Little is known about the composition of tropical epiphytic communities and the influence of environmental variables on community composition. In this thesis I quantify the diversity and biomass of bromeliads, and other vascular epiphytes along an altitudinal transect on the eastern slope of the southeast Peruvian Andes and then look for species’ adaptations related to patterns of diversity and biomass. I compare patterns with those of woody species. Bromeliad species, like tree species, were found to form ecological zones related to climate. The lowest altitude ecological zone (below 1250 m) is the lowland rainforest (LRF), which has the warmest climate and highest evapotranspiration. In LRF vascular epiphytes are less prominent than other ecological zones, with the lowest bromeliad species richness and lowest vascular epiphyte biomass. However, low water-availability gives rise to most variable shoot morphology of bromeliads. The tropical montane forest (TMF), between 1250 m and 2250 m, is intermediate in climate between the LRF and the tropical montane cloud forest (TCF). The TMF has the highest α-diversity, but species richness is lower than the TCF. The shoot morphology of bromeliads is intermediate between TCF and LRF. The highest altitude ecological zone with forest is the TCF (above 2250 m). The TCF has the highest bromeliad species richness, and lowest diversity of shoot forms. The low diversity of shoot forms represents the need for a large phytotelm (water-impounding shoot) to intercept and store precipitation. The TCF has the highest vascular epiphyte biomass, although the biomass is variable as a consequence of the natural disturbance caused by landslides. Along the transect bromeliad species with CAM photosynthesis are only present in the LRF. Terrestrial bromeliad distribution records covering the Neotropics show CAM photosynthesis is more prevalent in drier environments showing that CAM photosynthesis is primarily an adaptation to drought. Epiphytic bromeliads, pre-adapted to a water-stressed environment show no differences in presence along rainfall gradients, but species with CAM photosynthesis occupy warmer environments.

Centrosomes are cellular organelles present in most animal cells, and are formed of two main components: the centrioles and the pericentriolar material (PCM). Centrosomes perform a variety of functions: they are the main microtubule organising centre in the cell, and are important localisation hubs for kinases involved in regulating the cell cycle. Hundreds of proteins are thought to localise to centrosomes, but work in the last decade has narrowed down this list to a handful of proteins that are thought to be essential for centrosome structure and function in Drosophila. Asl, Ana2, DSas-4, DSas-6 and Sak have been identified as essential components for centriole duplication, while Cnn and DSpd-2 are thought to be important in establishing the PCM. However, the relative position of these 7 components in the pathway of centrosome assembly in Drosophila embryos remains elusive, as a genetics analysis of this process is hampered by the absence of centrioles in most mutant embryos for these proteins. In this thesis I elucidate the pathway of centrosome assembly in Drosophila by using SAPs (DSas-6/Ana2 particles that form in Drosophila unfertilised eggs upon moderate expression of DSas-6 and Ana2) as proxy models of centrosomes. I show SAPs are very similar to centrosomes in composition and dynamics but unlike centrosomes are able to form even in the absence of some of the essential centriolar components. SAP analysis in the absence of each of the main centrosome components reveals that: Sak is not required for the recruitment of downstream components; DSas-4 is necessary for Ana2 and DSas-6 to interact; Asl is the most upstream component of the PCM recruitment pathway, followed by DSpd-2; it is likely that there is an additional PCM recruitment pathway. I then take advantage of some of these results to examine how centrosome formation is potentiated after egg activation. My work allows me to propose an improved description of the pathway of centrosome formation in Drosophila.

The purpose of this thesis was to study mitosis in Drosophila, from a network biology perspective. The primary aim was to develop and test a network-based prediction model that could integrate available data in public databases (like Flybase) and, based on that, predict potential mitotic proteins. The approach taken to design the protein interaction network included the use of a priori knowledge about the microtubule composition of the mitotic spindle and the higher likelihood of microtubule-associated proteins (MAPs) to have a putative mitotic function. The design also included the integration of different complementary datasets, from gene expression and functional RNAi screens to cross species conservation of MAPs for fitting a network-based model for predicting mitotic proteins. I begin with the creation of the MAP interactome based on a MAP dataset in Drosophila. This initial network was extended by transferring homologs and interologues of MAP datasets from four other species, i.e. human, mouse, rat and Arabidopsis. These proteins were then used as seed proteins to conduct a virtual pull-down experiment, by adding indirect interactors into the network, i.e. proteins that directly bind to two or more MAPs within the network, which completed the MAP interactome. Data from genome-wide studies in Drosophila were gathered for each node in the MAP interactome. These ‘layers’ of data were then used as features to fit a prediction model that could score each node in the network, based on the likelihood of its role in mitosis. The final model performed with 96% accuracy after 10-fold cross validation and was used to rank all the proteins in the MAP interactome. By analysing the top 100 high scoring predicted mitotic proteins, a highly connected cluster of 33 proteins was identified that was subject to experimental validation in the lab. The first approach was to conduct an in vitro analysis using an RNAi screen to test for any spindle, chromosome or centrosome phenotypes upon gene knockdown. After two independent RNAi screens, around 80% of the proteins produced mutant mitotic phenotypes strongly supporting the results of the MAP prediction model. The second approach was to conduct an in vivo analysis by expressing GFP- fusion constructs of selected genes from the subcluster. These were expressed in Drosophila early embryos to study their subcellular localization during interphase and mitosis. A variety of localizations were observed ranging from chromatin and microtubules to more generic cytoplasmic localizations. These results suggested not all predicted proteins were co-localizing with microtubules, and therefore might not necessarily be microtubule associated proteins but can possibly be functioning as microtubule associated regulator proteins. Proteomics analysis of a subset of these genes showed a large proportion of false positive interactions but also picked new interactions between member proteins that highlighted a module within the subcluster. The RNAi hits from the in vitro analysis and the members of the module within subcluster-16 from the in vivo analysis provide interesting subjects for further characterization.

Carbonic anhydrase IX (CA9) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. The function of CAIX is to catalyze the reversible hydration of CO2 to bicarbonate and a proton. This helps hypoxic tumours to maintain a more neutral intracellular pH (pH_i) promoting survival, but produces a more acidic extracellular (pH_e) which promotes invasion and metastasis. Recent evidence has expanded on the role of hypoxia and CAIX by relating them to stem cell niches. In this study, taking advantage of the transmembrane location of CAIX, we show for the first time, a direct marked heterogeneity in response to hypoxia within each tumour cell population studied, associated with major biological differences. Based on CAIX expression pattern under hypoxic conditions, we identify, isolate and characterize two distinct populations of tumour cells, one that express CAIX and the other that does not. Interestingly, we discover that the CAIX positive population is enriched with cells expressing cancer stem cell markers. These include ALDHA1, IGF1, LIN28 and genes involved in epithelial-mesenchymal transition (EMT) and multi-drug resistance (i.e. WNT2, TWIST1, and ABCC2). Accordingly, CAIX+ve cells show higher self-renewal capacity and form tumours significantly faster compared to the CAIX-ve population. Importantly, functional suppression of CAIX in vitro and in vivo, in two breast cancer cell lines resulted in the downregulation of breast cancer stem cell signatures, suggesting that CAIX is not just a marker of stemness but also a regulator of stemness. The molecular mechanism underlying the differential expression of CAIX in the two populations is not HIF-1α-dependent, but instead driven by hypoxia-induced reorganization of chromatin structure. In line with this, we provide experimental evidence showing that the genomic locus encoding CA9 has a more “open” and transcriptionally active chromatin structure in CAIX+ve cells, and a condense and transcriptionally silent chromatin structure in the CAIX-ve cells. Given that HIF induces the transcription of CA9 by binding to hypoxia response elements (HREs) in its promoter we show a significant reduction in binding of HIF to the CA9 promoter of the negative population. We suggest that the reduce HIF binding is a result of the compact chromatin structure of CA9 promoter of the negative cells. Analysis of the transcriptome of the positive and negative populations suggests a symbiotic relationship between these two subpopulations and their environment, likely required to promote tumour growth. This is based on the following observations: Firstly, we identified that CAIX-ve cells express high levels of cytokines and based on this, we suggest that the cytokines secreted by CAIX-ve cells may transmit paracrine signals that regulate the CAIX+ve cells, thus providing a wider hypoxia tolerant microenvironment to protect the stem cell population. Secondly, we identified a metabolic heterogeneity between the CAIX+ve and CAIX-ve cells. The CAIX+ve cells show an upregulation of genes implicated in oxidative phosphorylation, TCA cycle and fatty acid synthesis. Whereas in CAIX-ve cells there is an upregulation of genes implicated in autophagy and mitophagy. Given the above together with the upregulation of oxidative phosphorylation and TCA cycle in the CAIX+ve cells, we proposed the existence of a metabolic symbiosis between the CAIX+ve and CAIX-ve cells. We postulate that the catabolic process such as autophagy and mitophagy in the CAIX-ve cells may results in the overproduction of high-energy metabolites such as lactate, glutamine and ketone bodies which in turns they are been utilized by CAIX+ve cells to fuel mitochondria respiration. Finally, we also demonstrated that in the CAIX+ve cells mTORC1 signaling is upregulated, and contributes to the regulation of CAIX expression. Given the role of mTORC1 in stem cell maintenance and EMT as well as the interdependence of mTORC1 and CAIX expression in the CAIX+ve cells we suggest that mTORC1 signaling may be the critical factor by which CAIX regulates stemness. Interestingly, the subpopulations show a differential sensitivity to HDAC inhibitors, NaBu and SAHA as treatment of MCF7 breast cancer cell line and HCT116 colon cancer cell line leads to elimination of the CAIX+ve population. This is not driven by the downregulation of HIF-1α, the major transcriptional regulator of CAIX. In contrast, we demonstrate that SAHA causes downregulation mTORC1. This suggests that SAHA-induced downregulation of CAIX expression could be due to its effect on mTORC1 pathway. Of wider significance, our findings show that tumours are not homogenous in their response to hypoxia, and distinct signal transduction networks regulate different populations of cells within the tumour. This highlights the need for the utilization of biomarkers, which reveal distinct functional hypoxia profiles of human cancers, and permit the stratification of tumours. Furthermore, the identification of epigenetic regulation of the histones in response to hypoxia for highly selective gene regulation, provides a connection between the epigenetic mechanisms under environmental stress and cancer progression, and is model for development of novel epigenetic cancer therapeutic drugs.

This thesis examines the urban topography of Berlin’s working-class districts, as seen in the art, architecture and other images produced in the city between 1924 and 1930. During the 1920s, Berlin flourished as centre of modern culture. Yet this flourishing did not exist exclusively amongst the intellectual elites that occupied the city centre and affluent western suburbs. It also extended into the proletarian districts to the north and east of the city. Within these areas existed a complex urban landscape that was rich with cultural tradition and artistic expression. This thesis seeks to redress the bias towards the centre of Berlin and its recognised cultural currents, by exploring the art and architecture found in the city’s working-class districts. The thesis adopts Henri Lefebvre’s premise that each society creates its own space in which it lives, works, and sustains its cultural identity. On this basis, working-class culture and the spaces in which it was practiced, are treated with equal weight. The thesis begins by examining how the laissez-faire economics of the German Empire (1871–1914), combined with a massive influx of rural migrants into Berlin, creating a complex industrial landscape, whose working-class inhabitants retained many pastoral traditions. The thesis moves on to study the works of a number of artists active in Berlin between 1924 and 1930, using examples of their work to examine the unique nature of the working-class districts, and the culture and traditions that took place within them. The second half of the thesis explores the working-class districts from an explicitly political perspective. The extensive house building programme that took place across Berlin throughout the twenties is explored in all its varied and conflicting political perspectives. What emerges is a picture of a growing schism between Berlin’s Social Democratic government, and Communist supporters in the working-class districts. 1929 emerges as a critical year in which political contestations of space between the two parties and their supporters reached new levels of hostility, as working-class culture clashed against Social Democratic urban policy.

Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. The evolution of these ciliary functions may be inferred from the distribution of the molecular components from which these organelles are composed. By linking protein distribution in 45 diverse eukaryotes with organismal biology, I define an ancestral ciliary inventory. Analysis of these core proteins allows the inference that the cenancestor of the eukaryotes possessed a cilium for motility and sensory function. I show that the centriolar basal body function is ancestral, whereas the centrosome is specific to the Holozoa, and I use this information to predict a number of roles for proteins based on their phylogenetic profile. I also show that while remarkably conserved, significant divergence in ciliary protein composition has occurred in many lineages, such as the unusual centriole of Caenorhabditis elegans and the transitional changes throughout the land plants. I exemplify this divergence through ultrastructural studies of the fern Ceratopteris richardii and the liverwort Marchantia polymorpha both of which have cilia that exhibit a number of distinctive morphological features, the most conspicuous of which is a general breakdown of canonical microtubule arrangements. Cilia have also been lost multiple times in different lineages: at least twice within the land plants. During these evolutionary transitions proteins with ancestral ciliary functions may be lost or co-opted into different functions. I have interrogated genomic data to identify proteins that I predict had an ancestral ciliary role, but which have been maintained in non-ciliated land plants. I demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes and I use expression data correlation to predict potential non-ciliary plant roles.

A key question in developmental biology is how form is generated. The model species Arabidopsis thaliana produces simple leaves with marginal outgrowths termed serrations. Serration development in A. thaliana requires both the transcription factor CUP-SHAPED COTYLEDON2 (CUC2) and the auxin efflux facilitator PIN-FORMED1 (PIN1), which regulates polar auxin transport by forming convergence points (Hay et al., 2006; Nikovics et al., 2006; Scarpella et al., 2006). In Chapter 3, I investigate how CUC2, PIN1 and auxin interact to control serration development. I demonstrate that CUC2 promotes PIN1 convergence point and auxin activity foci formation along the margin of the leaf, whilst high auxin activity represses CUC2 expression. Furthermore, interspersed peaks of CUC2 and auxin activity pattern serration development along the proximo-distal axis of the leaf. Thus, auxin, PIN1 and CUC2 form a negative feedback loop that patterns serration development. CUC genes and PIN1 are required for leaflet development in Cardamine hirsuta (Barkoulas et al., 2008; Blein et al., 2008), a close relative of A. thaliana that produces compound leaves subdivided into units termed leaflets. However, it is unclear how CUC and PIN1 interact to control leaflet development. In Chapter 4, I demonstrate that similar to A. thaliana, CUC genes promote PIN1 convergence point and auxin activity foci formation at the C. hirsuta leaf margin, whilst high auxin activity represses CUC2 expression. These genetic interactions likely create interspersed peaks of CUC2 and auxin activity that pattern leaflet development. Thus, the same negative feedback loop between CUC, PIN1 and auxin patterns both leaflet development in C. hirsuta and serration development in A. thaliana. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are expressed in C. hirsuta leaves, and interact with ChCUC and PIN1 in positive and negative feedback loops, respectively, to control leaflet development (Barkoulas et al., 2008; Blein et al., 2008). KNOX genes are not expressed in A. thaliana leaves, but deeply lobed margins reminiscent of leaflets develop in association with ectopic KNOX expression in leaves (Chuck et al., 1996; Hay et al., 2006). However, it is unclear whether regulatory interactions of PIN1, CUC and KNOX which occur in C. hirsuta leaflets are employed during KNOX-induced lobe development in A. thaliana. In Chapter 5, I demonstrate that CUC2 and polar auxin transport are required for ectopic KNOX expression. Conversely, I show that KNOX misexpression up-regulates CUC2 expression in A. thaliana leaves. Thus, interactions between KNOX, CUC and PIN1 that occur in leaflet development in C. hirsuta also occur in association with KNOX-induced lobe development in A. thaliana. In addition to investigating the regulatory interactions between known components of leaf development pathways, I sought to identify novel genes that mediate CUC2-dependent serration development in A. thaliana. In Chapter 6, I identify a suppressor of the smooth margin phenotype of cuc2 leaves that partially restores PIN1 localisation in the absence of functional CUC2. Finally, in the General Discussion I evaluate how interlinking feedback loops between CUC, KNOX and auxin pattern serration and leaflet development. I then discuss why interlinking feedback loops may have been deployed to control outgrowths in both plant and animal systems.

The case-control design is one of the most commonly used designs in genome- wide asociation studies. When we increase the sample size of either the controls or, more importantly, the cases, the power of whatever test we use will certainly increase. However increasing the sample size, means that addi- tional individuals need to be genotyped and this implies extra financial costs. However, nowadays with the emergence of genetic studies, a large number of genetic data are available at low or no extra cost. Even though those data may not be completely relevant to the current study, they can still be used to increase the probability to identify true associations. Furthermore, additional information, non-necessarily genetic, can also be used to improve the power of a method. In this thesis we extend the case-control design in order to take ad- vantage of such types of additional data and/or information. We discuss three designs; the case-cohort-control, the kin-cohort and the super-case– case–control–super-control designs. For each of these, we present methods that are adjusted or modified versions of standard case-control methods but we also propose novel ones developed with those extended designs in mind. Ultimately, we describe how those methods can be used in order to increase the power of association tests, especially compared to similar methods of the case-control design.

This thesis is a study of the detection of separation among the sample points in binary logistic regression models. We propose a new algorithm for detecting separation and demonstrate empirically that it can be computed fast enough to be used routinely as part of the fitting process for logistic regression models. The parameter estimates of a binary logistic regression model fit using the method of maximum likelihood sometimes do not converge to finite values. This phenomenon (also known as monotone likelihood or infinite parameters) occurs because of a condition among the sample points known as separation. There are two classes of separation. When complete separation is present among the sample points, iterative procedures for maximizing the likelihood tend to break down, when it would be clear that there is a problem with the model. However, when quasicomplete separation is present among the sample points, the iterative procedures for maximizing the likelihood tend to satisfy their convergence criterion before revealing any indication of separation. The new algorithm is based on a linear program with a nonnegative objective function that has a positive optimal value when separation is present among the sample points. We compare several approaches for solving this linear program and find that a method based on determining the feasibility of the dual to this linear program provides a numerically reliable test for separation among the sample points. A simulation study shows that this test can be computed in a similar amount of time as fitting the binary logistic regression model using the method of iteratively reweighted least squares: hence the test is fast enough to be used routinely as part of the fitting procedure. An implementation of our algorithm (as well as the other methods described in this thesis) is available in the R package safeBinaryRegression.

Although the history of Virtual Reality (VR) is only about half a century old, all kinds of technologies in the VR field are developing rapidly. VR is a computer generated simulation that replaces or augments the real world by various media. In a VR environment, participants have a perception of “presence”, which can be described by the sense of immersion and intuitive interaction. One of the major VR applications is in the field of sports, in which a life-like sports environment is simulated, and the body actions of players can be tracked and represented by using VR tracking and visualisation technology. In the entertainment field, exergaming that merges video game with physical exercise activities by employing tracking or even 3D display technology can be considered as a small scale VR. For the research presented in this thesis, a novel realistic real-time table tennis game combining immersive, interactive and competitive features is developed. The implemented system integrates the InterSense tracking system, SwissRanger 3D camera and a three-wall rear projection stereoscopic screen. The Intersense tracking system is based on ultrasonic and inertia sensing techniques which provide fast and accurate 6-DOF (i.e. six degrees of freedom) tracking information of four trackers. Two trackers are placed on the two players’ heads to provide the players’ viewing positions. The other two trackers are held by players as the racquets. The SwissRanger 3D camera is mounted on top of the screen to capture the player’s