VictorJuiz/thesis huggingface dataset:
Structural analysis of the cattle antibody repertoire

Cattle present an extreme immunological model, wherein immunoglobulin sequence diversification is achieved not through recombinational diversity, as in humans and mice, but by great heterogeneity in the length of the complementarity determining region #3 of the heavy chain (CDR3H), and by somatic hypermutation independent of antigenic contact. Bovine CDR3H lengths span 5 - 72 residues, with those containing >48 labelled as ‘ultralong’ and assumed to interact with epitopes typically invisible to paratopes with shorter CDR loops. Cattle also demonstrate preferential combination of V(D)J rearrangements without antigenic stimulus, inferring a structural relationship between bovine immunoglobulins that is not yet understood. This project aims to advance understanding of these relationships from a structural and functional context.
The structures of several chain-exchanged ultralong antibodies were solved by X-ray crystallography, or predicted by AlphaFold, revealing an intimate contact between the CDR3L and the CDR3H. Varying the CDR3L sequence imparts an axial twist about the ultralong stalk domain, with minor accommodating atomic shifts at the distal, antigen-interacting knob domain, together with a subtle change in binding affinity by biolayer interferometry. Electron cryomicroscopy was used to assess whether chain-exchange resulted in any changes to the recognised epitope, but no significant differences were observed at the attained resolutions. Initial efforts with a non-ultralong antibody-antigen system are also described.
These observations imply bovine light chains principally contribute a structural role stabilising ultralong CDR3H, but their contribution in non-ultralong antibodies remains enigmatic. The limited impact of light chain selection on antibody function provides little insight to bovine B cell development. Bioengineers of the ultralong CDR3H may wish to consider the light chain sequence should their targeted epitope lie within deep groves of known geometry, however at present, light chain selection is only a minor consideration in the development of bovine ultralong CDR3H antibodies as diagnostic, therapeutic or research tools.

Keywords: bRSV
crystallography
biolayer interferometry
FMDV
antigen
cryogenic electron microscopy
single particle analysis
X-ray diffraction
foot-and-mouth disease virus
BLI
antibody
XRD
chain-exchange
bovine respiratory syncytial virus
cryo-EM
SPA
ultralong
Advisor: Owens, R
Stuart, D
Hammond, J
Measurement of the Higgs boson mass and production cross section and calibration of the muon momentum with the ATLAS detector at the LHC

The ATLAS and CMS experiments at the Large Hadron Collider, located in CERN in Geneva, Switzerland announced the discovery of the Higgs boson on the 4th of July, 2012. Measurements of the Higgs boson properties provide stringent consistency tests for the Standard Model, and offer critical input parameters for theoretical models both within and beyond the Standard Model. Such measurements therefore require a precise understanding of the underlying detector throughout its operation. This thesis contributes to both aspects: measurements of the Higgs boson mass and cross-section, and studies of ATLAS detector performance through muon momentum calibration.
As the Higgs can decay to muons, improvements to the muon detector performance directly translate to the corresponding systematic uncertainties in the measurement of Higgs properties. This motivates the inclusion of new techniques in muon momentum calibration, including the addition of negative log-likelihood scans and generator reweighting. The calibration procedure is performed for both 2015-2018 and 2022 datasets, and reduces the leading systematic uncertainty in the Higgs mass measurement by 50\%.
The measurements of Higgs boson properties are performed for the Higgs decaying into a pair of Z bosons, and subsequently decaying into two pairs of electrons or muons. This is a fully reconstructible final state with a high signal-to-background ratio, thus an ideal final state for precision measurements. The Higgs boson mass measurement utilises proton-proton collision data collected between 2015 - 2018 at the centre-of-mass energy
√
s
= 13 TeV. A number of techniques, including per-event mass uncertainty, Z-mass constraint, and kernel estimation for background modelling, are included to reduce the statistical uncertainty of the measurement. The measured Higgs mass is 124.99±0.18 (stat.)±0.04 (sys.) GeV using the 2015-2018 data. The Higgs boson fiducial cross-section measurement utilises proton-proton collision data collected in 2022 at the centre-of-mass energy
√
s
= 13.6 TeV. The fiducial cross-section for its decay into a pair of Z bosons is measured to be σfid=2.80±0.74 fb, and σtot=46±12 pb once extrapolated to the full phase-space.

Keywords: Higgs physics
collider physics
particle physics
muon physics
detector performance
Advisor: Shipsey, I
Bortoletto, D
Fluorescence imaging and modelling of virus structure and infection

Diseases caused by viruses represent a serious global health concern. In this thesis, I present novel methods for labelling, imaging and analysing virus particles. I also describe mathematical and computational models that can be used to describe biological structures, such as viruses.
In studies of viral structure, pleomorphism, and articularly filamentous morphologies, are frequently overlooked.To characterise and better understand viral filaments, I developed and optimised an imaging protocol for individual virus particles that captured the heterogeneity of viral shape. I also produced analysis pipelines that took this image data to study the structure of viruses at the individual virion level in order to find an overall picture of the populations of virions that exist. These pipelines investigated virus size, patterning of surface proteins and the location of viral genome. I also demonstrated a novel virus labelling strategy that specifically labels virus that will aid in the future study of viral assembly and infection.
I also used mathematical modelling to further understand the role of viral morphologies. I modelled virus particles as a pressure vessel to study engineering stress in relation to virus, bacteria, and cellular morphology.
Together these advancements will further our ability to study viruses and our knowledge of virus structure.

Keywords: filamentous viruses
SIR model
fluorescence microsopy
Light-activated ATP synthesis in droplet networks

The fabrication of synthetic tissues that mimic the complex collective functionality of living tissues is the central objective of synthetic biology. A key step in this endeavour is the construction of an energy production system. Energised synthetic tissues could produce therapeutic oligonucleotides and proteins on demand, or transport substrates to modulate the behaviour of interfaced living tissues. This thesis describes the assembly of synthetic tissues with an encapsulated light- activated adenosine triphosphate (ATP) production system. The synthetic tissues comprised droplet networks, assemblies of aqueous droplets in a lipid-containing oil separated and stabilised by droplet interface bilayers (DIBs). Two membrane proteins were reconstituted into proteoliposomes to create a synthetic organelle able to generate ATP on demand: a modified light-activated proton pump, proteorhodopsin-mCherry and E. coli F1FO ATP synthase. An ATP synthesis rate of 53.2 nmol ATP.min-1.mgF1FO-1 was achieved in bulk studies. Transfer of the energy generating proteoliposomes to pL-sized droplets created a system capable of light-activated transcription of a ribonucleic acid (RNA) aptamer, which was detected with a novel hydrophilic analogue of the cognate fluorophore, DFHBI. To facilitate small molecule communication between compartments of the synthetic tissue, DIBs were permeabilised with a pore forming protein, alpha-hemolysin (aHL). ATP synthesised under control by light could then be transferred to and used in a neighbouring compartment.

Advisor: Bayley, J
Ishmukhametov, R
Single cell analysis of breast cancer response to hypoxia

Triple-negative breast cancer (TNBC) is the subtype of breast cancer that has the worst clinical outcome due to the absence of specific targeting agents. TNBC is highly heterogeneous and within the tumour microenvironment, hypoxia emerges as a pivotal factor contributing to its aggressive biology. Solid tumours experience a fluctuating oxygen supply, leading to the formation of zones with prolonged oxygen deprivation.
There have been few studies describing the response to chronic hypoxia [7-14 days] and this project investigates how chronic hypoxia contributes to tumour heterogeneity in a temporal manner. Single-cell RNA sequencing (SCS) was performed on MDA-MB-231 cells subjected to a 14-day hypoxia treatment at 1% oxygen. Samples from both normoxia and hypoxia conditions on Days 1, 2, 7, and 14 were collected. A total of 14,000 cells were sequenced. There were 6 hypoxic cell clusters which evolved over the time course.
Chronic hypoxia induces various transient subpopulations. Cells in hypoxia Day 7 grew more slowly, before recovering growth at Day 14, marking a crucial adaptation point at Day 7 in chronic hypoxia. A higher expression of cancer stem cell markers [CD44, CXCR4 and KLF4] in the chronic hypoxia subpopulations was observed. Notably, a distinct CD24+/CD49b-high population, comprising the largest proportion of the hypoxic Day 7 samples which disappears by Day 14 was identified. This population is characterised by the upregulation of cystatin family genes (CST1, 2, 3 and 4) which are inhibitors of cathepsin proteases. These findings may have important implications for strategies in the treatment of breast cancers by providing insights on the tumour heterogeneity induced by chronic hypoxia. Several small molecule inhibitors of cathepsin proteases have been tested in clinical trials for osteoporosis, immune disorders, and cancers with mixed success. The heterogeneity found in this study may explain this, with the existence of a transient population.
In summary, my work provided insights on how hypoxia induces protective translational reprogramming and drives tumour heterogeneity. Understanding these processes may facilitate the development of targeted treatments to combat therapy resistance in TNBC.

Keywords: ScRNA-seq
hypoxia
breast cancer
Advisor: Buczacki, S
Arora, A
Buffa, F
‘Search and social digital platforms, democracy and citizen exposure to speech pluralism in Australia.’

The business success of search and social digital platforms relies on an atomised approach to content dissemination. Algorithms rank and prioritise content to engage users and sell targeted advertising opportunities. Scholarship has produced divided results on the question of whether platform algorithms are harming democracy by limiting citizens’ exposure to a plurality of speech in several respects (including, though not limited to, by way of creating echo-chambers). Arguably, this division is a product of two methodological issues. Firstly, scholars struggle to obtain quantitative data measuring whether platforms limit exposure to speech; and secondly differences in their theoretical understandings of the purpose and structures of democracy have led to the absence of a uniform standard across scholarship against which harm to democracy is measured. In light of this, the Australian Government has suggested regulation cannot be justified. This thesis further considers this question. Its object is to yield a practical result for Australian lawmakers and as such, it looks to navigate the existing methodological issues. It does this firstly by employing doctrinal analysis – considering whether ‘exposure to speech pluralism’ is a structural requirement of Australia’s democracy as established by the Constitution. It finds that although no such requirement exists, the Legislature has, to some degree, sought to establish it. Specifically, the Legislature has made two value judgements about the extent to which this should be established. This thesis then assesses platform markets and products against these value judgements – finding that their concentrated market structure, and feed of content, personalised for (not by) the citizen, fall short. As such, it suggests Australian democratic standards require platform algorithms to facilitate a greater availability of plural political communication; and consequently, that regulation is warranted. This paper concludes by briefly recommending areas of law which may successfully regulate this aspect of platforms, for further research.

Keywords: Meta
constitutional law
Australia
speech
implied freedom of political communication
digital platforms
Australian constitutional law
competition law
free speech
Advisor: Khaitan, T
Catalytic enantioselective 6π photocyclization of acrylanilides and enantiomerisation processes of indolyl-napthyl atropisomers

This thesis is composed of two parts. The first part describes the development of a dual catalytic enantioselective 6π photocyclization (Scheme A1).
We go on to describe the work carried out to validate the predicted reaction pathway and determine whether the photocatalytic portion of the cycle operates by photoredox catalysis or energy transfer catalysis (Figure A1).
The second part describes the observation of non-first order racemisation of an indolyl-naphthyl atropisomer and the work carried out to reproducibly observe the effect and gather data across a number of concentrations (Figure A2).
We then describe the combination of experimental probes and modelling carried out in the hopes of determining the underlying cause of the observed behaviour (Figure A3).

Advisor: Smith, M
Quantitative methods for MRI-microscopy comparisons

Magnetic resonance imaging (MRI) is a powerful tool for the in-vivo diagnosis and assessment of neurodegenerative disorders. While numerous MRI techniques have yielded parameters sensitive to microstructural changes in the brain, MRI parameters are infamously non-specific. Different changes in brain tissue structure may result in the same change in MRI signal, making it challenging to pinpoint the exact source of these signal changes.
Biophysical modelling aims to achieve better biological specificity by relating dMRI signals to biologically interpretable tissue parameters. Yet, the fitting of these complex models with many parameters to unremarkable dMRI signals is challenging, often resulting in a degeneracy where multiple combinations of parameters explain the dMRI signal equally well.
Conversely, microscopy offers high biological specificity by targetting specific aspects of microstructure. By acquiring and relating MRI and microscopy metrics for the same tissue section, one can therefore leverage microscopy’s specificity to elucidate the microstructural basis of MRI signal change. However, microscopy is incredibly time-intensive, restricting examination to a few tissue sections at a time. A significant gap remains in the lack of a standardised pipeline for MRI-microscopy comparisons, with existing methods necessitating substantial manual intervention.
This thesis delves into methods that enhance MRI-microscopy comparisons. Specifically, we introduce an automated pipeline that rapidly and reliably extracts multiple quantitative microscopy parameters from sections that are histologically-stained. Utilising this pipeline alongside high-quality MRI-microscopy co-registrations, we performed whole-slide voxelwise comparisons between multimodal MRI- and microscopy-derived metrics. Finally, we present an alternative analysis method designed to relate degenerate biophysical model parameters to a continuous metric (e.g. from microscopy).
Overall, the techniques outlined in this thesis are intended to facilitate a more precise interpretation of microstructural change from MRI parameters and encourage a more systematic approach to processing microscopy data when relating them to MRI data.

Keywords: brain imaging
histology
diffusion modelling
microscopy
quantitative MRI
diffusion MRI
MRI-microscopy
microstructure imaging
Advisor: Miller, K
Howard, A
Jbabdi, S
Adaptation to chronic hypoxia in triple-negative breast cancer

Low levels of oxygen (i.e. hypoxia) are often observed in solid tumours. Hypoxia is able to drive several of the cancer hallmarks and promotes resistance to both radio and chemotherapy, resulting in its association with poor prognosis. Triple-negative breast cancer has the worst prognosis of all breast cancer subtypes with a 5 year survival rate of only 76.7%, and only 10.8% if the cancer has already metastasised. In line with this, triple-negative tumours often have higher levels of hypoxia than their hormone receptor expressing counterparts.
The length and severity of hypoxia is heterogeneous both within and between tumours, meaning cells within the same tumour may experience both acute and chronic hypoxia. Acute hypoxia (⩽ 24 hours) is relatively well studied, whereas research into chronic hypoxia (⩾ 7 days) is lacking. This thesis therefore aimed to characterise the transcriptional response to chronic hypoxia using RNA sequencing. It also aimed to identify proteins or microRNAs which are necessary for adaptation to chronic hypoxia using genome-wide CRISPR screening. This method enables specific vulnerabilities in hypoxic cells to be identified, meaning drugs can be designed to target these “Achilles heels”.
The major pathways altered under chronic hypoxia were related to epithelial-mesenchymal transition (EMT) and cell adhesion, with triple-negative breast cancer cells undergoing an atypical form of EMT. However, none of the genes which were upregulated in response to chronic hypoxia were found to be essential for their survival. Instead, hypoxic cells had a greater dependence on general control nonderepressible 2 (GCN2), an instigator of the integrated stress response usually activated under amino acid starvation. GCN2 was found to be active under chronic hypoxia, and chemical inhibition of GCN2 reduced cell growth in vitro. Previous work has shown that GCN2 knockout mice are healthy unless fed a diet restricted in essential amino acids, meaning targeting GCN2 is unlikely to result in severe toxicities. Together, this means GCN2 constitutes a promising therapeutic target for hypoxic tumours. No microRNAs essential for survival under hypoxia were identified in this study.

Keywords: miRNA
hypoxia
CRISPR
chronic hypoxia
EMT
oncology
tumour microenvironement
breast cancer
Advisor: Buffa, F
Harris, A
Sheldon, H
Developing light-activated nucleic acids for gene knockdown in cell-free and living systems

Nucleic acids encode the information of all biological functions, which are regulated through the expression of genes. Today, various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) structures can be reliably synthesized and manipulated to recreate naturallyoccurring phenomena or introduce new functions in cell-free and living systems. Controlling the expression of a gene to protein has become a fundamental tool for therapeutic and synthetic biology research. One way of controlling gene expression is by using specific wavelengths of light. Light is an ideal stimulus as it can be externally applied to a system with high spatiotemporal precision, and it is bioorthogonal to most biological processes.
The synthesis of a protein can be inhibited by degrading or deactivating the corresponding gene, a process called gene knockdown. The two nucleic acids mainly used for gene knockdown are antisense oligonucleotides (ASOs), short single stranded DNA sequences, and small interfering RNAs (siRNAs), short double stranded RNA sequences. ASOs and siRNAs can be synthesized and modified to be activated in response to light irradiation.
This work presents the development of an orthogonal two-wavelength control of cellfree gene expression. Light-activated (LA)-ASOs and LA-siRNAs were generated from commercially available amino modified sequences. The amine groups were functionalized with UV or blue light-activatable photocages, which inactivated the nucleic acids by steric hinderance. Upon irradiation with a specific wavelength of light, the knockdown activity was restored, as the photocages were cleaved off, uncaging to the initial amino-modified nucleic acid structure. Cell-free protein synthesis (CFPS) was controlled with UV lightactivated ASOs, by triggering RNase H-mediated mRNA degradation on demand. Additionally, by combining UV LA-ASOs to a previously developed blue LA-DNA template, transcription was activated with UV and translation halted orthogonally with blue light. Finally, ASOs targeting two different genes were modified with orthogonal photocages for precise and selective two-wavelength gene knockdown in CFPS.
Progress towards light-activated gene knockdown in mammalian cells using siRNA will also be presented. Two different strategies for generating LA-siRNA will be discussed. The siRNA photocages were decorated with a cell-penetrating peptide and a fluorophore, for self-delivery to cells and fluorescent tracing of the nucleic acid. Images demonstrating cellular uptake without the use of transfection reagents will be finally shown.

Advisor: Booth, M
Bayley, J
Design and simulation of reversible molecular mechanical logic gates and circuits

In this thesis, I outline a framework for constructing molecular mechanical circuits from logic gates and components that I have designed and optimised, and describe the results of simulations conducted to determine the thermodynamic properties of these circuits with a coarse-grained rigid body simulator. The molecular mechanical logic gates I have designed can be used to construct logically reversible circuits, which have the potential to be far more efficient than their irreversible counterparts due to the fact that they can easily be operated in a thermodynamically reversible manner.
Using the rigid body simulator, I first demonstrate Landauer's principle, a fundamental principle of information theory which relates the change in information entropy of a given process to the work required to complete it, thus showing that my rigid body simulator is thermodynamically self-consistent. I then simulate a novel molecular mechanical NAND gate design and use it to construct complex logically and thermodynamically reversible combinatorial circuits, including a half-adder. Subsequently, I investigate the use of pipelining to increase the throughput of a circuit at the cost of potential logical irreversibility, and show that it is possible to perform pipelining efficiently under certain conditions. Finally, I use a genetic algorithm to optimise various circuit components, demonstrating a novel technique to reduce the number of costly simulations that must be run in order to calculate the fitness function.

Advisor: Doye, J