VictorJuiz/thesis huggingface dataset:
Structure, Catalytic Mechanism, and Membrane Interaction of the mTOR Activator Rheb
Keywords: Crystallography
GTPase
mTOR
Nanodisc
NMR
Rheb

The activator of mammalian target of rapamycin complex 1 (mTORC1), Ras homolog enriched in brain (Rheb), is a membrane-associated protein belonging to the Ras subfamily of small GTPases. Rheb's slow GTPase activity is stimulated by the GTPase activating protein (GAP) domain of tuberous sclerosis complex 1 and 2 (TSC1/2). Rheb hyperactivation, through its overexpression or loss of TSC1/2 GAP function, results in hyperactivated mTORC1 signaling culminating in tumourigenesis. The molecular details of Rheb GTP hydrolysis and the effect of membrane association on Rheb structure, dynamics and its GTPase function are currently not fully understood. The studies presented in this thesis focus on two key determinants of Rheb function i) the mechanism of Rheb GTP hydrolysis and ii) the structural and functional consequence of Rheb-bilayer membrane interaction. Through studies of fluorescent nucleotides, we revealed that the conserved G2-box residue Tyr35 auto-inhibits GTP hydrolysis in Rheb. We demonstrated that a non-canonical catalytic residue, Asp65, in the switch II region of Rheb, contributed more to the GTP hydrolysis rate than Gln64, which corresponds to the canonical Ras Gln61. This non-canonical auto-inhibited mechanism of GTP hydrolysis was required for optimal mTORC1 regulation. These structural insights were then used to guide the design of novel gain- and loss-of function mutants by substitutions of the ultra-conserved G3-box Gly63 of Rheb. Finally, using solution NMR spectroscopy, we monitored the Rheb GTPase cycle and characterized its nucleotide-dependent membrane orientations on nanodisc-based phospholipid bilayers. Rheb was shown to sample two orientations in which its C-terminal helix was semi-perpendicular or semi-parallel with respect to the bilayer plane. The semi-parallel orientation, where switch II residues critical for mTORC1 communication are accessible, was favored in the GTP bound conformation, suggesting that membrane-tethering modulates Rheb function. These structural insights into the catalytic machinery of Rheb and its membrane interface suggest new approaches to modulate these key determinants of Rheb function through small molecules towards development of therapeutic avenues for Rheb-mediated pathogenesis such as tuberous sclerosis or cancer.
Investigation of Amorphous Solid Dispersions of Poorly Water-soluble Drugs in Poly (2-hydroxyethyl Methacrylate) Hydrogels for Enhanced Solubility and Controlled Release
Keywords: amorphous solid dispersion
crosslinked PHEMA hydrogels
crystallization
kinetic solubility
poorly water-soluble drugs
supersaturation

The purpose of this study was to investigate the potential of applying amorphous solid dispersions (ASD) in crosslinked PHEMA hydrogels to enhance the dissolution behavior of poorly water-soluble drugs. The first part of the study identifies physicochemical properties affecting the solid state and physical stability of ASD of the model drug indomethacin (IND) in PHEMA hydrogels. The results of the second part show that ASD based on water-insoluble crosslinked PHEMA can maintain a high level of supersaturation over a prolonged duration via a diffusion-controlled feedback mechanism, thus avoiding the initial surge of supersaturation followed by a sharp decline in drug concentration, which is typically encountered with ASD based on water-soluble polymers (e.g., PVP, HPMCAS) under nonsink dissolution conditions. A subsequent study examines the effect of supersaturation generation rate on the resulting kinetic solubility profiles of amorphous pharmaceuticals and delineates the interplay between dissolution and precipitation processes from a mechanistic viewpoint. In the absence of any dissolved polymer to inhibit drug precipitation from the supersaturated state, both our experimental and predicted results confirm that the faster rise of the kinetic solubility profile of an amorphous drug will inevitably lead to an earlier but higher maximum kinetic solubility and a sharper decline in the de-supersaturation phase, and vice versa. The relationship between the achievable maximum supersaturation and the rate of supersaturation generation in the observed kinetic solubility profiles has been described for the first time by our comprehensive mechanistic model taking into account the role of supersaturation in both the nucleation and crystallization processes as well as the associated competitive particle growth and ripening effects. Finally, this theoretical framework was further employed to semi-quantitatively predict the evolution of supersaturation of amorphous pharmaceuticals generated from nonlinear dissolution profiles. The effects of initial degree of supersaturation, dissolution of amorphous drug and that from the IND-PHEMA ASD under nonsink dissolution conditions were subsequently examined in detail. The comparison of dissolution behaviors between amorphous IND and IND-PHEMA ASD demonstrates the advantage of the diffusion-controlled feedback mechanism that makes crosslinked PHEMA a unique and desirable carrier for amorphous drug delivery systems.
Mechanism of Repression by the Sequence-Specific mRNA-Binding Protein Vts1p
Keywords: Smaug
Vts1
post-trancriptional regulation

Vts1p is a member of the Smaug family of sequence-specific RNA binding proteins that interact with stem-loop sequences to degrade and/or translationally repress target transcripts. To identify the molecular mechanism by which Vts1p represses target transcripts, I have investigated the role of one of Vts1p’s conserved domains, the SSR1 domain, in Vts1p-mediated repression. In vitro SSR1 functions as a dimerization domain and I provide evidence that SSR1 mediates dimerization in vivo. I have created Vts1p mutants which abolish dimerization and found that dimerization is required for full Vts1p-mediated repression and robust destabilization of a reporter containing two SREs. My data supports a model whereby dimerization of Vts1p functions to enhance binding of Vts1p to a target transcript carrying two or more SREs. Analysis of putative Vts1p mRNA targets suggests that dimerization may be important for proper repression of many target transcripts since a majority contain multiple SREs. I have also shown that Vts1p interacts with the Ccr4p/Pop2p/Not deadenylase in an RNA independent fashion. This is consistent with the model that Vts1p recruits the Ccr4p/Pop2p/Not deadenylase to target transcripts to induce deadenylation and subsequent degradation. Finally, by tethering Vts1p to a reporter construct, I have shown that two separate regions of Vts1p, the N-terminus and the C-terminus, are sufficient for repression, suggesting that Vts1p employs multiple mechanisms to regulate the expression of target mRNAs. I have also shown that the N-terminus contains a conserved motif that functions in Vts1p-mediated translational repression while playing no role in transcript decay. This is the first evidence that Vts1p mediates the translational repression of a target transcript.
Verse and Lore: ‘Poems on History’ (yongshi shi) from the Selections of Refined Literature (Wen xuan)
Keywords: poems on history
Six Dynasties

This dissertation discusses how cultural memory and nostalgia are demonstrated and negotiated in poems through investigating the section of “Poems on History” in the Selections of Refined Literature (Wen xuan 文選). Chapter One lays a solid foundation for further discussion, mainly exploring previous Chinese and English scholarship on poems on history in the early medieval period (220-580) and providing a brief history of this subgenre up to this point. Chapter Two examines why the Wen xuan editors chose certain poems in the section of Poems on History through a case study of the reception of Zuo Si’s poems. The reception and canonization process of his poems serve as a good example to illustrate how the memory of literary past is shaped and mediated by the intellectual and cultural zeitgeist of this period. Chapter Three deals with the different approaches by which the poets connect historical lore with poetry, and shows the sophistication of poems on history in Chinese literary tradition. The last two chapters discuss two case studies: one is from Zuo Si, an earlier case and the other on Yan Yanzhi, a case closer to the Wen xuan editors’ time period. Chapter Four explicitly discusses how poets use literature as a way to create a poetic-self as an alternative to images transmitted in standard histories, a represented self under their control through which poets influence their contemporaries and later readers’ perception and memories about them. A good case study of this process is Zuo Si’s “Poems on History”. Chapter Five talks about the formation of poets’ reputation in history, not based on their political achievements and contributions, but on the highly admirable moral values and ideals expressed in their poetry, which become models for later literati. Yan Yanzhi’s “Poems on the Five Lords” is a case study that shows the continuing tradition of character appraisal, dealing with five historical figures who were remembered because of their musical, literary, and spiritual cultivation. Thus poems on history--their composition, their reception, their transmission through anthologies--are shown to have been a vital means of interpreting and evaluating the past, and to have played an important role in shaping Chinese identity and character through literature.
Transgression in Texts: Literary Quality of Qi from Tang Tales of Marvels to Wang Xiaobo’s Tang Tales
Keywords: Chinese fiction
the marvelous in literature
intertextual and intergeneric transgression

The modern Chinese writer, Wang Xiaobo (1952-1997), retold three tales from the Tang dynasty (618-907) twice during the last two decades of the twentieth century. In this dissertation I examine the intertextual and intratextual relations between these tales and find that Wang Xiaobo’s efforts at literary adaptation belong to a long tradition of Chinese fiction writing in which the writer’s perceptions of the world are displayed through transformative experiments with language, genre, and reader’s expectations, experiments based on the pursuit of qi 奇 (“the marvelous”) in Chinese narrative literature. The literary quality of qi was first associated with Tang chuanqi 傳奇, or Tang tales of marvels. The Tang literati made use of shared literary knowledge in writing the Tang tales, and by engaging a literary spirit of you 遊 (“freedom of roaming”) in transgressing generic restrictions imposed by their classical education, they were able to present new perspectives on their own world. I argue that it is the pursuit of the literary quality of qi in Tang tales that makes transgression possible for the Tang literati in textual, generic, and thematic terms. Even though the literary quality of qi was domesticated and waned after the Tang dynasty, it persisted through the next twelve hundred years. I take an intertextual and intergeneric approach in analyzing selected texts by writers active in the centuries between the Tang literati and Wang Xiaobo, and find that writers who espouse the literary values of fiction help to preserve and enrich the literary quality of qi in their work. Wang Xiaobo continues the tradition of experimenting with shared literary and generic knowledge to achieve a sense of qi in his adaptations, adding to it a touch of postmodern irony. Wang Xiaobo revitalizes the literary power of qi as a means of two-fold transformation through which writing changes the writer, and reading changes the reader.
The Role of TNFR Family Members, TNFR2 and CD30, in the T cell Response Against Viral Infection
Keywords: T cells
viral infection
TNFR
TNF
CD30
influenza
LCMV

TNFR family members tightly regulate the threshold, magnitude and duration of T cell responses against respiratory viral infections. In this thesis, I examine the role of two TNFR family members, TNFR2 and CD30, in the CD8 and CD4 T cell responses against influenza virus. Therapeutic blockade of TNF is the gold standard treatment for certain chronic inflammatory diseases, consistent with the key role of TNF in many dysregulated inflammatory responses. TNF is produced by many cell types and is also involved in anti-viral immunity. In order to address the role of intrinsic TNF/TNFR2 interactions in CD8 T cell responses against respiratory influenza infection in mice, we used an adoptive transfer model in which only the transferred T cells lack TNF or TNFR2. During mild influenza infection, the capacity of the antigen-specific CD8 T cells to produce TNF increases throughout the infection, beyond the time of viral clearance. Autocrine TNF/TNFR2 interactions are dispensable for the initial expansion of the CD8 T cells, but potentiate contraction of the response in the lung, thus limiting a potentially damaging and excessive CD8 T cell response. However, peak CD8 T cell effector functions are enhanced by intrinsic TNF/TNFR2 interactions, illustrating a complex dual role for TNF in fine-tuning the adaptive immune response to influenza infection. CD30 was originally discovered as a marker highly expressed on Reed-Sternberg cells of Hodgkin’s lymphoma, and is currently being explored as a target for cancer therapy in clinical trials. However, the role of CD30 in regulating T cells in response to viral infection is poorly understood. Therefore, we investigated whether CD8 and CD4 T cell responses against influenza infection involve CD30. During both mild and severe influenza infections, CD30 is dispensable for the accumulation of CD8 and CD4 T cells and their cytokine production. On the other hand, preliminary data suggest CD30 plays a more significant role during a persistent virus infection. In summary, the studies conducted in this thesis reveal divergent roles for TNFR family members in controlling T cell responses against viral infection.
The Presynaptic Transmitter Release Site Within the Calcium Nanodomain: Calcium Channel and Synaptic Vesicle Tethering
Keywords: presynaptic
calcium channel
synaptic vesicle
tethering
tether
Cav2.2
transmitter release
PDZ
SV-PD

Fast neurotransmitter release (NTR) is triggered when the presynaptic membrane is depolarized, opening voltage-sensitive calcium channels (Cavs) and allowing calcium (Ca2+) ions into the terminal to trigger the fusion of synaptic vesicles (SVs). Tight spatial and temporal control of NTR suggests that the Cav and SV are tightly coupled and linked by a molecular tether. However, it is unclear how Cavs interact with SVs and the molecular identity of the tether has yet to be elucidated. Using a novel biochemical assay called SV pull-down, or SV-PD, we demonstrate that Cav2.2 channels can capture SVs in vitro. Pull-down experiments using chick Cav2.2 long splice variant fusion proteins demonstrate that the distal C-terminal is also important in SV capture. C-terminal postsynaptic density-95 (PSD-95)/Discs-large/Zonula-Occludens-1 (PDZ) ligand mutant fusion proteins and mimetic peptides of conserved binding sites on the distal C-terminal did not block SV-PD, suggesting that SV capture can occur in a PDZ ligand-independent manner.
‘Things Invisible to Mortal Sight’: Literary and Experimentalist Applications of Perspective in Seventeenth-Century England
Keywords: Milton, John
Cavendish, Margaret
optics
Hooke, Robert
perspective

This thesis investigates the dialogue between imaginative literature and experimentalist philosophy in Restoration England with a focus on the topics of vision and perspective. I examine Margaret Cavendish’s and John Milton’s contributions to this dialogue by reading Cavendish’s Blazing World and Milton’s Paradise Lost alongside optical writings by Johannes Kepler, René Descartes, Robert Hooke and Isaac Newton. Where current assessments of the relationship between early modern science and literature have tended to make content-based comparisons, my approach charts new territory by examining the structural, rhetorical, and stylistic components that shape and inform the content of these works. To lay a foundation for this analysis, I first demonstrate that, contrary to the prevalent view of experimentalist writers as anti-figuration, these writers relied on the rhetorical tropes of analogy and simile as a literary means of making invisible things visible to the reader’s imagination. I argue that in experimentalist literature, “similitudes” are conceived as literary analogues to the mechanical technology of the lens. Like lenses, they can serve both to clarify and distort the objects they describe. Thus experimentalist anxiety over the use of figuration is connected to the broader debate occurring in seventeenth-century England about the epistemological status of perspectival technologies and indeed of vision itself. Having laid this groundwork, I then explore what the textual “lenses” of similitude reveal in the works included in this study. I show that in addition to a mutual interest in the ways in which figuration can serve to manipulate perspective, these texts are part of a larger discussion about the epistemological, theological, and political implications of the new information made available through the application of perspectival technologies such as telescopes and microscopes to the natural world. It is my position that these concerns are expressed not only through content, but also through form. By analyzing linguistic technologies as an aspect of the response to the optical science of the period, I aim to provide a more complete picture of the relationship between imaginative literature and experimentalism than that which currently exists.
Constructing the Cámara Santa: Architecture, History, and Authority in Medieval Oviedo
Keywords: art history
medieval art
architectural history
historiography of art

My dissertation examines the Cámara Santa of the Cathedral of Oviedo as both a medieval and modern monument, shaped by twelfth-century bishops and twentieth-century restorers. I consider the space as a multi-media ensemble, containing manuscripts, metalwork, and sculpture, arguing that we must view it as a composite—if fragmented—whole. My analysis focuses on the twelfth century, a crucial period during which the structure, decoration, and contents of the Cámara Santa were reworked. A key figure in this story is Bishop Pelayo of Oviedo (d. 1153), who sought to enhance the antiquity and authority of the see of Oviedo by means of the cult of its most important reliquary: the Arca Santa. I argue that this reliquary shapes the form and function of the twelfth-century Cámara Santa, considering the use of the space in the context of liturgy and pilgrimage. Finally, I consider the sculpture that lines the walls of the space, arguing that it animates and embodies the relics contained within the Arca Santa, interacting with the pilgrims and canons who used the space. Thus, this sculpture represents the culmination of the long twelfth-century transformation of the Cámara Santa into a space of pilgrimage focused around the Arca Santa and the memory of the early medieval patrons of the Cathedral of Oviedo, a memory which abides to this day. What is importantly new about my project is this historiographic frame, combined with a theoretical approach that challenges traditional methods and interpretations. I foreground the importance of the Cámara Santa for both regional and national identity. My reflexive reading of the medieval and modern histories of the Cámara Santa offers alternative interpretations for a space whose meaning has long been presented as ideologically fixed.
Dissenting Shadows: A History of Film Policy and Production in Newfoundland and Labrador, 1933-1997
Keywords: film
Newfoundland and Labrador

This thesis examines the development of film policy and production in Newfoundland and Labrador between the vote to suspend Representative Government in the Dominion of Newfoundland in 1933 and the establishment of the Newfoundland and Labrador Film Development Corporation in the province of Newfoundland in 1997. Both the Commission of Government, established by Britain in 1934, and the Provincial Government, established upon Canadian Confederation in 1949, attempted to use film to rehabilitate what was widely understood to be abject space, rendered so by its malfunctioning political and economic systems. With the development of these systems under the auspices of the Canadian state, a range of filmmakers began to use the medium to interrogate, challenge, and at times actively contravene both those corrective representations that had come before as well as the modes of governance they espoused. Part one considers the development of film policy and production on the Island of Newfoundland, the seat of both the Dominion and Provincial Governments. Beginning with a reading of the Commission of Government’s general mandate, this section traces the manners in which that mandate came to bear upon the development of film exhibition policy as it was articulated by the Department of Education and how, in turn, that exhibition policy came to affect the film production practices by agents of institutional and local culture. It forwards the idea that film was utilized to develop contesting ideas of spatial capability. Part two examines the development of that same policy articulated in Newfoundland and how it came to affect film production practices in Labrador. Historically conceived of as a region meant to benefit Newfoundland, here the primary conflict between the agents of institutional and local culture was not one of spatial capacity but spatial ownership. What emerges are two distinct yet intimately bound histories of film’s unique ability to represent, rehabilitate, manufacture and reclaim space. They reveal as much about the difficult nature of Newfoundland and Labrador’s relationship to one another as they do the province’s relationship to Canada.
The (Proto-)Modernist Subject in Time: Fugue and the Fourth Dimension
Keywords: Fugue, Counterpoint, Subjectivity, Alterity, Time, Non-Euclidean Geometry, Intermediality, Melopoetics, Enigma, Fourth Dimension, Epiphany, Parallax

In such curiously compelling historical proximity, French symbolist poets and European modernist writers experimented with a particular musical form in literature: fugue, a musical style that reaches its height in the Baroque period, with the composition of Bach’s unfinished work, Die Kunst der Fuge. This dissertation analyzes the peculiar melopoetics of fugal writing in fin-de-siècle poets as well as in their modernist counterparts, who write in French, English and Italian. On a formal level, this project demonstrates how – within an intermedial methodological framework – fugue functions as a hybrid, experimental narrative, in its representation of polyphony and counterpoint, as well as both a horizontal and – a nearly impossible feat within linear narrative – vertical simultaneity in literature. Above and beyond the manner in which a fugal poetics manifests itself in the works of Verlaine, Mallarmé, Joyce, Pound, Gide and Vittorini, the fugue – defined, in part, as the variations of a single subject in time and space – becomes a vessel for authors to represent a fragmented modern subjectivity, and a crisis of language and communication, at a specific historical juncture, and within diverse aesthetic and political contexts. The enigmatic and resistant fugal form not only subverts language in these authors’ works – by colliding two distinct semiotic media, in which only one is mimetic –, thus creating a reading experience of “hostipitality” (Derrida), but it also performs the desire for wholeness, by deconstructing the ‘subject’ – with all of its polysemic ambiguity – in relation to the self, to objects, and to alterity. Finally, fugue’s interdisciplinarity goes beyond breaching the gap between music and literature, and comes into dialogue with the distinct notions of four-dimensionality, from relativity theory to non-Euclidean geometry.