Genetics of litter size and prenatal survival in pigs

Female reproductive performance is a critical component of sustainable pig production systems. There is abundant evidence of genetic variation in these traits among pig breeds. The aims of this study were to identify quantitative trait loci (QTL) affecting reproductive traits and to identify and characterise positional candidate gene(s) underlying the QTL. A Large White - Meishan F2 population was scanned for QTL with effects on reproductive traits. This analysis revealed 13 putative QTLs on seven different chromosomes with effects on five different traits: ovulation rate (OR), teat number (TN), prenatal survival (PS), total born alive (TBA) and litter size (LS). QTL for PS and LS on chromosome 8 were fine mapped and Secreted Phosphoprotein 1 (SPP1) confirmed as a candidate gene. A genome-wide association study was performed on a diverse population of different breeds and crosses lines, for reproductive traits including LS, TBA, number of stillborn piglets, and number of mummified piglets. Fourteen SNPs were found significantly associated with reproductive traits. The functional study of SPP1 examined the hypothesis that differences in foetal growth may be associated with the effectiveness of conceptus attachment, as measured by SPP1 expression. Patterns of SPP1 mRNA and protein expression in placental and uterine tissues supplying the smallest and a normal-sized foetus from the same uterus were examined in Large White-Landrace (LW-LR), Large White (LW) and Meishan (MS) females 40 and 45 of pregnancy. The smallest LW-LR foetuses tended to have a higher level of SPP1 mRNA in endometrium tissue compared to the normal-sized foetuses. However, placenta expression was higher in the normal-sized foetuses compared to the smallest ones. SPP1 protein levels in normal sized foetuses were significantly higher than in the smallest litter mates for all the tissues. Significantly higher levels of SPP1 mRNA and protein were found in MS compared to LW. In both breeds, significant differences between sizes were found in some tissues, with similar expression patterns in respect to size, for both mRNA and protein in endometrial tissues when compared to contemporary LW. In placenta, the direction of the expression differed between breeds, with a higher expression of mRNA and protein in the normal-sized MS foetuses and in the smallest sized LW foetuses. The comparison of SPP1 expression between different foetal sizes and different breeds revealed associations between breed, foetal size, and SPP1 protein, factors implicated in PS and LS. These results together with the genetic evidence indicate that the potential role of SPP1 in placental and foetal development merits further investigation

\u3ci\u3eAMBYSTOMA\u3c/i\u3e: PERSPECTIVES ON ADAPTATION AND THE EVOLUTION OF VERTEBRATE GENOMES

Tiger salamanders, and especially the Mexican axolotl (Ambystoma mexicanum), are important model organisms in biological research. This dissertation describes new genomic resources and scientific results that greatly extend the utility of tiger salamanders. With respect to new resources, this dissertation describes the development of expressed sequence tags and assembled contigs, a comparative genome map, a web-portal that makes genomic information freely available to the scientific community, and a computer program that compares structure features of organism genomes. With respect to new scientific results, this dissertation describes a quantitative trait locus that is associated with ecologically and evolutionarily relevant variation in developmental timing, the evolutionary history of the tiger salamander genome in relation to other vertebrate genomes, the likely origin of amniote sex chromosomes, and the identification of the Mexican axolotl sex-determining locus. This dissertation is concluded with a brief outline of future research directions that can extend from the works that are presented here

High resolution physical and comparative maps of horse chromosomes 14 (ECA14) and 21 (ECA21)

In order to identify genes or markers responsible for economically important traits in the horse, the development of high resolution gene maps of individual equine chromosomes is essential. We herein report the construction of high resolution physically ordered radiation hybrid (RH) and comparative maps for horse chromosomes 14 and 21 (ECA14 and ECA21). These chromosomes predominantly share correspondence with human chromosome 5 (HSA5), though a small region on the proximal part of ECA21 corresponds to a ~5Mb region from the short arm of HSA19. The map for ECA14 consists of 128 markers (83 Type I and 45 Type II) and spans a total of 1828cR.Compared to this, the map of ECA21 is made up of 90 markers (64 Type I and 26 Type II), that segregate into two linkage groups spanning 278 and 760cR each. A total of 218 markers provide on average one marker every 0.9Mb along the length of the two equine chromosomes. This represents a 5-fold improvement over the previous maps. Of greater significance is the ~8-fold increase in the density of Type I loci that provide a comprehensive and finely aligned map for the two chromosomes in relation to homologues in a range of evolutionarily distantly related species, viz., human, chimpanzee, mouse, rat, dog, cattle, pig, cat and chicken. The orientation and alignment of the linkage groups was strengthened by 28 new FISH localizations, of which 27 are gene-specific (22 from HSA5 and 5 from HSA19). Comparative analysis between the horse and human reveals that the order of genes on HSA5 is remarkably well conserved in the horse, with an evolutionary break/fusion point that could be correlated to a ~2Mb region between 68.5  70.9Mb positions on HSA5. Among the species analyzed to date, the HSA5 and 19p neighboring segment combination is unique to Perissodactyls and Cetartiodactyls, but, in the Perissodactyls, the portion of HSA5 that corresponds to this combination is HSA5p  q13, while in the Cetartiodactyls, it is HSA5q13  qter. This leads us to postulate that this neighboring segment combination arose as separate events during the divergence of Perissodactyls and Cetartiodactyls from a common ancestor

Ansätze zur Untersuchung der genetischen Ursachen für den Erbfehler Stülpzitze beim Schwein

Der Gesäugekomplex ist bei Säugetieren für die Versorgung der Nachkommen mit Nährstoffen und für die Immunisierung über die Kolostralmilch von Bedeutung. Die ersten Anlagen der Zitzen sind in der fötalen Entwicklung früh erkennbar. Zum Zeitpunkt der Geburt sind die Zitzenkuppen bei Neugeborenen bereits erkennbar. Ab der Pubertät entwickelt sich das eigentliche Drüsengewebe bei den weiblichen Säugern. In der Trächtigkeit beginnt ein Zyklus von Wachstum, funktioneller Differenzierung und Regression. Die Funktionsfähigkeit der Zitze kann durch endogene und exogene Faktoren beeinflusst werden. Störungen der Entwicklung der Zitze können zu unterschiedlich ausgeprägten Defekten führen. Der wichtigste angeborene Zitzendefekt beim Schwein ist die Stülpzitze, die mit Frequenzen zwischen 3 und 30 % in kommerziellen Schweinerassen gefunden wird. Dieser Erbfehler hat erhebliche negative ökonomische Auswirkungen auf die Schweineproduktion. Der Vererbungsmodus und die Anzahl der Gene, die an dieser Fehlentwicklung des Zitzenkomplexes beteiligt sind, sind noch unbekannt. Es werden verschiedene Methoden wie Assoziationsanalysen, Kopplungskartierung und Expressionsanalysen zur Detektion der Ursachen des Defektes angewandt. In der vorliegenden Arbeit wurde im Rahmen einer Kopplungskartierung eine QTLAnalyse über achtzehn Chromosomen in Schweinen durchgeführt. Damit sollten die in einer Versuchspopulation detektierten QTL in kommerziellen Familien der Schweinerassen Deutsche Landrasse, Deutsches Edelschwein und deren Kreuzungstiere bestätigt werden. Darüber hinaus wurden die Chromosomen 1, 2, 3, 4, 6 und 14 mit zusätzlichen Markern feiner kartiert. Dabei konnten Regionen auf den Chromosomen 2, 3, 4, 6 und 11 bestätigt werden. Auf Chromosom 11 und Chromosom 6 konnten bei der Analyse aller kommerziellen Familien signifikante non parametric likelihood (NPL) Werte gefunden werden. Verschiedene positionelle und funktionelle Kandidatengene wurden in den Regionen sowie in den vergleichenden Regionen auf den humanen Chromosomen bestimmt. Eine Assoziationsanalyse der Marker bestätigte teilweise die gefundenen QTL. Bei weiteren signifikant assoziierten Markern konnten übereinstimmende Positionen zu Kandidatengenen deren Einfluss auf die Entwicklung der Stülpzitze bestätigen.Detection of the genetic causes for the heritable inverted teat defect in pig The mammary gland plays a role for the feeding of the offspring with nutrients and for the immunisation via colostrum in mammals. The first appearance of the mammary gland is seen in the early embryonal development. At birth, the nipple of the offspring is already visible, whereas the development of the ultimate mammary gland tissue starts during puberty in the female. During pregnancy a cycle of growth, functional differentiation and regression starts. The functional capability of the teat can be affected by endogenous and exogenous factors. Dysfunction of teat development may lead to different expressed defects. The most important heritable teat defect in pigs is the inverted teat, which is found with frequencies between 3 to 30% in commercial pig breeds. This heritable defect has a considerable negative impact for the economy of the pig production. The mode of inheritance and the number of genes involved in this aberration of the mammary gland are still unknown. Different methods such as association analysis, linkage mapping and expression analysis were used to detect the cause of the defect. The aim of this study was to detect QTL by linkage mapping. A QTL analysis was performed over eighteen chromosomes, to confirm QTL found in an experimental population in animals of the commercial breeds German Landrace, Large White, and their crossbreeds. Moreover, the chromosomes 1, 2, 3, 4, 6, and 14 were fine mapped with additional markers. Regions on the chromosomes 2, 3, 4, 6, and 11 could be confirmed. Significant non-parametric likelihood (NPL) scores were detected on the chromosomes 6 and 11 by analysis of all commercial families. A number of positional and functional candidate genes was detected in the regions and the comparative regions on human chromosomes. Some of the detected QTL could be confirmed by conduction of an association analysis. Some significant associated markers were mapped in consistency with earlier investigated candidate genes for which the detected significant association to the inverted teat defect could be confirmed as well

Glaucoma

This book addresses the basic and clinical science of glaucomas, a group of diseases that affect the optic nerve and visual fields and is usually accompanied by increased intraocular pressure. The book incorporates the latest development as well as future perspectives in glaucoma, since it has expedited publication. It is aimed for specialists in glaucoma, researchers, general ophthalmologists and trainees to increase knowledge and encourage further progress in understanding and managing these complicated diseases

Investigating the effect of chronic activation of AMP-activated protein kinase in the liver

Obesity and its associated complications, are an increasing global problem. Non-alcoholic fatty liver disease (NAFLD), the hepatic component of the Metabolic Syndrome currently affects an estimated 25% of the world’s population, with 2% of those affected dying from a NAFLD liver related cause, such as hepatocellular cancer (HCC). AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis that regulates anabolic and catabolic pathways in response to ATP depletion has received substantial attention as a therapeutic target in treatment of the Metabolic Syndrome. More recently, a growing number of studies have focussed on the role of AMPK in cancer. This study has characterised a novel, activating mutation in the γ1 subunit of AMPK (D316A). Mice which express this mutation specifically in the liver (D316-Tg) have been used to investigate the effects of chronic hepatic AMPK activation under basal conditions and in response to the metabolic stresses of hypercaloric, high fat and lipogenic, high fructose diets. D316A-Tg mice have also been crossed with mice with liver specific Phospatase and tensin homologue (Pten) loss to evaluate the role of AMPK in NAFLD related HCC. Fatty acid synthesis was reduced in hepatocytes isolated from D316A-Tg mice and these mice were protected from hepatic steatosis under lipogenic conditions. Fatty acid oxidation in hepatocytes was unaffected by increased AMPK activity and correspondingly D316A-Tg mice were not protected from hepatic lipid accumulation, following high fat feeding. Increased hepatic AMPK activity also attenuated steatohepatitis and had a marked inhibitory effect on tumourgenesis in mice with liver specific Pten loss. Further work is required to elucidate the precise role of AMPK in tumour development. However, the findings of this study suggest that activation of AMPK in the liver may inhibit NAFLD progression and tumourgenesis and support the development of pharmacological, specific AMPK activators for use in these settings.Open Acces

Investigating the effects of platelet derived growth factor on the microRNA cargo content and function of vascular smooth muscle cell-derived extracellular vesicles

Invasive revascularisation procedures such as coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are used to treat advanced atherosclerosis. The CABG procedure is the most common type of cardiac surgery performed in the UK and the great saphenous vein (SV) remains the most widely used conduit largely due to availability and length. However, recent reports suggest that approximately 50% of all SV grafts fail at 10 years following CABG and the incidence of arterial restenosis, following drug-eluting stent (DES) implantation remains around 10%. Vascular smooth muscle cells (VSMCs) proliferation- and migration- driven neointimal formation post CABG- and/or PCI- induced vascular injury has been considered a major pathological driver of both vein graft disease (VGD) and in-stent restenosis (ISR) ultimately leading to treatment failure. Accumulating evidence suggests that extracellular vesicles (EVs) play a significant role in intercellular communication in both physiological and pathophysiological conditions, and due to their promising therapeutic potential, EVs are currently being extensively studied as disease mediators and therapeutic delivery vehicles. Understanding the different mechanisms that may be involved in the regulation of VSMC-driven neointimal formation remains an important step towards successfully developing therapeutic strategies that could improve clinical outcomes associated with both CABG and PCI. Therefore, the primary aim of this thesis was to study the effect of VSMC-derived EVs on recipient cell responses with a particular focus on EV-mediated autocrine regulation of VSMC. Since abnormal signalling mediated by platelet-derived growth factor (PDGF), has been implicated in the development of neointimal formation post vascular injury, prolonged PDGF stimulation of human SV smooth muscle cells (HSVSMCs) was used to model the pathological conditions under which neointimal lesions develop in an in vitro setting. Following the optimisation of an EV purification method, EVs were obtained from the conditioned culture media (CCM) of HSVSMCs +/- PDGF stimulation and successfully characterised in terms of size, concentration, protein content and morphological appearance. It was found that while EV size and morphology remained unaltered, PDGF stimulation of HSVSMCs resulted in increased EV secretion. Further studies determined that PDGF was not packaged into EVs after prolonged PDGF treatment of HSVSMCs. Next, following small RNA sequencing (RNAseq) analysis, it was found that PDGF stimulation of HSVSMCs induced significant changes in their EV cargo. Six known differentially expressed miRNAs: miR-24-3p, miR-409-3p, miR-21-5p, let-7A-5p, miR-1-3p and miR-224-5p, were found to be significantly upregulated in PDGF EVs compared to control EVs. Four out of six differentially expressed miRNAs (miR-24-3p, miR-224-5p, miR-409-3p and, let-7A-5p) were also successfully validated by qRT-PCR analysis. Gene set enrichment analysis (GSEA) revealed that miR-24-3p and miR-224-5p miRNAs may be involved in the regulation of biological processes such as cell proliferation, migration, and apoptosis - all previously implicated in the development of neointimal formation. Next, the effects of miR-24-3p or miR-224-5p EVs on HSVSMC proliferation, migration and cell viability were assessed. It was found that, while neither miR24-3p EVs nor miR-224-5p EVs had any significant effect on PDGF-induced HSVSMC proliferation and cell viability compared to naïve EVs, miR-224-5p EVs significantly inhibited EV-depleted foetal bovine serum (FBS)-induced HSVSMC migration compared to both naïve EVs and miR-24-3p EVs through an unknown mechanism. Finally, it was found that serum EVs from mice with carotid artery ligation induced vascular injury do not significantly differ compared to EVs from control mice in terms of size and concentration. However, qRT-PCR analysis of miR-24- 3p and miR-224-5p suggested that the expression of both miRNAs was significantly upregulated in serum EVs isolated from injured mice at day 14 and day 5 after surgery respectively compared to control mice. Overall, these studies provide evidence that prolonged PDGF signalling in HSVSMCs significantly alters the EV population secreted by those cells in terms of concentration of particles released and miRNA expression profile. Additionally, the demonstrated ability of miR-224-5p EVs to supress HSVSMC migration compared to naïve EVs and miR-24-3p EVs provides valuable insights into an alternative mechanism of EV-mediated regulation of VSMCs with promising potential for future therapeutic studies

No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases

Since the successful conquest of many acute, communicable (infectious) diseases through the use of vaccines and antibiotics, the currently most prevalent diseases are chronic and progressive in nature, and are all accompanied by inflammation. These diseases include neurodegenerative (e.g. Alzheimer's, Parkinson's), vascular (e.g. atherosclerosis, pre-eclampsia, type 2 diabetes) and autoimmune (e.g. rheumatoid arthritis and multiple sclerosis) diseases that may appear to have little in common. In fact they all share significant features, in particular chronic inflammation and its attendant inflammatory cytokines. Such effects do not happen without underlying and initially 'external' causes, and it is of interest to seek these causes. Taking a systems approach, we argue that these causes include (i) stress-induced iron dysregulation, and (ii) its ability to awaken dormant, non-replicating microbes with which the host has become infected. Other external causes may be dietary. Such microbes are capable of shedding small, but functionally significant amounts of highly inflammagenic molecules such as lipopolysaccharide and lipoteichoic acid. Sequelae include significant coagulopathies, not least the recently discovered amyloidogenic clotting of blood, leading to cell death and the release of further inflammagens. The extensive evidence discussed here implies, as was found with ulcers, that almost all chronic, infectious diseases do in fact harbour a microbial component. What differs is simply the microbes and the anatomical location from and at which they exert damage. This analysis offers novel avenues for diagnosis and treatment

Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years, and this has lead to the development of novel therapeutic strategies for these patients. In this book a review of the options available for the clinical management of patients with these tumors are outlined. In addition advances in radiology both for pre-operative diagnostic purposes along with surgical planning are described. Furthermore a review of newer developments in chemotherapy along with the evolving field of photodynamic therapy both for intra-operative management and subsequent therapy is provided. A discussion of certain surgical management issues along with tumor induced epilepsy is included. Finally a discussion of the management of certain unique problems including brain metastases, brainstem glioma, central nervous system lymphoma along with issues involving patients with a brain tumor and pregnancy is provided