VictorJuiz/thesis huggingface dataset:
The role of two-pore channels 2 (TPC2) in melanoma tumourigenesis and metastasis
Keywords: TPC2
melanoma
CRISPR-Cas9
Two-Pore Channel 2 (TPC2)

Melanoma is an exceedingly aggressive and resistant to treatment form of cancer. Therefore, the identification of novel therapeutic approaches continues to be a crucial task. Understanding the pathophysiological role of potential drug targets for this disease enables the discovery of new therapeutic strategies. The two-pore channel 2, TPC2, is located on late endosomes, lysosomes, and melanosomes. Here, we evaluated the effect of TPC2 knockout (KO) on human metastatic melanoma (CHL-1) and murine primary melanoma (B16) cells. TPC2- depleted melanoma cell lines were created using CRISPR-Cas9 gene editing. CHL-1, an amelanotic metastatic melanoma cell line, exhibited in vitro induced migratory traits after TPC2 depletion. In contrast, B16 TPC2 KO cells displayed a diminished ability to migrate in vitro. TPC2 KO activated genes regulated by YAP/TAZ, which are important regulators of tumorigenesis and metastasis, in CHL-1. TPC2 KO and RNA interference knockdown decreased the expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-II, a component of the HIPPO pathway that adversely affects YAP/TAZ activity. These findings are explained by a cellular mechanism mediated by ORAI1/ Ca2+/PKC-II.
Furthermore, CHL-1 and B16 TPC2 KO proteomic signatures were examined to WT cells. The differential expression of proteins between WT and KO cells enables us to evaluate TPC2's role in various crucial pathways and processes. Accordingly, the bioenergetic phenotype of TPC2-deficient cells was assessed. TPC2 depletion boosted ATP synthesis in CHL-1 and B16 TPC2 KO cells relative to WT cells, but increased lactate generation in B16 cells. TPC2 deficiency enhanced anaerobic respiration in B16 cells but reduced it in CHL-1 cells. OCR/ECAR analysis confirmed TPC2-KO cells' metabolic shift. CHL-1 TPC2 KO cells are more aerobic due to a reduced OCR/ECAR ratio. OCR/ECAR was elevated in B16 KO cells in complete media. B16 KO cells increased oxygen utilisation and lactate production without glucose. In addition, the cytoskeleton and synthesis and release of melanin were altered in TPC2 KO cells.
In vivo evaluation of B16 TPC2 KO melanoma cell lines following subcutaneous injection revealed a significantly reduced tumour volume compared to WT cells. However, histopathological examination of mice injected with KO cells revealed that the hepatic and splenic structures were drastically changed.
Clinical association is vital to determining correlations between molecular discoveries and cancer patient survival. TPC2 high-expression patients had a shorter overall survival time than low-expression patients. Furthermore, high TPC2 expression is associated with lower survival in BRAF mutant melanoma. In summary, the work presented in this thesis indicates that TPC2 plays vital functions in melanoma's progression, invasion, and metastasis.
Women's comprehensive health care in contemporary Tunisia
Keywords: health care
women's health services
Tunisia
health services

This dissertation explores the strengths and limitations of national health policy in Tunisia. The achievements of the Tunisian women’s health program, as reflected in both statistical and elite-centered historical analyses, include anv expansive health infrastructure, near universal primary health care coverage, and pioneering reproductive health initiatives. Yet the dominant historical narrative divorces women’s health from the broader social, economic, and political context and largely excludes the experiences and perspectives of women from its analysis. Furthermore, research dedicated to women’s health has historically concentrated almost exclusively on maternal and reproductive health, thereby precluding a sophisticated appreciation of other aspects of women’s health and the full ramifications of health policy. This dissertation moves beyond this limited focus and instead incorporates a more comprehensive vision of women’s health into the assessment of national health services. By approaching women’s health as social history, this dissertation explores the contemporary lives, experiences, and needs of women in Tunisia and chronicles the involvement of women, individually and collectively, in the development, evolution, and implementation of the national women’s health program. Highlighting three specific areas of women’s health in Tunisia -traditional health services, domestic violence, and young women’s health and sexuality- this research reveals both lacunae in the national program and arenas for future improvement. To examine the history and development of the women’s health program in Tunisia, identify limitations and strengths of the program, and consider a wide array of women’s health issues, this dissertation combines qualitative and quantitative research methods. Based on fourteen months of fieldwork in Tunisia, this dissertation draws from the fields of medical anthropology, public health, and feminist studies. In using a variety of extractive and participatory techniques, this interdisciplinary approach yields a more complete and nuanced understanding of the history, evolution, and consequences of the Tunisian national women’s health program and offers a framework by which to examine comprehensive women’s health in other parts of the Middle East and North Africa region.
Pulmonary cell signals mediating resident memory B cell responses to influenza rechallenge
Keywords: Chemokine signalling
tissue-resident immunity
resident memory B cells
influenza virus
pulmonary immunity

The capacity of the mammalian adaptive immune response to generate immunological memory has for years been exploited by systemic vaccination strategies to protect large populations from infectious diseases. Recently, evidence has emerged for the establishment of memory B cells in tissues prone to antigen exposure. These tissue-resident B cells (Brm) may be adapted toward rapid reactivation at local sites – reducing the need for systemic intervention when fighting recurring pathogens.
Here, I utilize cytometry, microscopy, and transcriptomic sequencing approaches to explore the dynamics of Brm residence in the lung and their response to secondary influenza virus infections. I propose that the induction of C-X-C motif chemokine ligands 9 and 10 (CXCL9, CXCL10) early after rechallenge are important for Brm migration and accumulation at sites of infection. Innate immune cells, including alveolar macrophages, bridge the inflammatory and adaptive immune systems in part by inducing the expression of these two chemokines.
The recruitment of Brm at sites of viral infection is associated with the rapid appearance of antibody-producing cells in the same niche. This enables the prompt delivery of antibodies in a highly localized manner at sites of viral replication. Defining the protection granted by Brm towards recurring antigens has important implications for developing immunization strategies against respiratory viruses such as influenza.
Monetary policy, 1833-9 : with special reference to the Anglo-American crisis of 1837
Keywords: foreign economic relations
banks
nineteenth century
Bank of England
Great Britain
monetary policy
19th century
Anglo-American crisis
United States
banking

This is a study of the monetary policy followed by the Bank of England between the years 1833 and 1839 - the instruments that it used to try to achieve the objective of specie convertibility chosen by Parliament.
The approach adopted is non-quantitative, because of data limitations, and institutional. This has the advantage of allowing study of a sub-sector of the financial system usually bypassed in monetary histories - the seven London and Liverpool merchant banks which were at the centre of Anglo-American trade and finance. A second theme is to emphasise the importance of divisions over policy among the Directors of the Bank of England. These led to frequent changes of direction which did much to diminish the Bank's reputation among contemporaries and historians.
The conclusion is broadly favourable to the Bank of England, or at least that part of it led by Horsley Palmer which was responsible for the evolution of 1830's policy. In particular, the acknowledgement by the Bank of its duty to act as a lender of last resort attracts praise, as having prevented the complete collapse of the British financial system during the crises of 1837 and 1839.
With the benefit of hindsight, some criticisms can be made: the most important is that the Bank was too rigid in its response to external developments and it allowed itself too little discretion in the formation of policy. But it was acting within the context of the monetary philosophy of the time, which was strongly antipathetice to giving too much power to any one institution. And, if a more discretionary policy had been adopted, it must be doubted whether this would have been much more successful, for the lesson of the monetary history of the 1830's is that monetary control is very difficult to achieve in a sophisticated financial system.
SeroTracker: synthesising seroprevalence data to map the true extent of COVID-19 infection and immunity
Keywords: antibodies
seroprevalence
biostatistics
dashboard
COVID-19
global health
statistical modelling

Effective decision-making during pandemics requires understanding the true extent of infection and immunity. Seroprevalence studies measure the population prevalence of antibodies, providing key insight into these quantities. However, the results from these studies are dispersed, hindering their use. We developed SeroTracker, a dashboard and data platform for SARS-CoV-2 seroprevalence studies. Through its ongoing systematic review, SeroTracker identified over 3,800 seroprevalence studies and made their data available to global health, government, and academic stakeholders. Meta-analysing these studies, we show that global SARS-CoV-2 seroprevalence was 59.2% in September 2021 (95% confidence interval 56.1 − 62.2%), and rose steeply during 2021 due to infection in some regions and vaccination in others. We also identify limitations of seroprevalence studies: their timeliness, where the median study is published 154 days after being conducted; measurement bias, where assay manufacturers systematically overestimate sensitivity by 5.4% and specificity by 2.8%; and coverage, where seroprevalence studies are sparse in time and space. To address these challenges, we develop a Bayesian modelling framework that synthesises information from case and seroprevalence data to provide up- to-date estimates of true infections. We validate this model on simulated data and apply it to Canadian data, showing that only approximately 20% of infections were detected as cases at the start of the pandemic, and revealing substantial geographical heterogeneity in infection dynamics. Returning to SeroTracker’s evidence synthesis process, we develop an automated tool for risk of bias assessment with excellent reliability compared to manual review (intraclass correlation 0.77), and implement a natural language processing-based process to screen abstracts for our systematic review that reduces screening time by approximately 70%. Finally, we reflect on the increased risk posed by pandemics and discuss future work applying engineering to public health to reduce these risks, including the development of a central data repository for respiratory virus surveillance data.
New methods to study macrophage biology in vitro and in vivo
Keywords: cre recombinase
microfluidics
macrophage
mouse model
chemotaxis

Macrophages are heterogeneous and plastic cells, which populate almost every organ in the body and migrate towards sites of inflammation. However, tools designed for studying macrophage biology, particularly genetic models and chemotaxis assays, are standardised to investigate a broad range of leukocyte populations. They fail to capture the unique properties of macrophages and distinguish them from other ontologically related populations. In this thesis, I set out to create novel in vitro and in vivo tools that are adjusted for studying macrophage biology. To address the in vivo arm of this project, I phenotyped a novel tamoxifen-inducible hCD68-CreERT2 mouse line. Having established an optimal protocol for tamoxifen dosing, I detected robust expression of Cre recombinase activity in tissue-resident macrophage populations in peritoneum, spleen and liver. Importantly, the expression was restricted to tissue-resident macrophages with marginal effects in blood leukocyte populations and splenic non-macrophage populations. When compared to other ‘macrophage-specific’ lines, hCD68-CreERT2 mice had fewer ‘off-target’ effects. Additionally, I was able to successfully use hCD68-CreERT2 mice to target macrophages present in atherosclerotic lesions. To address the in vitro arm of this project, I used the Freestyle Fluidics (FF) platform to design and optimise circuits that can be used to study macrophage migration. I created a passive diffusion-based system and an active pumping system that created stable controllable gradients. I showed that both chemotaxis and chemokinesis can be readily detected and quantified in both systems. Using the FF chemotaxis assay, I observed novel properties of macrophage behaviour such as population-level coordination of chemotaxis and the ability to sense the gradients both spatially and temporally. In summary, this project advanced the macrophage-specific technology platforms available to researchers.
Design and modular self-assembly of nanostructures
Keywords: self-assembly
nanotechnology
DNA origami
polycube

As nucleic acid nanostructures grow larger and more complex, new tools and methods are needed to facilitate their design. DNA origami structures, for example, are limited by the lengths of their scaffolds but larger assemblies can be bound together by interactions between multiple components. This thesis presents two projects, each approaching the design of such modular structures at a different level of abstraction.
Project 1 presents the polycube self-assembly model, where building blocks assemble stochastically using bindings between complementary patches. The assembly of both 2D polyominoes, as well as 3D polycubes, is considered. First, the mapping between input rules (defining the set of available species) and output shapes is investigated, revealing a clear bias toward low--complexity structures. Frequent shapes also tend to be highly modular and symmetric. Secondly, the reversed mapping is explored, presenting a method to find the minimal rule that assembles a specified output shape. Differences in assembly kinetics between possible solutions are investigated using patchy particle simulations, showing that minimal rules can assemble as well as fully addressable rules.
Project 2 presents a more detailed approach to the design of modular structures and individual modules: the oxView toolkit for the design, analysis, and visualisation of DNA, RNA and protein nanostructures. While many other design tools exist, oxView makes it easy to import and connect their designs into complete assemblies. Furthermore, oxView allows for free-form editing and rigid-body manipulation. Designs can then be interactively simulated using the oxDNA model, providing a more intuitive understanding of the resulting dynamics.
In conclusion, the design of self-assembling nucleic acid nanostructures has been investigated on both an abstract and a more detailed level. The presented projects have resulted in tools and methods for creating, simulating and analysing modular structures with minimal complexity, potentially containing building blocks created in multiple design software.
CRISPR/Cas9-mediated engineering of subtype-specific cell lines for iPSC-derived cardiomyocyte phenotyping
Keywords: selective disease modelling
iPSC-CM subtype purification
iPSC-CM population's heterogeneity
TTNtv
CRISPR/Cas9-assisted genome editing
fluorescent reporter line generation

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent a useful tool for cardiovascular research. However, the mixed subtypes, including atrial-like, ventricular-like and nodal-like, that result from differentiation protocols present a challenge for selective studies. This thesis exploited the emerging genome engineering technology “CRISPR/Cas9” to generate subtype-specific fluorescent reporter systems to label specific CM subtypes, facilitating subsequent isolation for use in subtype-specific disease modelling.
Atrial-specific myosin light chain 7 (MYL7)-mClover and pituitary homeobox 2 (PITX2)-mClover, ventricular-specific myosin light chain 2 (MYL2)-mClover and nodal-specific hyperpolarisation activated cyclic nucleotide-gated potassium channel 4 (HCN4)-mScarlet iPSCs were established by targeted insertion of the fluorophores at the stop codons of the target genes. Each fluorophore was preceded by a P2A linker, allowing bicistronic expression. Unfortunately, the PITX2-mClover and MYL2-mClover lines failed quality control and could not be used for fluorescent reporter iPSC-CM derivation. The MYL7-mClover and HCN4-mScarlet lines, however, exhibited faithful reporter systems where the expression of the fluorophores mirrored that of their respective target genes.
Heterozygous TTN c.59926+1 G>A, a truncating mutation of the titin gene, is associated with atrial fibrillation and dilated cardiomyopathy. This mutation was introduced into parental iPSCs for preliminary characterisations. Subsequently, the mutation was engineered in the MYL7-mClover iPSCs for exploring the application of subtype-specific reporter systems in disease studies. The fluorescent sorting of the cells could not be optimised in the available time, however, the engineered mutant MYL7-mClover line was differentiated towards the atrial and the ventricular lineages.
No prominent consequences in heart failure-associated foetal gene reprogramming, overall TTN expression, myofibrillar protein isoform switching, sarcomere organisation, sarcomeric protein localisation were identified in the heterozygous mutant model. Interestingly, the Ca2+ transients were altered specifically in the atrial-like heterozygous model. This alteration may underlie irregular, fast atrial rhythm in atrial fibrillation. The atrial-specific phenotype of TTN c.59926+1 G>A may be further clarified when using purer atrial-like iPSC-CMs obtainable by fluorescence-activated cell sorting of the differentiated MYL7-mClover iPSC-CMs.
Vernacular queer and shifting power in Russia: from the late Imperial era up to the 1930s
Keywords: gender
Soviet history
queer
Russia
homosexuality

This thesis explores the development and complexities of queer existence shown ‘from below’, that is to say, focusing on the direct speech of non-elite homosexual men and sexual and gender non-conformists from the late imperial era up to the 1930s. The current historiography of homosexuality provides a somewhat limited view, emphasizing the importance and overarching influence of medical and legal discourses on queer subjects. The thesis offers a different perspective, shifting the analysis of hegemonic discourses of experts to the self-expression of homosexuals and gender and sexual non-conformists in the tumultuous time of the 1920s. More broadly, the thesis seeks to answer the question of when a modern homosexual identity emerges not as a scientifically constructed type but as a self-accepted category. The thesis attempts to reorient our understanding of the difference between the sodomite and the homosexual, demonstrating the process of self-fashioning as a crucial element of modern sexuality. It has been previously acknowledged that the Russian Revolution of 1917 and the abrogation of the anti-sodomy article greatly influenced the understanding of homosexuality by medical professionals and triggered the proliferation of new approaches to it.
Following this assessment, the thesis demonstrates how homosexuals and gender and sexual non-conformists inventively absorbed new knowledge to fashion themselves and made sense of their ‘abnormal’ sexuality. Even those who continued self-pathologizing were not passive objects of expert indoctrination. They conflicted with the doctors and sought new options to be cured. Others overcame the limited frame of ‘deviant sexuality’, insisting on their inclusion in the new society on their terms (‘no conventions will convince us that our actions are criminal and abnormal’). Being primarily focused on self-identified homosexuals and those males who experienced same-sex relations, this thesis also recognizes that emancipatory impulses were not restricted to them and greatly influenced a gamut of sexual and gender non-conformists who cannot be easily defined in modern terms of LGBTQ nomenclature.
Investigating the function of coding polymorphisms within the TBX15-WARS2 locus associated with waist-hip ratio as a risk factor for metabolic syndrome
Keywords: GWAS
in vivo models
adipose biology

As prevalence of obesity and metabolic disorders increase, it is necessary to elucidate mechanisms driving body mass, body composition and energy homeostasis. Elevated waist-hip ratio (WHR) increases type 2 diabetes and cardiovascular disease risk. The TBX15-WARS2 locus contains several independent association signals for WHR adjusted for BMI (WHRadjBMI). However, whether these risk variants are functional remains to be determined. The aim of my DPhil was to characterise two potentially functional TBX15-coding polymorphisms, rs10494217 (p.H156N) and rs61730011 (p.M566R), at a cellular and organismal level to investigate their impact on TBX15 function in adipose tissue biology.
Firstly, I prioritised the mechanism-of-action of these two polymorphisms at either a DNA-regulatory or protein-coding level in silico. I showed the most likely mode-of-action was at the protein-coding level with p.H156N predicted to alter a loop structure in the T-box of TBX15, further implicated in altered protein-protein dimerisation. However, this was not recapitulated in vitro (within assay sensitivity limits).
TBX15-WARS2 signals were associated with differential TBX15 and WARS2 expression, and several Tbx15 and Wars2 mouse models had altered body composition phenotypes. I metabolically characterised heterozygous knock-out (Tbx15+/-) mice and mice carrying orthologous human polymorphisms p.H156N (Tbx15H156N) or p.M566R (Tbx15M566R) and showed all three alleles conferred resistance to fat mass gain.
Finally, to evaluate the effect of TBX15 on gene expression, I generated a TBX15 regulatory network using ChIP-sequencing of preadipocyte cell lines, and RNA-sequencing of adipose tissues from homozygous knock-out (Tbx15-/-) and Tbx15H156N mice. Functional enrichment of differentially expressed genes implicated TBX15 in regulating lineage determination, adipogenesis of committed preadipocyte cells and immune response. In summary, these results suggest TBX15 regulates adipose tissue biology and body composition and shows a novel functional role for two human WHRadjBMI risk variants. Further work is needed to elucidate underlying mechanisms of action that can be targeted therapeutically.
On the projective normality of an embedding of the moduli space of stable marked rational curves into projective space
Keywords: projective normality
moduli space
log canonical
rational curves
moduli
stable rational curves

We study an embedding of the fine moduli space of stable rational curves with n marked points into projective space for each n > 4. This embedding is developed from work of A. Gibney and D. Maclagan. It is related to the embedding given by the log canonical system, which is shown to be projectively normal in a paper by S. Keel and J. Tevelev using methods in sheaf cohomology. In this thesis, we prove the projective normality of our embedding for n = 5 and n = 6 using combinatorial methods. From this we can give, in a new way, a description of equations defining
¯
M
0,6 as a projective variety. We begin work on extending these ideas to obtain analogous results for each n > 6, as well as give conjectures on equations defining
¯
M
0,n as a projective variety in this general case.