The high mobility group of nucleosome binding proteins (HMGNs) are chromatin architectural proteins that bind specifically to nucleosomes and influence chromatin structure and DNA-dependent functions. However, the mechanisms underlying these events remain largely unknown. HMGN1 and HMGN2 are highly expressed by embryonic stem cells and are downregulated as differentiation proceeds. Nevertheless, embryonic and adult neural stem cells retain elevated levels of these proteins. Chromatin plasticity is essential for the pluri- and multipotency of stem cells and it is achieved by maintaining an open and dynamic chromatin conformation. Conversely, developmental potential seems to be restricted by chromatin condensation. The present work shows that loss of HMGN1 or HMGN2 in pluripotent embryonal carcinoma cells leads to increased spontaneous neuronal differentiation, which is accompanied by a reduction in pluripotency markers and higher gene expression of lineage-specific transcription factors. Inhibition of signalling pathways relevant for neurogenesis does not re-establish the phenotype observed in Hmgn2-knockout cells. Withdrawal of the factors sustaining pluripotency in embryonal carcinoma cells results in higher induction of pro-neural factors in cells lacking HMGN1 or HMGN2. Neural stem cells derived from Hmgn-knockout cells also display higher gene expression of pro-neural transcription factors and increased spontaneous neuronal differentiation. Loss of HMGN2 disrupts the active histone modification landscape, and therefore affects the chromatin structure at local and global levels. The proposition is that the local changes directly influence the transcription rates of pluripotency and lineage-specific transcription factors, while the global changes may restrict chromatin plasticity. The present data support a hypothesis whereby HMGNs contribute to the chromatin plasticity of stem cells by promoting an active histone modification landscape and open chromatin conformation, which are essential for preserving the self-renewal and developmental potential of stem cells.

1. The aim of this project was to determine whether advancing gestational age and parturition were associated with alteration in the relative level of expression of genes encoding prostanoid receptors in key uterine and fetal tissues. I also sought to determine whether advancing gestational age and parturition were associated with alteration in the expression of genes encoding lipoxygenase (LOX) enzymes in key intra-uterine tissues. 2. Caesarean hysterectomy was performed on 15 pregnant baboons in the last third of pregnancy. Samples of myometrium (from multiple uterine sites), cervix, decidua and chorion were obtained. In addition, the ductus arteriosus was obtained from nine fetal baboons, 28 fetal lambs and 4 neonatal lambs. Expression of genes was studied using Northern blot analysis and in situ hybridization. Expression of genes was quantified by Northern analysis as a ratio of the signal for the gene of interest to each of three housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin and cyclophilin). Statistical comparison of the effects of gestational age and labour was performed using linear regression. Student's t-test, repeated measures analysis of variance and analysis of covariance, as appropriate. 3 Initial studies of animals not in labour using cDNA probes demonstrated transcripts of similar size to the human genes for prostanoid EP2, EP3, EP4, and FP receptor mRNA using Northern blot in myometrium. Myometrium from the lower uterine segment (LUS) had greater expression of EP2 receptor mRNA and less expression of EP3 mRNA compared with the fundus and corpus. However, similar levels of EP4 and FP receptor mRNA were observed comparing the fundus and LUS. Expression of EP2, EP3 and EP4 receptor mRNA were also detected in cervix, decidua and chorion. EP2 mRNA was most abundant in cervix, EP3 was most abimdant in myometrium and EP4 mRNA was most abundant in decidua. The variation in myometrial expression of genes encoding EP receptor sub-types paralleled the contractile responses of paired samples (reported elsewhere). 4 When expression of prostanoid receptor genes was studied in myometrium obtained from animals both in labour and not in labour and the techniques employed were optimized (principally the use of riboprobes), transcripts of similar size to the human genes were detected for prostanoid EP1, EP2, EP3, EP4, IP, FP and TP receptor mRNA using Northern blot. There were no gestational age related changes in expression of these genes. Expression of EP1, EP3 and IP receptor mRNA was significantly higher in myometrium from the fundus (compared with lower segment) whereas EP2 gene expression was significantly lower in the fundus. Labor was associated with a reduction in the regional variation of both EP2 and IP receptor gene expression, but not EP1 and EP3 expression. Labor was also associated with an overall lower level of expression of EP2 receptor mRNA. 5 When expression of prostanoid receptor genes was studied in cervix obtained from animals both in labour and not in labour, clear signals which were similar in estimated size to the human genes were detected by Northern analysis for EP1, EP2, EP3, EP4, FP, IP and TP receptors. Expression of the gene encoding the prostanoid EP? receptor increased with advancing gestational age prior to labor. Expression of the EP2, FP and TP receptor genes was much lower in animals that were delivered during spontaneous labor than in animals which were not in labor. 6 When expression of prostanoid receptor genes was studied in decidua and chorion obtained from animals both in labour and not in labour, expression of the genes encoding the EP1 and FP receptor in decidua and the EP4 receptor in chorion was lower with advancing gestational age. Expression of the EP? receptor gene was lower in labour in decidua, whereas expression of the IP receptor gene was higher in labour in both decidua (2-fold) and chorion (4-fold).

No description available.

No description available.

This PhD provides a novel design for a fluorescence-based biosensor and the associated immunoassay framework. It offers a solution for measuring progesterone concentration in bovine milk that can be used as an integral part of a dairy farm’s reproductive management, with a direct impact on the farm profitability. A cow needs to give birth in regular intervals in order to produce a healthy amount of milk. A high pregnancy rate requires an accurate estimate of the onset of oestrus to achieve an effective insemination and therefore a high conception rate. Progesterone concentration in bovine milk reaches a minimum at around 2 ng/ml, approximately 72 hours before oestrus, a characteristic that can be used to detect the onset of oestrus. The majority of methods currently employed for progesterone measurement require expensive laboratory-based instruments and time-consuming preparation stages. This thesis, however sets out the case for the design of a portable commercially-viable biosensor, capable of measuring progesterone concentration on-site at dairy farms. The proposed solution features a unique adaptive offset cancellation mechanism, which compensates for the effect of varying background LED light and provides a wide dynamic range. The design eliminates the need for the expensive optical components such as lens, collimator and dichroic mirror. Furthermore, the innovative design of the casing substantially reduces the effect of background light on the detector circuit, whilst improves the fluorescent excitation within the sample and subsequent detection by the sensor. This thesis also provides a novel framework for a competitive homogeneous immunoassay based on Fluorescence Resonance Energy Transfer (FRET). Three specific immunoassay procedures are proposed, offering a trade off on hardware cost versus running cost as well as procedural and algorithmic complexity. Following a thorough analysis of the hardware design and immunoassay framework and the optimisation of the key system parameters, a prototype biosensor was developed. Numerous experiments were carried out and the performance was verified against CLARIOstar spectrofluorometer. The outcome demonstrates the effectiveness of the immunoassay framework and the ability of the biosensor to measure fluorophore concentration with an accuracy of ±250 pM, which is suitable for detecting the onset of oestrus.

KIF1C is a kinesin-3 family motor protein that accumulates at the tip of the tail in migrating cells and transports α5β1-integrins into cellular protrusions and is therefore required for the regulation of cell adhesion structures. KIF1C also transports dense core vesicles (DCVs) in neurons. Consistent with its function in long distance transport, mutations in KIF1C causes hereditary spastic paraplegia (HSP) and cerebellar dysfunction in humans. Previous data generated in the Straube lab (Bachmann et al, unpublished) show that PTPN21, a scaffolding phosphatase restores KIF1C mediated α5-integrin transport when overexpressed in KIF1C-depleted cells. In this study, the hydrodynamic analysis of recombinant full-length KIF1C and crosslinking mass spectrometry reveals that KIF1C is a stable dimer that adopts an autoinhibited conformation by interaction of its tail with the motor domain. Next, we show that KIF1C is a processive plus-end directed motor in single molecule assays and confirm that the scaffold phosphatase PTPN21 is a positive regulator of KIF1C-mediated transport. We also investigate the structural domains of KIF1C contributing to the rear accumulation in cells and show that the proline rich domain at the C-terminus of KIF1C is required for the tail localisation of KIF1C, but that the binding of 14-3-3 proteins to the tail domain negatively regulates KIF1C. Finally, using optical trapping we show that a single KIF1C can generate forces up to 5.5 pN and the HSP-causing mutations are processive but weak motors.

Decidualization denotes the transformation of endometrial stromal cells into specialised secretory decidual cells, a process indispensable for pregnancy. Decidualization of the human endometrium is not dependent on an implanting embryo but initiated during the mid-luteal phase of the cycle by elevated progesterone levels and local paracrine signals. Consequently, decidualization is a reiterative process directly linked to menstrual repair and rapid oestrogen-dependent growth. The extraordinary regenerative ability of the endometrium depends on endometrial mesenchymal stem cells (eMSCs) with inexhaustible self-renewing and differentiation capacity. Cyclic regeneration and rapid proliferation also render the stroma intrinsically heterogeneous, harbouring not only eMSCs but also endometrial transit amplifying (eTAs), mature, and senescent fibroblast subpopulations. Several lines of evidence presented in this thesis demonstrated that imbalance in these subpopulations is associated with reproductive failure. Quantification of clonal (eMSCs/eTAs) cells in mid-luteal biopsies obtained in consecutive cycles revealed increased levels in the 2nd biopsy obtained from miscarriage but not infertile patients, indicating that the tissue response to injury (i.e. the 1st biopsy) differs between patient groups. Further, in-depth characterization of primary stromal cell cultures prior to in vitro fertilisation (IVF) treatment showed that disordered temporal changes in the secretome of decidualizing cultures are associated with subsequent implantation failure. Additional characterization of perivascular eMSCs, which drive endometrial regeneration, highlighted the unique properties of these cells in terms of gene expression, metabolism, clonogenic and angiogenic potential. Importantly, eMSCs also formed 3D structures that resemble the uterine mucosa when cultured in Matrigel. These novel organoids termed endometrial regenerative bodies (ERBs), epithelialize when co-cultured and decidualize in response to differentiation cues. In sum, I provided evidence that dyshomeostasis between stromal subpopulations, which may be caused by eMSCs deficiency or dysfunction, precedes reproductive failure. Further, the ability of eMSCs to form ERBs provides a powerful new tool to study physiological and pathological implantation events in vitro.

The adipose tissue has an important role in maintaining the energy homeostasis balance. Understanding its physiology is important for the development of treatments against diseases where this equilibrium is compromised, such as obesity and associated metabolic disorders. MicroRNAs (miRNAs) are important gene regulators and an increasing body of evidence suggests their involvement in adipogenesis and adipose metabolism. MiRNAs can also be secreted into the extracellular environment and be taken up by distal cells, mediating cell-to-cell communication. However, very little is known about adipose tissue-derived circulating miRNAs. Through miRNA PCR array analysis we identified several miRNAs that are differentially secreted among mouse undifferentiated and differentiated brown and white adipocytes, among which, miR-196a and miR-378a-3p showed a conservative pattern of secretion in different adipocyte models. MiR-138-5p was identified as the unique miRNA differentially secreted between human brown and white adipocytes. Bioinformatics target prediction revealed that these miRNAs are potentially involved in important processes regulating the functioning of adipose tissue and its cross-talk with distal cells. By ultracentrifugation of adipose conditioned media and Nanosight technology, we investigated vesicle and vesicle-free miRNA carriers and characterized adipose derived vesicles. Finally, through microRNA array and mRNA sequencing we identified genes, miRNAs and pathways differentially enriched in human brown and white adipocytes contributing to improve the knowledge on the nature of human adipocytes, hampered by the scarce availability of human brown adipose samples. Through integration of the two analyses, we identify poorly known or novel miRNAs, potentially involved in the pathways associated with the genes differentially expressed between human brown and white adipocytes. Among the significantly downregulated miRNAs in brown versus white adipocytes we highlighted miR-513a-3p, miR-4511 and miR-4328. While, among the significantly upregulated miRNAs in brown versus white we highlighted miR-4698, miR-4516, miR-4531, miR-29a-3p and miR-3915.

A working internal circadian clock allows a healthy organism to keep time in order to anticipate transitions between night and day, allowing the temporal optimisation and control of internal processes. The internal circadian clock is regulated by a set of core genes that form a tightly coupled oscillator system. These oscillators are autonomous and robust to noise, but can be slowly reset by external signals that are processed by the master clock in the brain. In this thesis we explore the robustness of a tightly coupled oscillator model of the circadian clock, and show that its deterministic and stochastic forms are both significantly robust to noise. Using a simple linear algebra approach to rhythmicity detection, we show that a small set of circadian clock genes are rhythmic and synchronised in mouse tissues, and rhythmic and synchronised in a group of human individuals. These sets of tightly regulated, robust oscillators, are genes that we use to de ne the expected behaviour of a healthy circadian clock. We use these “time fingerprints" to design a model, dubbed “Time-Teller", that can be used to tell the time from single time point samples of mouse or human transcriptome. The dysfunction of the molecular circadian clock is implicated in several major diseases and there is significant evidence that disrupted circadian rhythm is a hallmark of many cancers. Convincing results showing the dysfunction of the circadian clock in solid tumours is lacking due to the difficulties of studying circadian rhythms in tumours within living mammals. Instead of developing biological assays to study this, we take advantage of the design of Time-Teller, using its underlying features to build a metric, Θ, that indicates dysfunction of the circadian clock. We use Time-Teller to explore the clock function of samples from existing, publicly available tumour transcriptome data. Although multiple algorithms have been published with the aims of “time-telling" using transcriptome data, none of them have been reported to be able to tell the times of single samples, or provide metrics of clock dysfunction in single samples. Time-Teller is presented in this thesis as an algorithm that both tells the time of a single time-point sample, and provides a measure of clock function for that sample. In a case study, we use the clock function metric, , as a retrospective prognostic marker for breast cancer using data from a completed clinical trial. Θ is shown to correlate with many prognostic markers of breast cancer, and we show how could also be a predictive marker for treatment efficacy and patient survival.

The potential of a maleimide based fluorophore as well as the utilisation of simple methodologies to produce synthetic mimics of natural light harvesting complex (LHC) were explored. In Chapter 1, the inspiration for the current work is discussed, followed by a brief guide for the interpretation of the work presented in subsequent chapters. Finally, the ultimate aim and concept for the thesis is detailed. The general instruments and methodologies applicable to all experiments conducted are then described in Chapter 2. Chapter 3 presents the first set of results from the thesis work. This work focused on unravelling the previously unexplained fluorescence quenching observed in maleimide based fluorophores in protic solvent. This was achieved through the use of computational chemistry, ultrafast spectroscopy and synthetic chemistry. The work identified a photochemical process, in the form of electron driven proton transfer, as the fluorescence quenching mechanism. Such understandings should provide a much needed insight towards future designs of maleimide based fluorophores that fully realise the potential of this class of photoactive pigments. This was then followed by the attempt to produce the platform for which inter-chromophoric assemblies could occur in aqueous media, documented in Chapter 4. The main body of the work involved the selection of the most effective synthetic method readily reported in the literature: the synthesis of functionalised porphyrin through the condensation reaction between dipyrrolemethane and functionalised aldehyde; and the conjugation between porphyrin and polymers with the copper catalysed alkyne-azide cyclo addition click reaction. The resulting platform was successfully demonstrated to be capable of self-assembly, without the need of covalently linking the chromophores to one another. Furthermore, and most importantly, the photophysical properties of these chromophores were largely retained in the assembled structure. The results presented could lead to a new class of photoactive nanostructures that closely mimic natural LHCs, in which the properties of individual chromophores could be fully exploited and assembled without the need of covalent linkage. The potential of using the platform documented in Chapter 4 to produce functional natural LHC mimics was then explored in the final results chapter, Chapter 5, where a pseudo reaction centre (RC) was introduced. The spectroscopic experiments demonstrated that the photodynamics of the assembly was significantly altered in the presence of the RC. This was proposed to be based on a push-pull mechanism facilitated by the presence of metal to N donor coordinated complex. Furthermore, the N donor in the chosen RC proved to be instrumental for the assembly and spectral differences observed. Therefore, these presented properties of the platform described could likely produce much more complex, functional multi-chromophoric assemblies. The thesis then closed with a brief overview and an outlook which discussed two multi-chromophoric assemblies, based on the work presented in Chapter 3–5.