The structural and mechanical integrity of historic wood

Little is known about historic wood as it ages naturally. Instead, most studies focus on biological decay, as it is often assumed that wood remains otherwise stable with age. This PhD project was organised by Historic Scotland and the University of Glasgow to investigate the natural chemical and physical aging of wood. The natural aging of wood was a concern for Historic Scotland as traditional timber replacement is the standard form of repair used in wooden cultural heritage; replacing rotten timber with new timber of the same species. The project was set up to look at what differences could exist both chemically and physically between old and new wood, which could put unforeseen stress on the joint between them. Through Historic Scotland it was possible to work with genuine historic wood from two species, Oak and Scots pine, both from the 1500’s, rather than relying on artificial aging. Artificial aging of wood is still a debated topic, with consideration given to whether it is truly mimicking the aging process or just damaging the wood cells. The chemical stability of wood was investigated using Fourier-transform infrared (FTIR) microscopy, as well as wet chemistry methods including a test for soluble sugars from the possible breakdown of the wood polymers. The physical properties assessed included using a tensile testing machine to uncover possible differences in mechanical properties. An environmental chamber was used to test the reaction to moisture of wood of different ages, as moisture is the most damaging aspect of the environment to wooden cultural objects. The project uncovered several differences, both physical and chemical, between the modern and historic wood which could affect the success of traditional ‘like for like’ repairs. Both oak and pine lost acetyl groups, over historic time, from their hemicellulose polymers. This chemical reaction releases acetic acid, which had no effect on the historic oak but was associated with reduced stiffness in historic pine, probably due to degradation of the hemicellulose polymers by acid hydrolysis. The stiffness of historic oak and pine was also reduced by decay. Visible pest decay led to loss of wood density but there was evidence that fungal decay, extending beyond what was visible, degraded the S2 layer of the pine cell walls, reducing the stiffness of the wood by depleting the cellulose microfibrils most aligned with the grain. Fungal decay of polysaccharides in pine wood left behind sugars that attracted increased levels of moisture. The degradation of essential polymers in the wood structure due to age had different impacts on the two species of wood, and raised questions concerning both the mechanism of aging of wood and the ways in which traditional repairs are implemented, especially in Scots pine. These repairs need to be done with more care and precision, especially in choosing new timber to match the old. Within this project a quantitative method of measuring the microfibril angle (MFA) of wood using polarised Fourier transform infrared (FTIR) microscopy has been developed, allowing the MFA of both new and historic pine to be measured. This provides some of the information needed for a more specific match when selecting replacement timbers for historic buildings.

Investigating the role of N-WASP in the development of colorectal cancer

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.

Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.