VictorJuiz/thesis huggingface dataset:
Analysis methods for single molecule fluorescence spectroscopy

This thesis describes signal analysis methods for single-molecule fluorescence data. The primary factor motivating method development is the need to distinguish single-molecule FRET fluctuations due to conformational dynamics from fluctuations due to distance-independent FRET changes.
Single-molecule Förster resonance energy transfer (FRET) promises a distinct advantage compared to alternative biochemical methods in its potential to relate biomolecular structure to function. Standard measurements assume that the mean transfer efficiency between two fluorescent probes, a donor and an acceptor, corresponds to the mean donor-acceptor distance, thus providing structural information. Accordingly, measurement analysis assumes that mean transfer efficiency fluctuations entail mean donor-acceptor distance fluctuations. Detecting such fluctuations is important in resolving molecular dynamics, as molecular function often necessitates structural changes. A problem arises, however, in that factors other than donor-acceptor distance changes may induce mean transfer efficiency fluctuations. We refer to these factors as distance-independent FRET changes.
We present analysis methods to detect distance-independent photophysical dynamics and to determine their correlation with distance-dependent FRET dynamics. First, we review a theory of photon statistics and show how we can use the theory to detect FRET fluctuations. Second, we extend the theory to alternating laser excitation (ALEx) measurements and demonstrate how fluorophore stoichiometry, a measure of fluorophore brightness, reports on distance-independent photophysical dynamics. Next, we provide a measure to determine the extent to which stoichiometry fluctuations account for FRET dynamics. Finally, we use a framework similar to the preceding along with recent advances in the theory of total internal reflection fluorescence (TIRF) microscopy FRET measurements to detect TIRF FRET fluctuations which occur on a timescale faster than the measurement temporal resolution. We validate our methods with simulations and demonstrate their utility in delineating RNA polymerase open complex conformational dynamics.

Keywords: Time-Series Analysis
MVA
confocal
BVA
FRET
Photophysics
TIRF
SBVA
EBVA
Advisor: Kapanidis, A
Transition metal catalyzed alkylation and synthesis of biotin derivatives

Transition Metal Catalyzed Alkylation
We have reported methodology for the use of methanol as an alkylation reagent using catalytic rhodium or iridium species for the formation of branched products from methyl ketones. The synthetic utility of the dialkylated products was enhanced by performing a regioselective Baeyer-Villiger oxidation which allowed access to ester products. A range of different phosphine ligands were screened, and sterically hindered and electron rich phosphine ligands were found to favour the formation of enone and methoxy adducts under an O2 atmosphere. This interrupted hydrogen borrowing reaction enabled the in situ addition of a nucleophile to give more complex products. A range of tetrasubsitituted pyridines were then synthesized from 1, 5-dicarbonyl compounds formed in the methylenation/conjugate addition sequence. Finally, deuteration experiments suggest that the reaction proceeds via a monohydride mechanism, and the possibilities for the beneficial effect of O2 were discussed.
Synthesis of biotin derivatives
The streptavidin-biotin system was chosen for the studies of protein/ligand interactions at molecular level. A series of modified biotin ligands were designed and synthesized to introduce repulsive interations with streptavidin. The protein/ligand complexes were analyzed at high resolution by X-ray crystallography.

Keywords: catalysis synthesis alkylation biotin
Advisor: Donohoe, T
Characterising KMOS and scaling relations in the rich cluster MS0451.6-0305

This Thesis contains two parts. Part I details my work on KMOS, a new instrument for the VLT and Part II details my research on scaling relations for the massive galaxy cluster MS0451.6-0305.
Part I: The K-Band Multi-Object Spectrograph (KMOS) is a new near infra-red (NIR) instru- ment to be installed at the Very Large Telescope (VLT). KMOS is capable of spatially resolved kinematics via 24 deployable integral field units (IFUs), each with a 2.8′′ field of view. I describe my contribution to the construction and optical characterisation of the spectrograph modules and instrument as a whole.
Part II: GMOS-N spectroscopy has been used to obtain velocity dispersions (σ) and archival HST photometry has been used to determine the effective radii (Re) & average surface brightnesses within Re (⟨I⟩e) of 26 confirmed cluster members of MS0451.6-0305 at z = 0.55. The Kormendy Relation, Faber-Jackson Relation and Fundamental Plane have been produced for the cluster and have been compared to the results for a local reference sample in Coma. GMOS-N g′ and r′ band photometry has also been used to produce a Colour-Magnitude Relation for MS0451.6-0305.
It is found that the KR and FJR disagree at the 2.1σ level with respect to luminosity evolution since z = 0.55. When correcting for size-evolution (SE), the magnitude offsets agree and are consistent with passive evolution of the galaxy stellar populations from a single burst of star formation 9.8+4.5-1.7 Gyrs ago or z = 1.7+∞-0.6. The median offset in the FP relation for the SE corrected data is also consistent with this formation epoch. Evidence is found for the evolution of the FP tilt in MS0451.6-0305, where it is shown to be steeper than that of the Coma FP, when expressed as a relation between galaxy mass and M/L.

Keywords: KMOS
infrared
cluster
MS0451
spectrograph
IFU
Advisor: Thatte, N
Tecza, M
N-heterocyclic carbene stabilisation of low valent metal centres for the activation of E-H bonds

This thesis examines the effects of coordinating highly sterically demanding and strongly electron donating saturated N-heterocyclic carbenes (NHCs) at late transition metal centres.
Chapter III details the synthesis of a range of iridium complexes of the type (NHC)2IrHxCly [x = 1, 2; y = 0, 1], bearing the saturated NHCs 5-Mes, 6-Mes and 7-Mes. Unusually facile activation chemistry is observed in the reaction of [Ir(COE)2Cl]2 with 6-Mes and 7-Mes to form the doubly cyclometallated species (6-Mes')2IrH and (7-Mes')2IrH, which were fully characterised. The responses of these complexes to the addition of dihydrogen and HCl were studied, leading to the controlled synthesis of range of precursors to 14-electron iridium cations.
In Chapter IV the formation of low valent iridium cations with weakly coordinating anions is targeted. Isolation of the cationic complexes [(NHC)(NHC')IrH][BArf4] and [(NHC)2IrH2][BArf4] (NHC = 6-Mes, 7-Mes) showcases the stabilising power offered by these expanded ring systems. This allowed the study the interaction of these low valent species with a range of amine-borane substrates which are known to be readily dehydrogenated. Thermodynamic data on the C-H bond activation processes occurring at these iridium centres were able to be obtained due to facile, reversible oxidative addition of C-H bonds across the 14-electron iridium.
Chapter V focuses on the effects of increasing the steric bulk of these NHCs to limit the coordination of multiple ligands at the metal centre. Use of 2,6-diisopropyl-phenyl (Dipp) groups on the expanded ring NHCs, instead of mesityl groups, leads to an unprecedented mode of reactivity with [Ir(COE)2Cl]2. Activation and cleavage of C-N bonds in the carbene ring is observed, resulting in an open chain ligand chelating to the metal centre. Activation of the backbone in this manner has allowed the synthesis of saturated NHCs bearing a weakly coordinating anion on the ring. Here the first example of an anionic, saturated NHC is reported.
In Chapter VI these highly sterically demanding NHCs are exploited to stabilise active species in low valent gold chemistry. The extreme steric bulk of the 6-Dipp ligand disfavours reduction of Au(I) to Au(0), however the resulting cation is observed to interact strongly with the weakly coordinating anion, [BArf4]-. Thus, attempts were made to optimise the anion and conditions to isolate a catalytically relevant intermediate. The strong donating power of these expanded ring NHCs is also exploited to activate gold hydride complexes of the type (NHC)AuH (NHC = 6-Dipp, 7-Dipp). Analogues of [H3]+ containing gold atoms ([{LAu}2H]+ and [LAuH2]+) supported by expanded ring NHCs were also targeted.

Keywords: metal hydride
gold
iridium
bond activation
N-heterocyclic carbene
dehydrogenation
Advisor: Aldridge, S
Investigating the roles of zinc finger homeobox 3 in circadian rhythms

This thesis describes a program of investigation carried out by the author at MRC Harwell that shows, for the first time, that the gene Zfhx3 plays a role in modulating the circadian clock. The work has used the short circuit (Sci) mouse model to assess the molecular and genetic role of Zfhx3 in circadian behaviour and visual processing.
The Sci mutant was identified in an ENU mutagenesis dominant screen at MRC Harwell. The causative mutation was found in a highly conserved region of zinc finger homeobox 3 (Zfhx3), previously not known to play a role in the function of the circadian clock.
This study has shown that Zfhx3 RNA is specifically expressed in the adult SCN. Additionally, co-immunoprecipitation has shown that ZFHX3 can interact with core clock proteins, CRY1, CRY2 and PER2 and the Sci mutation causes differential effects on the RNA expression of Cry1, Cry2 and Per1. Genetic interaction experiments identified an interaction between Sci and Cry2 under constant darkness and a potential interaction between Sci and Cry1 and Cry2 under constant light. Together, these data indicate that Zfhx3 has a role in modulating the core circadian oscillator, in particular through cryptochromes.
A role for Zfhx3 has also been identified in visual processing and retinal function. Zfhx3 RNA was shown to be highly expressed in the ganglion, inner and outer nuclear layers of the retina. Sci mutants have increased retinal sensitivity by 20 fold, which may be attributed to a decrease in GABA neurotransmission in the retina.
From this study, novel functions for Zfhx3 within the circadian and visual systems have been identified. This exemplifies that molecular components of the circadian oscillator may remain to be identified and forward genetic approaches are facilitating advancements in this field.

Keywords: Mutagenesis
Zfhx3
ENU
Behaviour
Circadian
Genetics
Mouse
Molecular
Advisor: Nolan, P
Foster, R
Transcranial direct current stimulation (tDCS) as an adjunct intervention in stroke rehabilitation

Stroke is the leading cause of physical disability worldwide and patients are often left with substantial impairments in motor function. Intensive physiotherapy is the most widely used rehabilitative intervention following a stroke, but the potential for motor recovery with physiotherapy alone is limited by the activity of the residual cortex. Consequently there is increasing interest in combining traditional rehabilitation techniques with adjunct interventions to improve outcomes. Transcranial direct current stimulation (tDCS) is a promising tool for rehabilitation. Anodal stimulation of primary motor cortex increases cortical excitability and in stroke patients this translates into transient behavioural improvements after a single session of stimulation. This thesis aimed to investigate whether repeated sessions of tDCS delivered in conjunction with an intensive motor training program would result in long-lasting improvement of motor abilities in chronic stroke patients.
Twenty-four patients received motor training for two weeks for the paretic upper limb; patients were randomised to receive anodal or sham tDCS and were then examined over the subsequent three months to investigate behavioural improvements. All patients experienced a significant improvement in motor function following the motor training intervention. Patients who had received anodal tDCS experienced additional behavioural improvements that were more enduring over time. Baseline characteristics such as the presence of motor evoked potentials in the paretic hand and lesion volume were found to be significantly associated with initial motor impairment, and the degree of initial impairment and allocation to the anodal tDCS condition was found to predict therapy-mediated improvements in motor function.
The results presented in this thesis provide important information regarding the improvement in motor abilities associated with repeated sessions of tDCS, and in particular they suggest that tDCS may be utilised as a clinically useful tool for motor rehabilitation in chronic stroke. Furthermore, they suggest that baseline characteristics could be used to target interventions to patients most likely to benefit.

Keywords: rehabilitation
stroke
tDCS
brain stimulation
Advisor: Johansen-Berg, H
Anarchist organization

This thesis approaches anarchist organisation in practical and revolutionary terms. Its theme is Kropotkin's conception of the relationship between the end of anarchy and the process of anarchist transformation. The thesis examines this relationship as a continuous theoretical development, questioning the existing interpretations of his thought which identify a revision of his ideas in his formulation of the theory of mutual aid. It finds that these interpretations of his work are mistaken and based on a misunderstanding of his use of Darwinian evolutionary theory. Rejecting the beliefs that Kropotkin's scientific anarchism is based either on a desire to prove the necessity of anarchy or to replace revolution with a process of gradual evolutionary reform, the thesis suggests that questions of strategy have a secondary importance in his work. Kropotkin's anarchism is directed toward securing moral behaviour by restructuring society.
Rejecting the alleged discontinuity of his thought, the thesis acknowledges that there are differences between Kropotkin's early and late writings. In his early work, Kropotkin's understanding of anarchist organisation is based on a commitment to communism and on an expectation of revolution. In the theory of mutual aid Kropotkin subordinates communism to an ideal of community and resolves the problem of change by the force of his scientific ideology. But his conrmitment to anarchy is affirmed. Formulating the concept of mutual aid, the thesis finds that Kropotkin uses science as a theoretical incentive, promising practical and spiritual well-being, for the masses to hasten the realisation of the anarchist society.
In conclusion the thesis reviews the existing interpretations of Kropotkin's commitment to Victorian positivism and suggests that his adherence to the standards of natural scientific research are compromised by the radicalism of his liberatory desires.
Viable project or wishful thinking? The European Union (EU) policy in the fight against terrorism: quest for strong human rights safeguards and enhanced security

This thesis examines whether and how the EU counter-terrorism (CT) policy reconciles the demands of security with adequate protection of human rights. It starts from the assumption that human rights and security are mutually reinforcing and interdependent objectives in the CT fight where the erosion of one objective leads automatically to negative consequences in respect of the other objective. It specifically argues that the reconciliation of these two objectives has to be addressed at two distinct levels: first, in the framework of the EU primary law and, second, in the content of each EU CT provision. Consequently, the thesis examines both levels in order to respond to the research question. In the course of this investigation, the research has demonstrated that the legal framework resulting from the Treaty of Lisbon provides a basis for better addressing the human rights protection and security objectives of the EU CT policy. However, the analysis of three specific instruments – two in force and one at the level of a legislative proposal – provided contrasting results regarding the simultaneous fulfilment of the two imperatives outlined above. Moving beyond questions pertaining to the advancement of preventive criminal law and the possible reinforcement of a surveillance society, the thesis advances the hypothesis that, in the field, we are confronted more and more with what we could term ‘grey laws’ – following Dyzenhaus, Lynch and Reilly – due to their frailties as regards the tests of proportionality and legal certainty. Moreover, the thesis explores the EU’s stance as an actor in the field and the applicability of Wallace’s ‘pendulum model’ for CT decision-making, as well as the position of an individual subject to all the three measures indicated above. Solutions in order to overcome the identified shortcomings as well as further potential areas of research are also explored.

Keywords: human rights
EU
terrorism
Advisor: Zedner, L
Ziegler, K
The characterisation of intestinal dendritic cells and the control of immune responses towards the microbiota

Dendritic cells (DCs) are regulators of the immune response and are thought to be critical in maintaining tolerance towards the intestinal microbiota. Recent data have identified distinct subsets of DCs with specific functional properties. The objective of this thesis was to further define CD103⁺ and CX3CR1⁺ DCs in the intestine and to determine how DCs and regulatory T (Treg) cell responses are influenced by the microbiota.
Using multicolour flow cytometry, we identified two CD103⁺ DC subsets with differential aldehyde dehydgrogenase (ALDH) activity and two populations of CX3CR1⁺ cells. In the mesenteric lymph node CD103⁺ALDH⁺ DCs were highly mature (CD86hi, MHCIIhi), likely migratory (CCR7⁺) and enhanced Treg cell induction compared with ALDH⁻ DCs. CX3CR1int cells accumulated during bacterially-induced colitis suggesting a pro-inflammatory role whereas CX3CR1hi cells were associated with the production of the anti-inflammatory cytokine IL-10 during homeostasis.
We also assessed the generation of CD103⁺ DCs from bone marrow progenitors. Although only small proportions of CD103⁺ DCs were detected in culture with FLT3L or GM-CSF alone, the combination of FLT3L and GM-CSF induced CD103⁺ DCs with a phenotype similar to those found in the small intestine. Using this system we showed that TLR ligands and retinoic acid induce ALDH enzyme activity in vitro.
In order to assess how DCs and Treg cells respond to changes in the microbiota we employed broad-spectrum antibiotic treatment to deplete endogenous bacteria and also analyzed the impact of colonization with the model organism Helicobacter hepaticus. Interestingly, we did not detect alterations in the proportions of different DC subsets following antibiotic treatment or H. hepaticus infection. However, using a novel FoxP3huCD2-IL-10GFP reporter mouse, we found that IL-10 production by Treg cells was ablated following antibiotic treatment and significantly elevated following H. hepaticus infection. Preliminary investigation of the mechanism underlying this effect suggests a role for IL-27. In summary, this thesis provides further detail on the phenotype of intestinal DCs and shows that Treg cell IL-10 production is sensitive to the composition of the microbiota.

Keywords: immunology
intestine
pathology
Advisor: Powrie, F
Population genetics genealogies under selection

In the presence of selection and mutation, the genealogy of a given sample configuration can be described by two classes of ancestral processes, namely the coalescent-in-a-random-background model of Kaplan et al. (1988) and the dual process with typed lines of Etheridge and Griffiths (2009). These two processes are based on the same forwards population genetics model. However, in the former model, selection is reflected in the ancestral frequencies in the population, while in the latter model, there are branching events that generate virtual ancestral lines. We simulate the dual processes with typed lines and derive the limits of the two ancestral processes under strong selection and under selection-mutation balance to address the question of to what extent the genealogy is distorted. The two ancestral processes generate the same limiting genealogy. In a two-allele population under strong selection, the disfavoured individuals in the sample are instantaneously converted to a random number of favoured individuals, and the limiting genealogy is governed by the usual Kingman’s coalescent. Under selection-mutation balance, all disfavoured individuals in the sample are instantaneously converted to the favoured type, and the limiting genealogy is determined by a time-changed Kingman’s coalescent. The proofs of these limiting processes are based on the convergence result of Mohle (1998, Lemma 1). The studies of selection-mutation balance are then extended to an additive selection model, where each individual is composed of L diallelic loci. In the corresponding dual process with typed lines, the evolution of the virtual lines on a faster timescale can be approximated by a deterministic process, while the evolution of the real lines is independent of the virtual lines. The structure in the limiting genealogy collapses to Kingman’s coalescent. We also let L tend to infinity, and obtain a full description of the limiting genealogy in the background selection model.

Keywords: selection-mutation balance
strong selection
strong mutation
additive selection
Advisor: Griffiths, R
Network communities and the foreign exchange market


Many systems studied in the biological, physical, and social sciences are composed of multiple interacting components. Often the number of components and interactions is so large that attaining an understanding of the system necessitates some form of simplication. A common representation that captures the key connection patterns is a network in which the nodes correspond to system components and the edges represent interactions. In this thesis we use network techniques and more traditional clustering methods to coarse-grain systems composed of many interacting components and to identify the most important interactions. This thesis focuses on two main themes: the analysis of financial systems and the study of network communities, an important mesoscopic feature of many networks. In the first part of the thesis, we discuss some of the issues associated with the analysis of financial data and investigate the potential for risk-free profit in the foreign exchange market. We then use principal component analysis (PCA) to identify common features in the correlation structure of different financial markets. In the second part of the thesis, we focus on network communities. We investigate the evolving structure of foreign exchange (FX) market correlations by representing the correlations as time-dependent networks and investigating the evolution of network communities. We employ a node-centric approach that allows us to track the effects of the community evolution on the functional roles of individual nodes and uncovers major trading changes that occurred in the market. Finally, we consider the community structure of networks from a wide variety of different disciplines. We introduce a framework for comparing network communities and use this technique to identify networks with similar mesoscopic structures. Based on this similarity, we create taxonomies of a large set of networks from different fields and individual families of networks from the same field.