A state of chronic metabolic inflammation and activation of the cell stress response in organs such as liver and adipose tissue are important pathogenic adaptations with the onset of obesity and the metabolic syndrome. The extent to which these processes are modulated by the early life nutritional experience is not well established, especially in large animal models. The overall aim of this thesis was to identify whether nutritional programming during prenatal and postnatal development enhances metabolic inflammation and cell stress response of obesity. A nutritional model of fetal growth restriction achieved by maternal nutrient restriction (NR) to 60% of requirements during late gestation (110 days to term at 147 days) in twin bearing sheep was used. Combination of prenatal and postnatal nutritional interventions were studied with the following three study protocols: 1. Offspring of twin bearing sheep born to mothers nutrient restricted or fed to appetite were separated after weaning at 3 months of age and then exposed to either restricted physical activity leading to obesity or to unrestricted activity and remained lean. 2. Following maternal NR, both twins or only one twin were reared on their mother’s milk during suckling period in order to achieve a relatively faster growth rate in the latter. 3. Twin offspring of sheep randomised to NR or feeding to requirement during late gestation were separated after birth and randomised to either formula feeding or being fed by the mother until weaning followed by obesogenic rearing. Total body weight of sheep in the obese group was raised by ~30% and was unaffected by any intervention. Obesity led to an increased insulin response to the glucose tolerance test, together with hepatic triglyceride deposition, and adipocyte hypertrophy with macrophage infiltration in omental adipose tissue. NR exacerbated obesity associated hepatic triglyceride deposition and upregulated gene expression of hepatic autophagy and omental unfolded protein response. Formula feeding of sheep offspring following NR was associated with slower weight gain and decreased gene expression for MTOR. Sheep offspring fed by mother as singleton gained weight at faster rate during suckling period as compared to offspring fed by their mothers as twins. Neither postnatal interventions exacerbated the state of obesity associated metabolic inflammation and cell stress response. It is possible that the increased hepatic autophagic gene expression is a reflection of defective autophagy and future work should include study of markers of autophagic function. Possible mechanisms of upregulated omental adipose UPR in offspring of sheep undergoing NR could include a programmed decrease in adipocyte number or selective survival of preadipocytes with effective ER stress response. Such adaptations followed by obesity would predispose the adipocytes to initiate inflammation and cell death pathways.

Stem cells may offer solutions for many health issues facing the world’s population. Early Biotech-led approaches are supporting novel mesenchymal stem cell (MSC) therapies through biomedical trials. However, their potential benefits are currently curtailed by challenges linked to high cell dose requirements which pose availability and manufacturing challenges, combined to suboptimal delivery methodologies. Whilst systemic delivery may be suitable for many pharmaceuticals, more complex and selective treatments such as emerging cell therapies require smarter targeting strategies on safety and cost/benefit grounds. Several groups are developing targeting strategies to guide stem cells to specific locations and hold them in situ whilst performing a repair. The targeting approach presented here uses superparamagnetic iron oxide microparticles (MPs) loaded within stem cells to facilitate control of the cells using magnets. Magnetic resonance imaging (MRI) can be used to monitor the loaded cells’ contribution to the repair process. Questions remain around MP safety and effects on both delivered cell therapies and the receiving patient. Presented data demonstrates labelling of MSC populations with a commercially available MP called SiMAG in two sizes (500 nm and 1000 nm). Particles were assessed for characteristics which influence their suitability for labelling and were found to have a non-uniform variable structure and size. Labelling was found to be both rapid and effective with low 10 µg/Fe/mL labelling doses able to distinguish cell populations by flow cytometry. Super-resolution microscopy, fluorescent microscopy and transmission electron microscopy were used to determine the location of particles within the cell and were noted to accumulate around the nucleus in large vesicles. Uptake into the cell was found to be influenced by serum with 10% serum resulting in a 75% drop in relative uptake over a 24 hour period. Potential sharing of MP between MSCs was investigated both qualitatively with fluorescent microscopy and quantitatively in a MSC co-culture experiment. No statistically significant sharing of MPs between MSCs could be seen to be taking place between populations. The fate of MPs within MSCs was investigated using pH nanosensors to interrogate the internal cell pH. A novel flow cytometry assay using pH nanosensors and MPs was used to examine the internal pH of large populations of cells. This yielded results which suggest a pH decrease over 4 days from pH 5.5 to 4.7 followed an increase to 5.4 by day 6. This effect is suggested to be caused by a complex pH mediated degradation of MPs followed by increase in pH due to the degraded iron overloading the cell. This degradation was carried out in simulated lysosomal conditions and found to act in a similar way. Macrophages were also used to test degradation and again they were found to reduce the fluorecense of the MPs rapidly over 7 days. The ability of MSCs to tolerate MPs without impacting cell health was probed with a range of assays. The metabolic assay Presto blue demonstrated doses of 10 µg/Fe/mL did not impact the metabolic status of the cell. This was tested with other potential surface chemistries of the same particle design and these were also found to be well tolerated. Membrane intergrity was assessed with flow cytometry for both 500 and 1000 nm SiMAG and was found to have no damaging effects present at 10 µg/Fe/mL. SiMAG 1000 nm was found to have no membrane compromising effect all the way up to 100 µg/Fe/mL. Cell identity was assessed with common MSC markers established by the Dominici position paper and no change to expression was found to occur even with repeated, high dose long-term (14 day) labelling strategies. As particles accumulate round the nucleus, deleterious effects of MP on DNA were tested using the comet assay and visual inspection with no statistically significant increase up to the maximum tested of 100 µg/Fe/mL. Similarly, no effect on cell cycle status was noticed for populations of MSCs. The retention of cell “function” was tested not only following labelling, but following hypothermic storage of cells to simulate shipping to a clinical setting. This was carried out for a range of clinically relevant cell types including mesenchymal stem cells (MSCs), cardiomyocytes (CaM) and ReNeuron neural stem cells (ReN). MSCs were found to freely differentiate to tri-lineage osteogenic, chondrogenic and adipogenic lineages. CXCR4 expression was measured as a marker of MSCs ability to home in on damage and was found to be raised in response to MP presence. CaM were found to resume beating both after hypothermic storage, as well as at high (1000 µg/Fe/mL SiMAG doses). ReN cells were found to be more sensitive to SiMAG with only 10 µg/Fe/mL doses tolerated although successful neural differentiation was still possible. The ability to culture MSCs and label them in a scalable manufacturing scenario was also examined and found to be possible. SiMAG was demonstrated to be a suitable labelling agent both for imaging as well as magnetic manipulation. Precise magnetic manipulation of labelled cells was demonstrated both as an individual cell and as a cell population moving through a simulated tissue gel. Entrapment of labelled cells from a simulated circulatory system was also shown to be possible with close to 100% of cells recruited in the first pass. The fluorophores on SiMAG were not strong enough to be visualised on their own and quantum dots were used to demonstrate successful retention of labelled MSCs in an ex vivo rat model. MRI was however shown to be a suitable method for assessing the location of labelled cell populations at even low cell concentrations ~1x106 and low SiMAG doses of 5 µf/Fe/mL. In conclusion, this study indicates that MPs represent a cell-safe and effective potential tool to better target cells. The key benefit of this cell labelling technology is in the high degree of understanding over the entire labelling process from entry through to degradation. In addition, this labelling technology has been shown to be cell-safe in a large number of cells both for physical health and basic function. Whilst this should be further explored for further, more specific applications, it makes a compelling case for SiMAG as a multi-functional tool for cell manipulation and tracking.

Thesis (M.A.)--Guo li Taiwan shi fan da xue, 1984. / Cover title. Reproduced from typescript, on double leaves. Bibliography: p. 193-195.

Worldwide populations are growing older. Thus, many countries fear economic and societal burden, but population aging can also be seen as an opportunity for a society. Which view prevails is very much dependent on the chosen measures of aging that are so far mainly based on chronological age. In the first part of this thesis measures of population aging are analyzed and new measures are proposed. The novel cognition adjusted dependency ratio (CADR) is introduced as an alternative to the old-age dependency ratio (OADR), which only considers a chronological age threshold. A CADR based ranking of certain countries across the world shows a completely different picture compared to an OADR based ranking of the same countries. Northern European countries and the United States are listed at the top according to their CADR, while they are at the bottom end considering their OADR. This difference is discussed in detail within this thesis. On the micro level, a characteristics based approach is proposed to measure and compare individual aging. More specifically, differences in aging are converted into single years of age, which highlights the magnitude of differences between groups such as socioeconomic subpopulations. The second part of this thesis addresses the huge variability in cognitive functioning, a dimension of healthy aging, between and within countries. The relationship between cognition and education (e.g. individual and national level) is investigated across countries that vary substantially in terms of their demography and level of economic and social development. The results indicate that increasing the national educational level is assigned with better individual cognitive performance in addition to the positive individual education effect. In the subsequent study, determinants associated with the differences in cognitive functioning across countries, cohorts, and gender are examined. Improvements in living conditions and education support better cognitive performance. Moreover, better living conditions and higher education will increase gender differences in some cognitive functions and decrease or eliminate the gender differences in other cognitive abilities. (author's abstract)

LIM domain containing protein 1 (LIMD1) is a tumour suppressor located at 3p21.3, a region that harbours multiple tumour suppressor genes and is commonly subject to homozygous deletions and loss of heterozygosity in many cancers. The mechanism of LIMD1 tumour suppressive activities are not fully elucidated, however to date it has been shown to bind to the retinoblastoma protein (pRb) and repress E2F driven transcription as well as being a critical component of miRNA mediated gene silencing. Recent work has also identified LIMD1 as a possible negative regulator of hypoxia inducible factor α (HIF1α) and the hypoxic response. In lung cancer, LIMD1 protein expression is down regulated in up to 79% of tumours when compared to normal tissue with gene deletion and loss of heterozygosity accounting for 32 and 12% respectively, leaving 30% of tumours with unexplained mechanism of LIMD1 protein loss. In an aim to identify other possible mechanisms of LIMD1 loss, scrutinisation of the LIMD1 promoter identified a CpG Island in the 5’ promoter region, within which a small region was found to be critical for transcriptional activation. This region was methylated in the non-LIMD1 expressing MDA-MB435 cell line, but became hypomethylated and LIMD1 expressed following treatment with the DNA methylation inhibitor 5-Aza-2’-deoxycytidine. In primary lung tumours, analysis of genomic DNA also identified increased methylation of this region as well as a reduction in LIMD1 mRNA levels when compared to matched normal lung tissue. Furthermore, in silico analysis identified a conserved binding motif for the Ets transcription factor PU.1. Experimentally PU.1 was verified as binding to the LIMD1 promoter with siRNA mediated depletion of PU.1 significantly reducing endogenous LIMD1 protein levels, thus identifying two possible novel mechanisms of LIMD1 silencing. Transcription of LIMD1, like that of other HIF1α regulatory proteins, was enhanced when cells were exposed to hypoxia (1% O2), facilitated by HIF1α binding a hypoxic responsive element (HRE) within the promoter. At the molecular level, in vivo LIMD1 forms an endogenous complex with proline hydroxylase 2 (PHD2) and the von Hippel-Lindau (VHL) protein, with LIMD1 loss decreasing the efficiency of HIF1α degradation and impeding the resultant cellular adaptation to chronic hypoxia. In summary these studies identified epigenetic silencing of LIMD1 as a possible explanation for LIMD1 protein loss in transformed cells. Furthermore, LIMD1 transcription was identified as being activated by PU.1 and enhanced by HIF1α, and a revised, LIMD1 integrated, model of HIF1α regulation is proposed.

The fatty acid amides (FAAs) family includes endocannabinoids, such as anandamide, as well as endocannabinoid-like molecules, such as N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA). Members of the FAA family show agonist activity at transmitter-gated channels (TRPV1), as well as peroxisome poliferator-activated receptors (PPARs). Given that FAAs appear to be hydrolysed principally through the action of the enzyme fatty acid amide hydrolase, inhibition of FAAH should lead to accumulation of a variety of FAAs. Therefore, in this study it was investigated whether pharmacological inhibition of FAAH could influence PPAR activity in SH-SY5Y human neuroblastoma cells or HeLa human cervical carcinoma cells. FAAH activity was assessed by monitoring liberation of [3H]-ethanolamine from labelled anandamide. FAAH protein and RNA expression were measured by immunoblotting and qRT-PCR respectively. Endocannabinoid levels were measured by LC-MS/MS. In order to evaluate PPAR activation, a PPRE-linked luciferase construct was co-transfected with expression plasmids for either PPAR α, β or γ. Binding to PPAR receptors was assessed with a competitor displacement assay (Invitrogen). In intact SH-SY5Y cells, sustained FAAH inhibition by URB597 (~75 %) led to accumulation of AEA, 2AG and PEA, but not OEA. Treatment with URB597, OL135 or PF750, three structurally and functionally distinct FAAH inhibitors, induced activation of endogenously expressed PPARs, while no activation was observed in FAAH-1 negative HeLa cells. Furthermore, exposure to URB597, OL135 or PF750 led to activation of over-expressed PPARs in SH-SY5Y cells. To rule out direct activation of PPARs by the FAAH inhibitors, cell-free binding assays showed that URB597, OL135 and PF750 could not bind to PPARα, PPARβ or PPARγ. Surprisingly, treatment with URB597 in HeLa cells led to intracellular accumulation of PEA but not AEA, OEA or 2AG. This might be due to inhibition of either FAAH-2 or NAAA, both of which are expressed in HeLa cells. Moreover, the presence of either URB597 or OL135 led to activation of PPARγ receptors over-expressed in HeLa cells. In conclusion, data in this study showed activation of PPAR nuclear receptors in vitro by inhibition of FAAH activity and subsequent augmentation of endocannabinoid tone. These data suggest that, at least in a model setup, it is possible to modulate the endocannabinoid tone without any previous external stimulus of their synthesis and trigger a functional effect.

A DNA double-strand break is an exceptionally toxic lesion that threatens the structural and functional integrity of the genome. In this thesis the repair of DSBs was investigated using the bacterium Escherichia coli, which repairs DNA breaks almost exclusively by homologous recombination. The studies described focus on the repair of damage induced by reactive oxygen species, but especially on the RecN protein, which is associated specifically with the repair of double-strand breaks. The RecN protein is highly conserved across bacterial species and in E. coli has been identified as a key factor in the repair of DNA breaks. In this thesis three RecN homologs were analysed. RecN from Haemophilus influenzae is shown to be capable of replacing the functions of E. coli RecN in vivo. However, homologs from Aquifex aeolicus and Bacteriodes fragilis cannot do so. Biochemical analysis of all three RecN homologs was undertaken. The H. influenzae RecN and A. aeolicus RecN were shown to have weak ATPase activity and an ability to interact with single-stranded DNA. ATPase deficient mutants of the RecN proteins were created and used to demonstrate the functional importance of the ATP hydrolysis. In the case of E. coli and H. influenzae, the ATPase defective mutants failed to function in vivo. In vitro, the ATPase deficient H. influenzae RecN mutant and a similar mutant of A. aeolicus RecN failed to interact with single-stranded DNA. These data are discussed in terms of a relationship between RecN and the structural maintenance of chromosome family of proteins. Finally, a model for RecN activity is presented based on those developed to explain the function of structural maintenance of chromosome proteins and the new data presented here. In this model, RecN is suggested to trap DNA molecules holding a break site and repair template in close proximity, facilitating the repair of DNA breaks by homologous recombination. The possibility of RecN acting as a global, damage induced cohesin is also discussed.

DNA is under constant attack from numerous damaging agents and our cells deal with thousands of lesions every day. With such constant damage it is inevitable that the template will not be completely cleared of lesions before the replication complex arrives. The consequences of the replisome meeting an obstacle will depend upon the nature of the obstacle. I have focussed upon replication in Escherichia coli and the effect of UV-induced lesions, which would block synthesis by the replicative polymerases. It is accepted that a UV lesion in the lagging strand template can be bypassed by the replisome complex, but the consequences of meeting a lesion in the leading strand template remain unclear. A lesion in the leading strand template could block replisome progression and the fork might require extensive processing in order to restart replication. However, it has also been proposed that the replisome could progress past these lesions by re‐priming replication downstream and leaving a gap opposite the lesion. The results of my studies revealed that all modes of synthesis are delayed after UV. I have demonstrated that when synthesis resumed, the majority reflected the combined effects of oriC firing and the initiation of inducible stable DNA replication. These modes of synthesis mask the true extent of the delay in synthesis at existing replication forks. The results also revealed that all synthesis after UV is dependent upon DnaC, suggesting that the replicative helicase and possibly the entire replisome, needs to be reloaded. A functional RecFOR system is required for efficient replication restart, without these proteins replication is capable of resuming but only after a long delay. My data support models proposing that replication forks require extensive processing after meeting a lesion in the leading strand template. Whilst I cannot exclude the possibility that replication forks can progress past some such lesions, my data indicate that they cannot progress past many before stalling. Overall, my results demonstrate the importance of measuring all modes of DNA synthesis when assessing the contribution of any particular protein to recovery after UV irradiation. Thus, although net synthesis in cells lacking RecG appears similar to wild type after UV, the mode of replication is in fact quite different. A dramatic increase in the level of stable DNA replication appears to account for much of the overall synthesis detected and coincides with a major chromosome segregation defect. The importance of stable DNA replication in irradiated recG cells has not previously been considered because the different modes of synthesis were ignored. The significance of this pathology and of the other findings reported in this thesis is discussed in relation to current models of DNA repair and replication restart.

This thesis aimed to define and manipulate epigenetic stability of human embryonic stem cells (hESCs). The allele-specific expression of 22 imprinted genes was examined in 22 hESC lines by distinguishing parental single nucleotide polymorphisms in genomic DNA and cDNA. Half of the genes examined (PEG10, PEG1, MESTIT1, IGF2, H19, GTL2, NESP55, PHLDA2 and ATP10C) showed variable allele-specific expression between cell lines, indicating vulnerability to disrupted imprinting. However, 8 genes (KCNQ1OT1, NDN, NDNL1, SNRPN, IPW, PEG3, KCNQ1 and CDKN1C) showed consistent monoallelic expression. Moreover, 4 genes (TP73, IGF2R, WT1 and SLC22A18) known to be monoallelically expressed or to exhibit polymorphic imprinting in human tissues were always biallelically expressed. MEST isoform 1, PEG10 and NESP55 showed an association between the variability observed in interline allele-specific expression status and DNA methylation at their imprinting regulatory regions. These evidences demonstrate gene-specific differences in the stability of imprinted loci in hESC lines and identify disrupted DNA methylation as one potential mechanism. hESOD1 (human embryonic stem cells overexpressing DNMT1) cell lines were established to manipulate epigenetic stability of hESCs. Of ~ 2,200 CpG loci examined by restriction landmark genomic scanning (RLGS), cell lines (cultured over 23 passages) having only endogenous DNMT1 showed in vitro culture induced DNA methylation alterations at 6 loci. However, hESOD1 cell lines showed DNA methylation alterations at only 1 or 2 loci, indicating that overexpression of exogenous DNMT1 resulted in increased epigenetic stability. Of 14 imprinting regulatory regions, 10 tumour-suppressor gene promoters and 3 repetitive sequences examined, 3 loci (DAPK-1, MGMT and TIMP-3) were indentified to be hypermethylated in hESOD1 cell lines, whereas other 21 loci showed normal methylation levels. These evidences demonstrate that overexpression of exogenous DNMT1 can prevent hESCs from accumulating DNA methylation changes upon in vitro culture and cause locus-specific hypermethylation.

MRSA is a significant pathogen, which can cause a range of minor and major infections both in the hospital and community environments. MRSA is developing resistance to many antibiotics, including vancomycin, which is now the first choice antibiotic to treat MRSA infections in the UK. This together with the dearth of new antibiotics being introduced could see the emergence of untreatable S. aureus strains. This has led to renewed interest in alternative antimicrobial agents. Lysostaphin is an endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, which cleaves the peptidoglycan cross-bridges of other staphylococcal species. Lysostaphin has been investigated as a potential therapeutic agent and has shown promise in in vitro and in vivo studies and in clinical trials. However, resistance to lysostaphin is likely to emerge and there will be a demand for second generation Iysostaphins and/or other similar novel antimicrobials that can counteract this resistance. This study describes the cloning, purification and assaying of an endolysin of the S. aureus P68 bacteriophage. Lys16 lysin has previously been shown to possess staphylolytic activity. This study demonstrates that the purified recombinant protein is poorly soluble and is inactive against live cells. The Atl autolysin of S. aureus was also investigated as a potential antimicrobial. This study confirmed the hydrolytic profiles of the enzymes, and a chimeric peptide incorporating the lysostaphin targeting domain with the Atl glucosaminidase was designed. This did not confer greater activity against S. aureus, although the targeting domains of each enzyme were shown to utilise different cell surface receptors. Finally, this study reports the development of a novel assay to measure the activity of antimicrobial peptides against S. aureus, using a bioluminescence reporter. This was shown to be a sensitive assay, able to distinguish small differences in the activity of antimicrobial peptides.