Cell Fate Specification and the Regulation of RNA-dependent DNA Methylation in the Arabidopsis Root Meristem

The Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.

Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.

Temporomandibular joint disorder (TMJD) is known for its mastication-associated pain. TMJD is medically relevant because of its prevalence, severity, chronicity, the therapy-refractoriness of its pain, and its largely elusive pathogenesis. Against this background, we sought to investigate the pathogenetic contributions of the calcium-permeable TRPV4 ion channel, robustly expressed in the trigeminal ganglion sensory neurons, to TMJ inflammation and pain behavior. We demonstrate here that TRPV4 is critical for TMJ-inflammation-evoked pain behavior in mice and that trigeminal ganglion pronociceptive changes are TRPV4-dependent. As a quantitative metric, bite force was recorded as evidence of masticatory sensitization, in keeping with human translational studies. In Trpv4(-/-) mice with TMJ inflammation, attenuation of bite force was significantly less than in wildtype (WT) mice. Similar effects were seen with systemic application of a specific TRPV4 inhibitor. TMJ inflammation and mandibular bony changes were apparent after injections of complete Freund adjuvant but were remarkably independent of the Trpv4 genotype. It was intriguing that, as a result of TMJ inflammation, WT mice exhibited significant upregulation of TRPV4 and phosphorylated extracellular-signal-regulated kinase (ERK) in TMJ-innervating trigeminal sensory neurons, which were absent in Trpv4(-/-) mice. Mice with genetically-impaired MEK/ERK phosphorylation in neurons showed resistance to reduction of bite force similar to that of Trpv4(-/-) mice. Thus, TRPV4 is necessary for masticatory sensitization in TMJ inflammation and probably functions upstream of MEK/ERK phosphorylation in trigeminal ganglion sensory neurons in vivo. TRPV4 therefore represents a novel pronociceptive target in TMJ inflammation and should be considered a target of interest in human TMJD.

The adaptive response of humans to exercise: Impact of exercise intensity, fibre-type specific responses and molecular patterns of response

In an attempt to improve the current understanding of the adaptive response to exercise in humans, this dissertation performed a series of studies designed to examine the impact of training intensity and mode on aerobic capacity and performance, fibre-type specific adaptations to training, and individual patterns of response across molecular, morphological and genetic factors. Project #1 determined that training intensity, session dose, baseline VO2max and total training volume do not influence the magnitude of change in VO2max by performing a meta-regression, and meta-analysis of 28 different studies. The intensity of training had no effect on the magnitude of increase in maximal oxygen uptake in young healthy participants, but similar adaptations were achieved with lower training doses following high intensity training. Project # 2 determined the acute molecular response, and training-induced adaptations in aerobic performance, aerobic capacity and muscle phenotype following high-intensity interval training (HIT) or endurance exercise (END). The acute molecular response (fibre recruitment and signal activation) and training-induced adaptations in aerobic capacity, aerobic performance, and muscle phenotype were similar following HIT and END. Project # 3 examined the impact of baseline muscle morphology and molecular characteristics on the training response, and if muscle adaptations are coordinated. The muscle phenotype of individuals who experience the largest improvements (high responders) were lower before training for some muscle characteristics and molecular adaptations were coordinated within individual participants. Project # 4 examined the impact of 2 different intensities of HIT on the expression of nuclear and mitochondrial encoded genes targeted by PGC-1α. A systematic upregulation of nuclear and mitochondrial encoded genes was not present in the early recovery period following acute HIT, but the expression of mitochondrial genes were coordinated at an individual level. Collectively, results from the current dissertation contribute to our understanding of the molecular mechanisms influencing skeletal muscle and whole-body adaptive responses to acute exercise and training in humans.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Vascular smooth muscle cell (VSMC) behaviour and phenotypic modulation is critical to vessel repair following damage, and the progression of various cardiovascular diseases. The second messenger cyclic adenosine monophosphosphate (cAMP) plays a key role in VSMC function under the synthetic/activated phenotype, which is typically associated with unhealthy cell behaviour. Consequently, cAMP signaling is often targeted in attempts to impact several pathological diseases, including atherosclerosis, restenosis, and pulmonary arterial hypertension (PAH). The cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of cAMP to an inactive form, and therefore directly regulate cAMP signaling. The PDE4D family dominates in synthetic VSMCs, and there is considerable interest in determining how distinct PDE4D isoforms affect cell function. Specifically, we are interested in the potential link between short isoforms of PDE4D and VSMC desensitization to pharmacological agents that impact cardiovascular disease via cAMP signaling. This study extends on previous work that assessed the expression of PDE4D splice variants in rat aortic VSMCs following prolonged challenge with cAMP-elevating agents. It was determined that PDE4D1 and PDE4D2 were uniquely expressed in synthetic VSMCs incubated with these agents, and that this upregulation impacted PDE activity and cAMP accumulation in these cells. Here, we report that PDE4D1 and PDE4D2 are markedly upregulated in synthetic human aortic smooth muscle cells (HASMCs) following prolonged challenge with cAMP-elevating agents. Using a combination of RNAi-based and pharmacological approaches, we establish that this upregulation is reflected in levels of cAMP PDE activity, and restricted to the cytosolic sub-cellular compartment. Our results suggest a role for localized PDE4D1 and PDE4D2 activity in regulating cAMP-mediated desensitization in HASMCs, and highlight their therapeutic potential in treating various cardiovascular diseases.

MiRNA 34a: a therapeutic target for castration-resistant prostate cancer

INTRODUCTION: Development of a therapy for bone metastases is of paramount importance for castration-resistant prostate cancer (CRPC). The osteomimetic properties of CRPC confer a propensity to metastasize to osseous sites. Micro-ribonucleic acid (miRNA) is non-coding RNA that acts as a post-transcriptional regulator of multiple proteins and associated pathways. Therefore identification of miRNAs could reveal a valid third generation therapy for CRPC. Areas covered: miR34a has been found to play an integral role in the progression of prostate cancer, particularly in the regulation of metastatic genes involved in migration, intravasation, extravasation, bone attachment and bone homeostasis. The correlation between miR34a down-regulation and metastatic progression has generated substantial interest in this field. Expert opinion: Examination of the evidence reveals that miR34a is an ideal target for gene therapy for metastatic CRPC. We also conclude that future studies should focus on the effects of miR34a upregulation in CRPC with respect to migration, translocation to bone micro-environment and osteomimetic phenotype development. The success of miR34a as a therapeutic is reliant on the development of appropriate delivery systems and targeting to the bone micro-environment. In tandem with any therapeutic studies, biomarker serum levels should also be ascertained as an indicator of successful miR34a delivery.

Obestatin as a key regulator of metabolism and cardiovascular function with emerging therapeutic potential for diabetes

Obestatin is a 23-amino acid C-terminally amidated gastrointestinal peptide derived from preproghrelin and which forms an alpha helix. Although obestatin has a short biological half-life and is rapidly degraded, it is proposed to exert wide-ranging pathophysiological actions. Whilst the precise nature of many of its effects is unclear, accumulating evidence supports positive actions on both metabolism and cardiovascular function. For example, obestatin has been reported to inhibit food and water intake, body weight gain, and gastrointestinal motility, and to also mediate promotion of cell survival and prevention of apoptosis. Obestatin-induced increases in β-cell mass, enhanced adipogenesis and improved lipid metabolism have been noted along with upregulation of genes associated with β-cell regeneration, insulin production and adipogenesis. Furthermore, human circulating obestatin levels generally demonstrate an inverse association with obesity and diabetes, whilst the peptide has been shown to confer protective metabolic effects in experimental diabetes, suggesting that it may hold therapeutic potential in this setting. Obestatin also appears to be involved in blood pressure regulation and to exert beneficial effects on endothelial function, with experimental studies indicating that it may also promote cardioprotective actions against, for example, ischaemia-reperfusion injury. This review will present a critical appraisal of the expanding obestatin research area and discuss the emerging therapeutic potential of this peptide for both metabolic and cardiovascular complications of diabetes. 

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

BackgroundThe recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear.Design and MethodsThe expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays.ResultsWe found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples.ConclusionsIn conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

Molecular Mechanism of the Response to Hydrogen Sulfide in C. elegans

Thesis (Ph.D.)--University of Washington, 2016-08

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone ‘Norgestrel’ as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1 μM) showed this rise to be PGRMC1-dependent, primarily utilising calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μM) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Regulatory T cells control strain specific resistance to Experimental Autoimmune Prostatitis

Susceptibility to autoimmune diseases results from the encounter of a complex and long evolved genetic context with a no less complex and changing environment. Major actors in maintaining health are regulatory T cells (Treg) that primarily dampen a large subset of autoreactive lymphocytes escaping thymic negative selection. Here, we directly asked whether Treg participate in defining susceptibility and resistance to Experimental Autoimmune Prostatitis (EAP). We analyzed three common laboratory strains of mice presenting with different susceptibility to autoimmune prostatitis upon immunization with prostate proteins. The NOD, the C57BL/6 and the BALB/c mice that can be classified along a disease score ranging from severe, mild and to undetectable, respectively. Upon mild and transient depletion of Treg at the induction phase of EAP, each model showed an increment along this score, most remarkably with the BALB/c mice switching from a resistant to a susceptible phenotype. We further show that disease associates with the upregulation of CXCR3 expression on effector T cells, a process requiring IFNγ. Together with recent advances on environmental factors affecting Treg, these findings provide a likely cellular and molecular explanation to the recent rise in autoimmune diseases incidence.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Macrophages and CD4 T-cells in rheumatoid arthritis and their modulation by JAK inhibitors

Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that causes significant morbidity and mortality and has no cure. Although early treatment strategies and biologic therapies such as TNFα blocking antibodies have revolutionised treatment, there still remains considerable unmet need. JAK kinase inhibitors, which target multiple inflammatory cytokines, have shown efficacy in treating RA although their exact mechanism of action remains to be determined. Stratified medicine promises to deliver the right drug to the right patient at the right time by using predictive ‘omic biomarkers discovered using bioinformatic and “Big Data” techniques. Therefore, knowledge across the realms of clinical rheumatology, applied immunology, bioinformatics and data science is required to realise this goal. Aim: To use bioinformatic tools to analyse the transcriptome of CD14 macrophages derived from patients with inflammatory arthritis and define a JAK/STAT signature. Thereafter to investigate the role of JAK inhibition on inflammatory cytokine production in a macrophage cell contact activation assay. Finally, to investigate JAK inhibition, following RA synovial fluid stimulation of monocytes. Methods and Results: Using bioinformatic software such as limma from the Bioconductor repository, I determined that there was a JAK/STAT signature in synovial CD14 macrophages from patients with RA and this differed from psoriatic arthritis samples. JAK inhibition using a JAK1/3 inhibitor tofacitinib reduced TNFα production when macrophages were cell contact activated by cytokine stimulated CD4 T-cells. Other pro-inflammatory cytokines such as IL-6 and chemokines such as IP-10 were also reduced. RA synovial fluid failed to stimulate monocytes to phosphorylate STAT1, 3 or 6 but CD4 T-cells activated STAT3 with this stimulus. RNA sequencing of synovial fluid stimulated CD4 T-cells showed an upregulation of SOCS3, BCL6 and SBNO2, a gene associated with RA but with unknown function and tofacitinib reversed this. Conclusion: These studies demonstrate that tofacitinib is effective at reducing inflammatory mediator production in a macrophage cell contact assay and also affects soluble factor mediated stimulation of CD4 T-cells. This suggests that the effectiveness of JAK inhibition is due to inhibition of multiple cytokine pathways such as IL-6, IL-15 and interferon. RNA sequencing is a useful tool to identify non-coding RNA transcripts that are associated with synovial fluid stimulation and JAK inhibition but these require further validation. SBNO2, a gene that is associated with RA, may be biomarker of tofacitinib treatment but requires further investigation and validation in wider disease cohorts.

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.