explain the multifocal distribution of atherosclerotic plaques. With the altered haemodynamics there is endothelial injury and phenotypic changes in the endothelium result, which in turn lead to endothelial cell dysfunction. These haemodynamic variables are difficult to measure directly in vivo. In this work a novel model is developed utilising human autologous vein bypass grafts as a surrogate vessel for the observation of pathological structural changes in response to altered haemodynamics. The influence of haemodynamic factors (such as wall shear stress) in the remodeling of the vein graft wall and the pathogenesis of Myointimal Hyperplasia (MIH) and resultant wall thickening in femoral bypass grafts is analysed. The haemodynamic determinants of MIH (which have been established in many animal models) are similar to those implicated in atherosclerosis. The accelerated responses of the vein (Intimal hyperplasia develops much more rapidly than atherosclerotic lesions in native vessels) make it an ideal model to expediently examine the hypothesised relationships prospectively in an in vivo setting. Furthermore, the utilisation of in vivo data acquired from non-invasive diagnostic methods (such as Magnetic Resonance Angiography (MRA) and Duplex ultrasound) combined with the application of state-of-the-art Computational Fluid Dynamic (CFD) techniques makes the model essentially non-invasive. The following hypotheses are examined: 1) regions of Low shear and High tensile stress should develop disproportionately greater wall thickening, 2) regions of greater oscillatory blood flow should develop greater wall thickening, and 3) regions of lower wall shear should undergo inward (or negative) remodelling and result in a reduction in vessel calibre. The conclusions reached are that abnormal haemodynamic forces, namely low Time-averaged Wall Shear Stress, are associated with subsequent wall thickening. These positive findings have great relevance to the understanding of vein graft MIH and atherosclerosis. It was also evident that with non-invasive data and CFD techniques, some of the important haemodynamic factors are realistically quantifiable (albeit indirectly). The detection of parameters known to be causal in the development of graft intimal hyperplasia or other vascular pathology may improve ability to predict clinical problems. From a surgical perspective this might be employed to facilitate selection of at-risk grafts for more focused postoperative surveillance and reintervention. On a broader stage the utilisation of such analyses may be useful in predicting individuals at greater risk of developing atherosclerotic deposits, disease progression, and the likelihood of clinical events such as heart attack, stroke and threat of limb loss.

Different species are separated by pre-zygotic reproductive barriers which impede gene flow between them. Rarely, when pre-zygotic barriers break down, interspecific hybrids are produced that display abnormal phenotypes, collectively called hybrid dysgenesis effects. Interspecies hybrid placental dysplasia (IHPD) in the genus Mus is a very consistent X-linked hybrid dysgenesis effect. Reproductive cloning and mutation of the gene Esx1 lead to placental hyperplasias with phenotypic similarities to IHPD. Comparative gene expression analysis of these three different models of placental hyperplasia showed that different mechanisms underlie these placental hyperplasias. We also identified several genes for which roles in placentation had not been studied earlier. We screened five of these genes, Car2, Ncam1, Fbln1, Cacnb3 and Cpe for their functions in placentation. Analysis of the spatio-temporal expression patterns of these genes during mouse placental development showed that they are ectopically expressed in IHPD placentas. Placental phenotype and gene expression was then studied in mice mutant for these genes. Our results show that complicated by the expression of functional counterparts, deletion of these genes failed to produce any consistent phenotype. Incompletely penetrant phenotypes were found in Cacnb3 and Cpe mutants. The Cpe mutant placentas recapitulated some IHPD phenotypes, despite co-expression of Cpd, a functionally redundant gene. Deregulated expression of Cpe and Cpd prior to manifestation of IHPD phenotype indicated that these are causally involved in IHPD and might be speciation genes in the genus Mus. We found that AT24 placentas also exhibit deregulated expression of these genes and could be used as a model to study IHPD. We tried rescuing the AT24 placental phenotype, by decreasing the expression of the over expressed genes. Normalization of transcript levels of these genes did not rescue the AT24 phenotype, thus indicating that up-regulation of these genes is a down-stream event in the generation of IHPD.

Placental development can be disturbed by various factors, such as mutation of specific genes or maternal diabetes. Our previous work on interspecies hybrid placental dysplasia (IHPD) and two additional models of placental hyperplasia, cloned mice and Esx1 mutants, showed that many genes are deregulated in placental dysplasia. Two of these candidate placentation genes, Cpe and Lhx3, were further studied. We performed in situ hybridization to determine their spatio-temporal expression in the placentas and placental phenotypes were analyzed in mutant mice. Our results showed that the placental phenotype in Cpe mutant mice mimics some IHPD phenotypes. Deregulated expression of Cpe and Cpd, a functionally equivalent gene, prior to the manifestation of the IHPD phenotype, indicated that Cpe and Cpd are potentially causative genes in IHPD. Lhx3 mutants lacked any placental phenotype. Deletion of Lhx3 and Lhx4 together caused an inconsistent placental phenotype which did not affect placental lipid transport function or expression of Lhx3/Lhx4 target genes. Down regulation of Lhx3/Lhx4 did not rescue the placental phenotype of AT24 mice and hence could be excluded as causative genes in IHPD. Analysis of placental development in diabetic mice showed that severe maternal diabetes resulted in fetal intrauterine growth restriction (IUGR) without any change in placental weight and lipid transport function. The diabetic placentas however exhibited abnormal morphology. Gene expression profiling identified some genes that might contribute to diabetic pathology. In another study, it was found that the heterochromatin protein CBX1 is required for normal placentation, as deletion of the gene caused consistent spongiotrophoblast and labyrinthine phenotypes. Gene expression profiling and spatio-temporal expression analysis showed that several genes with known function in placental development were deregulated in the Cbx1 null placenta.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Die Restenose stellt ein zentrales Problem der interventionellen Kardiologie dar und ist häufigste Komplikation nach perkutanen Angioplastieverfahren. Hauptursache dieser Wiederverengung des Gefäßes ist die Bildung einer Neointima durch die Proliferation transdifferenzierter vaskulärer glatter Muskelzellen und die Sekretion extrazellulärer Matrix. Die Entstehung reaktiver Sauerstoffspezies (ROS) und die Entzündungsreaktion nach der Gefäßverletzung werden als frühe, die Neointimabildung induzierende Prozesse diskutiert. Im Rahmen dieser Arbeit wurden mehrere Projekte bearbeitet, die Aufschluss über die während der Neointimabildung statt findenden Prozesse geben sollen. Mit Hilfe eines Verletzungsmodells der murinen Femoralarterie wurde der Einfluss der Entzündung und der ROS-Bildung auf die Neointimabildung in der Maus untersucht. Die Behandlung mit dem mitochondrialen Superoxiddismutase-Mimetikum MitoTEMPO verminderte die Bildung der Neointima besser, als die Behandlung mit dem globalen ROS-Fänger N-Acetylcystein. Die stärkste Hemmung der Neointimabildung wurde jedoch durch die Immunsuppression mit Rapamycin erreicht. Interferon-γ (INFγ) ist ein wichtiges Zytokin der Th1-Immunantwort, das in Folge der Gefäßverletzung freigesetzt wird und die proinflammatorischen Chemokine CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) und CXCL11 (I-TAC, Interferon inducible T cell-Chemoattractant) induziert. CXCL9, CXCL10 und CXCL11 sind Liganden des CXC-Chemokinrezeptors 3 (CXCR3) und locken chemotaktisch CXCR3 positive Entzündungszellen zum Ort der Gefäßverletzung. Daher wurde die spezielle Bedeutung des Chemokins CXCL10 in der Restenose untersucht. Dazu wurden CXCL10-defiziente Mäuse dem Femoralisverletzungsmodell unterzogen und die Gefäße nach 14 Tagen morphometrisch und immunhistologisch untersucht. CXCL10-Defizienz führte in Mäusen zu einer verminderten Neointimabildung, die mit einer verringerten Inflammation, Apoptose und Proliferation im verletzten Gefäß korrelierte. Neben der Inflammation beeinflusst aber auch die Reendothelialisierung der verletzten Gefäßwand die Restenose. Interessanterweise war im Vergleich zu Wildtyp-Mäusen in den CXCL10-Knockout-Mäusen auch die Reendothelialisierung erheblich verbessert. Offensichtlich ist das CXCR3-Chemokinsystem also in völlig unterschiedliche biologische Prozesse involviert und beeinflusst nicht nur die Bildung der Neoimtima durch die Förderung der Entzündung, sondern auch die Unterdrückung der Reendothelialisierung der verletzten Gefäßwand. Tatsächlich wird der CXCR3 nicht nur auf Entzündungszellen, sondern auch auf Endothelzellen exprimiert. Zur separaten Untersuchung der Rolle des CXCR3 in der Inflammation und der Reendothelialisierung wurde im Rahmen dieser Arbeit damit begonnen konditionelle CXCR3-Knockout-Mäuse zu generieren, in denen der CXCR3 entweder in Entzündungszellen oder in Endothelzellen ausgeschaltet ist. Zum besseren Verständnis der molekularen Mechanismen, mit denen der CXCR3 seine Funktionen vermittelt, wurde zudem untersucht ob dieser mit anderen G-Protein-gekoppelten Rezeptoren (GPCR) interagiert. Die Analyse von Coimmunpräzipitaten deutet auf eine Homodimerisierung der beiden CXCR3 Splicevarianten CXCR3A und CXCR3B, sowie auf die Heterodimerbildung von CXCR3A und CXCR3B mit sich, sowie jeweils mit CCR2, CCR3, CCR5 und den Opioidrezeptoren MOR und KOR hin. Die getestete Methode des Fluoreszenz-Resonanz-Energietransfers (FRET) erwies sich jedoch als ungeeignet zur Untersuchung von CXCR3, da dieser in HEK293T-Zellen nicht korrekt transient exprimiert wurde. Insgesamt deuten die Ergebnisse dieser Arbeit darauf hin, dass das CXCR3-Chemokinsystem eine zentrale Rolle in unterschiedlichen, die Neointimabildung beeinflussenden Prozessen spielt. Damit könnten der CXCR3 und insbesondere das Chemokin CXCL10 interessante Zielmoleküle in der Entwicklung neuer verbesserter Therapien zur Verhinderung der Restenose darstellen. / Restenosis represents a central problem after coronary angioplasty procedures and is caused by intimal hyperplasia, also called neointima, as a result of transdifferentiation, proliferation of vascular smooth muscle cells and secretion of extracellular matrix. Formation of reactive oxygen species (ROS) and inflammation after vascular injury caused by angioplasty are discussed as early inducers of neointima formation. In several projects the processes causing the development of intimal hyperplasia were investigated. First of all, the impact of inflammation and ROS in neointima formation was investigated using the mouse femoral injury model. The mitochondrial superoxide dismutase mimetic mitoTEMPO could reduce neointima formation better than the global ROS scavenger N-acetylcystein. However, the strongest reduction of neointima formation was achieved by the treatment with the immunosuppressant rapamycin. Interferon-γ(INFγ) is a major cytokine of the Th1 immune response. It is released as a result of vessel injury and induces the proinflammatory chemokines CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) and CXCL11 (I-TAC, Interferon inducible T-cell-Chemoattractant), which are ligands of the CXC chemokine receptor 3 (CXCR3) and by this chemotactically recruit CXCR3 positive cells to the site of vessel injury. In this work the special role of CXCL10 in restenosis was investigated. Therefore, CXCL10 decient mice underwent the mouse femoral injury model. The vessels were analysed morphometrically and immunohistologically 14 days after injury. CXCL10 deciency lead to decreased neointima formation that correlated with a reduced recruitment of inflammatory cells as well as diminished numbers of apoptotic and proliferating cells at the site of vessel injury. In addition to inflammation the reconstitution of the endothelium has also impact on the development of restenosis. Interestingly reendothelialisation was strongly improved in CXCL10 decient mice compared to wildtype mice. Obviously the CXCR3 chemokine system is involved in different biological prosesses and impairs neointima formation on one hand by the advancement of inflammation and on the other hand by the suppression of reendothelialisation. In fact the CXCR3 is not only expressed on inflammatory cells but also on endothelial cells. To investigate the role of CXCR3 in inflammation and reendothelialisation separatly the generation of conditional CXCR3 knockout mice with a CXCR3 knockout in T-cells or endothelial cells was started in an additional project. For a better understanding of the molecular mechanisms on which the CXCR3 mediates its biological functions the protein-protein interactions of the CXCR3 with other G-protein coupled recteptors (GPCR) was analysed. Coimmunoprecipitation showed homodimerization of the CXCR3 splice variants CXCR3A and CXCR3B, as well as heterodimerization of CXCR3A and CXCR3B with each other and with the chemokine receptors CXCR4, CCR2, CCR3, CCR5 and the opioid receptors MOR and KOR. The additional tested Fluorecence resonance energy transfer (FRET) method proved to be not suitable to measure interactions of CXCR3, since this receptor could not be expressed correctly on the cell surface after transient transfection. To summarise, the results indicate that the CXCR3 chemokine system plays a central role in different processes that mediate neointima formation. Thus, the CXCR3 and especially the chemokine CXCL10 could be interesting therapeutic targets in the development of new or improved treatments to reduce the risk of restenosis.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Vascular stents have become a standard for treating atherosclerosis due to distinct advantages in trauma and cost with other surgical techniques. Unfortunately, the therapy is hindered by the risk of a new blockage (termed restenosis) developing in the treated artery. Clinical studies have indicated that stent design is a major risk factor for restenosis, with failure rates varying from 20 to 40% for bare metal stents. Subsequently, there has been a significant effort devoted to reducing failure rates by covering stents in polymer coatings in which anti-proliferative drugs are embedded, however complications have arisen (e.g. incomplete endothelization, lack of success in peripheral arteries, lack of long-term follow-up studies) that have limited the success of this technology. It has been thought that restenosis is directly related to the mechanical conditions that vascular stents create. Moreover, it has been hypothesized that stents that induce higher non-physiologic stresses result in a more aggressive pathobiological response that can lead to restenosis development. In this study, a combination of computational modeling and in vivo analysis were conducted to investigate the artery stent-induced wall stresses, and subsequent biological inflammatory response. In particular, variations in stent design were investigated as a means of examining specific stent design criteria that minimize the mechanical impact of stenting. Collectively, these data indicate that stent designs that subject the artery wall to higher stress values result in significantly more neointimal tissue proliferation, therefore, confirming the aforementioned hypothesis. Moreover, this work provides valuable insight into the role that biomechanics can play in improving the success rate of this percutaneous therapy and overall patient care.

1. INTRODUCTION 1.1. Work actuality In recent years the number of males with urination disorders mostly induced by prostate pathology has significantly increased [Gas et al., 1998; Khan, Khan et al., 2005; Amaral, Coeli et al., 2004]. Benign prostate hyperplasia (BPH) affects males at a much younger age making them complain of urination disorders of different types and intensity. It is quite a common pathology among males (which is) conditioned by life style and nutrition [Saga & Sugimura, 2004; Shabbir & Mumtaz, 2004; Cambell, 2005]. Clinical symptoms of BPH are not restricted only to urination complaints. With an increase of age of males, the balance of androgens and estrogens undergoes changes in the male organism, affecting the power of libido. According to C.M.Porth, 2005, two theories related to senility have been created, trying to explain biological processes occurring with age: The first theory is related to the so called genetically programmed changes. This theory affirms that changes brought on by aging, are genetically predetermined. Another theory is called the stochastic theory which says that all changes are of accidental nature, i.e. the body undergoes accidental changes. There is one more theory, the so-called neuroendocrinic theory of senility. This theory involves three basic factors of the process of aging: 1) facilitated degradation of hormones, 2) decreased synthesis and secretion of hormones, 3) diminished sensitivity of “target” receptors to... [to full text]

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.