http://www.dspace.cam.ac.uk:80/handle/1810/221767 Wed, 22 May 2013 00:30:05 GMT 2013-05-22T00:30:05Z http://www.dspace.cam.ac.uk:80/handle/1810/244505 Title: Bleb-driven chemotaxis in Dictyostelium discoideum Authors: Zatulovskiy, Evgeny Abstract: Migrating cells have two basic ways of extending their leading edge: by dendritic actin polymerization beneath the membrane, or by fluid pressure, which produces blebs. Most cells are believed to move using actin-driven projections, but in more physiological conditions, blebbing motility is also apparent. It has been shown that certain cells even can switch between these two modes of motility, although it is not known how this switch is triggered. Besides, it is unclear whether blebbing can be regulated by chemotactic stimuli, and generally, how blebbing is controlled in the cell. In this study I employed a popular model organism – Dictyostelium discoideum – to investigate the role of blebbing in chemotaxis. Here I confirm that in standard conditions Dictyostelium cells move by a combination of F-actin-driven protrusions and blebs. Blebbing is characterized by the rapid projection of hemispherical patches of plasma membrane at 2-4 times the speed of an actin-driven projection, and leaves transient scars of F-actin marking the original cortex in the base of blebs. I demonstrate that Dictyostelium cells can adjust their mode of movement according to the conditions: in a resistive environment they switch almost entirely to “bleb mode”. I show that in chemotaxing cells, blebs are mainly restricted to the leading edge, and they often lead the way when a cell is forced to re-orientate. Bleb location appears to be controlled directly by chemotactic gradients. To investigate how chemoattractant induces blebbing, I have screened signal transduction mutants for altered blebbing. I have found that blebbing is unaffected in many chemotactic mutants, but unexpectedly depends on PI3-kinases and two downstream PIP3-binding proteins of unknown function – PhdA and CRAC. I conclude that Dictyostelium cells move using a hybrid motor in which hydrostatic pressure-driven bleb formation is as important as F-actin-driven membrane extension, and that cells can change the balance between modes as required. I propose that blebbing motility of Dictyostelium cells is a direct response to mechanical resistance of environment. More generally, bleb-driven motility may be a ‘”high-force” mode of movement that is suited to penetrating tissues. Blebs are chemotactic and their induction may involve branches of the chemotactic signal transduction pathway distinct from F-actin regulation. Tue, 12 Mar 2013 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/244505 2013-03-12T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/244503 Title: A cylindrical specimen holder for electron cryo-tomography Authors: Palmer, Colin Michael Abstract: The ‘missing wedge’ is a long-standing problem in electron tomography, caused by the use of slab-like flat specimens, which increase in thickness when tilted to high angles. Attempts have been made to reduce the undesirable effects caused by the missing wedge, but the problem remains, particularly for the radiation-sensitive frozen-hydrated specimens used for high resolution imaging. Specimens with cylindrical symmetry offer a way to overcome this problem, since the thickness remains constant at all viewing angles. However, while this has been suggested before, it has never been demonstrated for frozen-hydrated specimens. In this work, I present a way to make cylindrical specimens for electron cryo-tomography, using thin-walled carbon tubes as specimen holders. The tubes are made in a multi-step process, involving carbon deposition on glass micropipette templates and subsequent removal of the glass. Tube diameters are typically a few hundred nanometres, with a wall thickness of 10–20 nm. To make frozen-hydrated specimens, the tubes are filled with an aqueous sample and then plunge-frozen in liquid ethane. Electron images acquired from the tubes have equal quality at all viewing angles, with a tilt range restricted only by the physical limits of the microscope. Tomograms from specimens such as gold particles and liposomes show that the effects of the missing wedge are substantially reduced, with much improved resolution along the electron beam axis. Structural features oriented in all directions are visible in the reconstructions, in marked contrast to tomograms acquired over a more restricted angular range. These results are promising, however some technical challenges remain before this method can be used routinely. Tue, 12 Mar 2013 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/244503 2013-03-12T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/243944 Title: Fronto-parietal cortex in sequential behaviour Authors: Farooqui, Ausaf Ahmed Abstract: This dissertation investigates the fronto-parietal representation of the structure of organised mental episodes by studying its effect on the representation of cognitive events occurring at various positions within it. The experiments in chapter 2 look at the completion of hierarchically organized mental (task/subtask) episodes. Multiple identical target-detection events were organized into a sequential task episode, and the individual events were connected in a means-to-end relationship. It is shown that events that are conceptualized as completing defined task episodes elicit greater activity compared to identical events lying within the episode; the magnitude of the end of episode activity depended on the hierarchical abstraction of the episode. In chapter 3, the effect of ordinal position of the cognitive events, making up the task episode, on their representation is investigated in the context of a biphasic task episode. The design further manipulated the cognitive load of the two phases independently. This allowed for a direct comparison of the effect of phase vis-à-vis the effect of cognitive load. The results showed that fronto-parietal regions that increased their activity in response to cognitive load, also increased their activity for the later phases of the task episode, even though the cognitive load associated with the later phase was, arguably, lower than the previous phase. Chapter 4 investigates if the characteristics of the higher-level representations, like organization of task descriptions, have a causal role in determining the structure of the ensuing mental episode. Results show this to be true. They also confirm the results of earlier chapters in a different framework. Chapter 5 shows that the effect of episode structure is not limited to the elicited activity, but also affects the information content of the representation of the events composing the episode. Specifically, the information content in many regions of later steps is higher than that of earlier steps. Together, the results show widespread representation of the structure of organised mental episodes. Mon, 08 Oct 2012 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243944 2012-10-08T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/243852 Title: Understanding language and attention: brain-based model and neurophysiological experiments Authors: Garagnani, Max Abstract: This work concerns the investigation of the neuronal mechanisms at the basis of language acquisition and processing, and the complex interactions of language and attention processes in the human brain. In particular, this research was motivated by two sets of existing neurophysiological data which cannot be reconciled on the basis of current psycholinguistic accounts: on the one hand, the N400, a robust index of lexico-semantic processing which emerges at around 400ms after stimulus onset in attention demanding tasks and is larger for senseless materials (meaningless pseudowords) than for matched meaningful stimuli (words); on the other, the more recent results on the Mismatch Negativity (MMN, latency 100-250ms), an early automatic brain response elicited under distraction which is larger to words than to pseudowords. We asked what the mechanisms underlying these differential neurophysiological responses may be, and whether attention and language processes could interact so as to produce the observed brain responses, having opposite magnitude and different latencies. We also asked questions about the functional nature and anatomical characteristics of the cortical representation of linguistic elements. These questions were addressed by combining neurocomputational techniques and neuroimaging (magneto-encephalography, MEG) experimental methods. Firstly, a neurobiologically realistic neural-network model composed of neuron-like elements (graded response units) was implemented, which closely replicates the neuroanatomical and connectivity features of the main areas of the left perisylvian cortex involved in spoken language processing (i.e., the areas controlling speech output – left inferior-prefrontal cortex, including Broca’s area – and the main sensory input – auditory – areas, located in the left superior-temporal lobe, including Wernicke’s area). Secondly, the model was used to simulate early word acquisition processes by means of a Hebbian correlation learning rule (which reflects known synaptic plasticity mechanisms of the neocortex). The network was “taught” to associate pairs of auditory and articulatory activation patterns, simulating activity due to perception and production of the same speech sound: as a result, neuronal word representations distributed over the different cortical areas of the model emerged. Thirdly, the network was stimulated, in its “auditory cortex”, with either one of the words it had learned, or new, unfamiliar pseudoword patterns, while the availability of attentional resources was modulated by changing the level of non-specific, global cortical inhibition. In this way, the model was able to replicate both the MMN and N400 brain responses by means of a single set of neuroscientifically grounded principles, providing the first mechanistic account, at the cortical-circuit level, for these data. Finally, in order to verify the neurophysiological validity of the model, its crucial predictions were tested in a novel MEG experiment investigating how attention processes modulate event-related brain responses to speech stimuli. Neurophysiological responses to the same words and pseudowords were recorded while the same subjects were asked to attend to the spoken input or ignore it. The experimental results confirmed the model’s predictions; in particular, profound variability of magnetic brain responses to pseudowords but relative stability of activation to words as a function of attention emerged. While the results of the simulations demonstrated that distributed cortical representations for words can spontaneously emerge in the cortex as a result of neuroanatomical structure and synaptic plasticity, the experimental results confirm the validity of the model and provide evidence in support of the existence of such memory circuits in the brain. This work is a first step towards a mechanistic account of cognition in which the basic atoms of cognitive processing (e.g., words, objects, faces) are represented in the brain as discrete and distributed action-perception networks that behave as closed, independent systems. Mon, 12 Oct 2009 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243852 2009-10-12T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/243495 Title: Pattern codes for perceived gaze direction revealed by functional MRI Authors: Carlin, Johan D. Abstract: Perceiving the direction of another's attention is a critical component of normal social behaviour. Seminal electrophysiology studies demonstrated that single cells in macaque superior temporal sulcus (STS) are tuned to specific directions of social cues, including gaze direction, head view, and body posture. Furthermore, a subset of such neurons respond to a single direction across multiple cues, suggesting that the code is driven by the direction of another's social attention regardless of how this is conveyed. Attempts to reveal similar gaze representations in humans using fMRI have provided mixed results. This thesis describes research where multivariate pattern analysis (MVPA) methods are applied to fMRI data in order to better explain how the human brain and particularly STS codes perceived gaze direction. After describing the MVPA methods applied in this thesis, I first demonstrate that fMRI response patterns in anterior STS distinguish between the direction of dynamic head turns, but not between the direction of rotation in non-social ellipsoids. In subsequent work, anterior STS is found to code the direction of another's gaze in a head view-invariant manner, thus demonstrating a potential parallel to previous macaque evidence for single cells that code the direction of another's attention. However, comparisons that run both across species (macaque, human) and methods (electrophysiology, fMRI) are problematic. To overcome this limitation I next tested whether macaque STS distinguishes gaze direction and head view when responses are measured with fMRI. In conclusion, this thesis demonstrates the utility of applying MVPA to fMRI data to reveal socially-relevant representations of the direction of another's attention. The thesis particularly highlights anterior STS as a key region in supporting direction-specific representations of social cues. These results advance our understanding of how the brain codes socially-relevant information, and highlight possible similarities and dissimilarities between humans and macaques. Mon, 11 Jun 2012 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243495 2012-06-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/242018 Title: MYC transcriptional functions controlling epidermal stem cell self-renewal and differentiation Authors: Nascimento, Elisabete Abstract: The oncoprotein MYC has long been recognized as an important stem cell regulator, yet its direct biological contributions have been difficult to determine. MYC activation can induce pleiotropic phenotypes and mediates cellular functions as opposing as cell growth and proliferation, metabolism, differentiation and apoptosis. In addition, functional redundancy with MYCL and MYCN proteins as well as dose dependency, complicates the identification of the most relevant biological functions. Studies in tissues with high proliferative capacity and rapid turnover have shown that MYC is a key regulator of homeostasis by balancing stem cell self-renewal, proliferation and differentiation processes. In skin, MYC induces the exit of epidermal stem cells from their niche, increases proliferation of progenitor cells and subsequently stimulates lineage specific differentiation into interfollicular epidermis and sebaceous glands; yet the direct transcriptional roles of MYC in these processes remained elusive. To gain insight into the transcriptional roles of MYC in epidermal stem cell homeostasis, I performed chromatin immunoprecipitation on microarrays (ChIP-on-Chip) using mouse proximal promoter arrays combined with mRNA expression data that was generated using epidermal cells from wild-type and transgenic K14MycER mice, treated in a time-course from zero to six days with tamoxifen, to induce the ‘Myc’ transgene expression in the basal undifferentiated layers of the epidermis. Data analysis revealed that 2187 genes, which corresponds to 15% of the promoter regions covered, were directly regulated by MYC. To identify genes uniquely regulated by MYC in skin, I performed gene expression studies on mouse skin in which MYC was conditionally deleted in the basal layer of the epidermis. Remarkably, I found that 45% of all repressed genes were related to epidermal maintenance and differentiation. To better understand the mechanism of how MYC induces keratinocytes to differentiate specifically into lineages of sebaceous glands and interfollicular epidermis, I analyzed whether MYC might have directly regulated genes involved in skin differentiation. Here, I focused my studies on a single 2.2 Mb locus located on mouse chromosome 3 designated as the epidermal differentiation complex (EDC). To assess how activation of MYC could influence the expression of genes localized to the EDC, I performed ChIP-on-Chip for MYC, H3K4me3, H3K27me3, as well as transcription factors, which have been described to regulate terminal differentiation in skin, such as CEBPα, OVOL-1, KLF4, TCFAP2-γ and SIN3A, among others. I demonstrated that MYC recruits a specific set of tissue-specific transcription factors to the EDC, (e.g. KLF4 and OVOL-1) and thereby prevents binding of a different and distinct set of genomic regulators, (e.g. CEBPα , MXI1 and SIN3A). Using a combination of mouse models and systems biology tools, I then identified SIN3A as a key regulator in this MYC-dependent transcriptional network. I found that MYC and SIN3A form a negative feedback loop, which is required to balance proliferation and differentiation in epidermis, and both factors are essential to maintain skin homeostasis. Wed, 31 Aug 2011 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/242018 2011-08-31T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/236171 Title: Exploiting network-based approaches for understanding gene regulation and function Authors: Janga, Sarath Chandra Abstract: It is increasingly becoming clear in the post-genomic era that proteins in a cell do not work in isolation but rather work in the context of other proteins and cellular entities during their life time. This has lead to the notion that cellular components can be visualized as wiring diagrams composed of different molecules like proteins, DNA, RNA and metabolites. These systems-approaches for quantitatively and qualitatively studying the dynamic biological systems have provided us unprecedented insights at varying levels of detail into the cellular organization and the interplay between different processes. The work in this thesis attempts to use these systems or network-based approaches to understand the design principles governing different cellular processes and to elucidate the functional and evolutionary consequences of the observed principles. Chapter 1 is an introduction to the concepts of networks and graph theory summarizing the various properties which are frequently studied in biological networks along with an overview of different kinds of cellular networks that are amenable for graph-theoretical analysis, emphasizing in particular on transcriptional, post-transcriptional and functional networks. In Chapter 2, I address the questions, how and why are genes organized on a particular fashion on bacterial genomes and what are the constraints bacterial transcriptional regulatory networks impose on their genomic organization. I then extend this one step further to unravel the constraints imposed on the network of TF-TF interactions and relate it to the numerous phenotypes they can impart to growing bacterial populations. Chapter 3 presents an overview of our current understanding of eukaryotic gene regulation at different levels and then shows evidence for the existence of a higher-order organization of genes across and within chromosomes that is constrained by transcriptional regulation. The results emphasize that specific organization of genes across and within chromosomes that allowed for efficient control of transcription within the nuclear space has been selected during evolution. Chapter 4 first summarizes different computational approaches for inferring the function of uncharacterized genes and then discusses network-based approaches currently employed for predicting function. I then present an overview of a recent high-throughput study performed to provide a ‘systems-wide’ functional blueprint of the bacterial model, Escherichia coli K-12, with insights into the biological and evolutionary significance of previously uncharacterized proteins. In Chapter 5, I focus on post-transcriptional regulatory networks formed by RBPs. I discuss the sequence attributes and functional processes associated with RBPs, methods used for the construction of the networks formed by them and finally examine the structure and dynamics of these networks based on recent publicly available data. The results obtained here show that RBPs exhibit distinct gene expression dynamics compared to other class of proteins in a eukaryotic cell. Chapter 6 provides a summary of the important aspects of the findings presented in this thesis and their practical implications. Overall, this dissertation presents a framework which can be exploited for the investigation of interactions between different cellular entities to understand biological processes at different levels of resolution. Mon, 05 Jul 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/236171 2010-07-05T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/236123 Title: Human GINS, a conserved DNA replication factor and candidate cancer marker Authors: Marinsek, Nina Abstract: The GINS complex (a heterotetramer of Sld5, Psf1, Psf2 and Psf3) is a highly conserved DNA replication factor required for the initiation and elongation of DNA replication. GINS is believed to associate with Cdc45 and MCM proteins on replicating DNA. The interaction between GINS and MCM is also conserved in archaea. In my thesis, I explore the subcellular localisation of the GINS complex in relation to the MCM proteins and sites of DNA replication by high-resolution confocal microscopy. For these studies, I generated and carefully validated purified rabbit polyclonal and mouse monoclonal antibodies; these show a specific staining pattern by immunohistochemistry, immunoblotting and immunofluorescence. At high-resolution, all GINS antibodies produced a focal nuclear pattern, similar to that seen for the MCMs. However, confusingly, colocalisation between GINS and MCMs and between the GINS subunits themselves is poor. Investigations are continuing to understand this conundrum. Given the value of MCM proteins as specific and sensitive markers for cancer screening, I investigated whether GINS subunits also have potential diagnostic value. Sld5 and Psf3 expression is restricted to the proliferative compartment in normal tissue, but is found in the majority of cells in a wide range of dysplastic and malignant tissues, including cervix, colon and bladder. In vitro studies of tissue culture cells and cell lysates incubated in urine suggest that Sld5 protein is more stable than Mcm2 in harsh extracellular environments. In an ongoing pilot clinical study of Sld5 protein as a potential biomarker, Sld5 is readily and specifically detectable in the cellular fraction of the samples from prostate and bladder cancer patients. Work is ongoing to evaluate Sld5 protein levels in the supernatant portion of those same urine samples as an easy-to-screen diagnostic/prognostic marker for male urogenital cancers. Owing to their stability, GINS proteins hold promise as independent or complementary markers to the MCM proteins for cancer screening in harsh extracellular environments such as urine. Mon, 11 Oct 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/236123 2010-10-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/228700 Title: Structural and functional studies of mitochondrial NADH:ubiquinone oxidoreductase (complex I) Authors: King, Martin Abstract: NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme in the mitochondrial electron transfer chain. It catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of protons across the mitochondrial inner membrane, maintaining the proton motive force used for ATP synthesis. Complex I is the least understood of the respiratory enzymes; although the mechanisms of NADH oxidation and intramolecular electron transfer are gradually becoming appreciated, the mechanisms of quinone binding and reduction and proton translocation remain unknown. Complex I dysfunction has been implicated in a wide range of pathologies including mitochondrial diseases such as Leigh’s disease, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The work described in the first part of this thesis is aimed at elucidating the structure of either a subcomplex of mitochondrial complex I, or of the intact enzyme itself. A comprehensive investigation revealed that hydrophilic subcomplexes of complex I from bovine heart mitochondria are not suitable for use as models of the intact enzyme. Attempts to prepare intact complex I of sufficient quality for structural work were successful; however, results from a large set of crystallization trials were disappointing. The second part of this thesis describes three studies of the function and mechanism of complex I from bovine heart mitochondria. First, the flavin mononucleotide, the site of NADH oxidation, was identified as the site of the ‘inhibitor-insensitive’ NADH:ubiquinone oxidoreduction reaction. The formation of semiquinones initiates redox cycling reactions with oxygen, producing vast amounts of reactive oxygen species; further studies revealed that other oxidants, such as paraquat, also react at the flavin site and initiate redox cycling reactions. Second, kinetic studies showed that the reaction between NADH and positively charged oxidants such as HAR (hexaammineruthenium (III)) proceeds by an unusual ternary reaction mechanism at the flavin site of complex I. Finally, double electron-electron resonance spectroscopy was used to show unambiguously that iron sulphur cluster 4Fe[TY]1 gives rise to electron paramagnetic resonance signal N4; the data provide an alternating potential energy profile for electron transfer along the cluster chain between the flavin and the quinone-binding site. Tue, 16 Nov 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/228700 2010-11-16T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/217870 Title: Structural and biochemical studies of the regulation and catalytic mechanism of ATP synthase Authors: Bowler, Matthew William Abstract: ATP synthase (F1Fo-ATPase) catalyses the production of ATP from ADP and orthophosphate by using the proton motive force established across a membrane by photosynthesis or oxidative phosphorylation. The ATP synthase of eukaryotic mitochondria is located in the inner membrane and is comprised of two domains. The globular F1 domain protrudes into the matrix and it contains the catalytic sites for ATP synthesis. The membrane bound Fo domain contains a proton channel. The two domains are connected by central and peripheral stalks. When F1 is removed from the complex, it can hydrolyse ATP but not synthesise it. It is composed of nine subunits with the stoichiometry α3β3γδε . The α and β subunits are arranged alternately round the symmetric γ subunit, which, with the δ and ε subunits, forms the central stalk. Catalysis occurs by a rotary mechanism where rotation in Fo, induced by the passage of protons, is transmitted to F1 via the central stalk. The rotation of the γ subunit induces conformational changes in the catalytic β subunits that lead to the synthesis of ATP. The three catalytic sites proceed through three major and well defined conformations sequentially, and no two sites are the same at any one time. The peripheral stalk counters the tendency of the α3β3 subcomplex to rotate with the γ subunit. The ATP synthase of mitochondria is regulated by an inhibitor protein, IF1, that prevents hydrolysis of ATP when the proton motive force collapses. Saccharomyces cerevisiae has two inhibitor proteins, YIF1 and STF1. The states of oligomerisation of their active and inactive forms have been investigated. In contrast to bovine IF1, which is active as a dimer, the yeast inhibitors are active as monomers around pH 7.0. Like the bovine protein, they form inactive oligomers at higher pH values. While many of features of the mechanism of catalysis of the ATP synthase are well understood, it is now clear that there are many sub-steps within the cycle. Some of them have been revealed by analogues of phosphoryl transfer. Bovine mitochondrial F1-ATPase inhibited with ADP and magnesium fluoride forms a transition state analogue complex. Its structure was solved to 2.5 Å resolution. The βTP and βDP catalytic sites both contain ADP and MgF3 -. The βE subunit binds ADP, despite being in an essentially open conformation. The structure represents a new sub-step in the catalytic cycle just before the release of the substrates of ATP hydrolysis. Sat, 01 Jan 2005 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/217870 2005-01-01T00:00:00Z