published_or_final_version / Medicine / Master / Master of Philosophy

Influenza B virus is a major human pathogen causing highly contagious respiratory disease. It accounts for approximately ~30% of influenza virus infection per year. The effector domain of the NS1 protein of influenza B virus (NS1B protein), encompassing the carboxy terminal two thirds of the protein, suppresses interferon-β (IFN-β) synthesis in virus-infected cells by unknown mechanism(s). The induced IFN-β mediates innate immunity. To elucidate the mechanism by which the NS1B effector domain suppresses the production of IFN-β, we identified cellular proteins that interact with the NS1B effector domain. Two approaches were used. The approach that succeeded employed the transfection into cells of plasmids expressing the NS1B effector domain containing two affinity tags. After double affinity purification, co-purified cellular proteins were identified by mass spectrometry. We identified Brd2 as a cellular protein that interacts with the NS1B protein. We established that Brd2 specifically binds to the NS1B effector domain in vitro, in vivo, and in virus-infected cells. Serial mutagenesis experiments showed the phenylalanine at position 171 (F171) of the NS1B protein is essential for Brd2 binding. To determine the function of the interaction of Brd2 with the NS1B protein, we generated a recombinant virus encoding an NS1B protein in which F at position 171 was replaced by an alanine. The F171A mutant virus was attenuated, and unlike the wild-type virus, induced the synthesis of IFN-β mRNA. IRF3, a key transcription factor for transcription of the IFN-β gene, was activated in mutant virusinfected cells, but not in wild-type virus-infected cells. Transfection assays implicated the activation of the TBK1 kinase as the step in IRF3 activation that is induced in mutant virus-infected cells. We interpreted these results as showing that Brd2 binding to the NS1B protein is required for suppressing IRF3 activation and IFN-β induction. Attempts at further confirmation by depletion of endogenous Brd2 using RNA interference were not successful because of inefficient knock-down efficiency and nonspecific IFN-β induction. A further complication is that another bromodomain protein, Brd4, interacts with the NS1B protein and could compensate for depletion of Brd2. / text

B cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus. / text

The drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim was investigated using an in vitro culture system, an in vivo mouse model, and a clinical trial in HIV-infected patients. We hypothesized that combination exposure causes immune cell populations in the bone marrow to undergo apoptotic cell death, and that the toxicity would affect the host response to an infectious stimulus. Mice were dosed with zidovudine, sulfamethoxazole-trimethoprim, the combination of both drugs, or vehicle only control via oral gavage. Focusing on B-lineage cells in the bone marrow, we determined that cells of the rapidly cycling, early pre-B cell subset are targeted, as well as pro-B cells earlier in development. This toxicity was found to be cell cycle dependent, with an increase in percentage of cells in the S/G2/M phases of the cycle. In vitro experiments using the drugs in a bone marrow culture system demonstrated that the effect of cytotoxicity with combination exposure is synergistic and concentration-dependent. The mechanism of apoptosis that is induced appears to be caspase-independent. To measure host response in mice, animals treated with zidovudine plus sulfamethoxazole-trimethoprim were infected with Pneumocystis murina pneumonia, and the group that received the combination of agents had a blunted antigen-specific IgG response, possibly due to a decreased number of B cells and activated B cells in the draining lymph nodes of the lungs. A clinical trial was conducted in HIV-infected patients, dividing subjects into groups receiving zidovudine, sulfamethoxazole-trimethoprim, the combination of both, or neither agent. Upon vaccination with the influenza vaccine, the combination treatment group had a blunted humoral response, with reduced antigen-specific serum IgG titers as compared to the control group. We conclude that the drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim is clinically-significant, and clinicians must consider this toxicity when treating patients with these agents concurrently.

Dans cette thèse nous présenterons un travail relatif à la théorie des (a,b)-modules. Nous nous intéresserons en particulier à trois problèmes liés à la dualité des (a,b)-modules: l'existence de formes hermitiennes, la symétrie des suites de Jordan-Hölder et la relation avec les "higher residue pairings" de K. Saito. Dans la première partie on étudie les équivalents des concepts de conjugué, adjoint et de forme hermitienne dans le contexte des (a,b)-modules. Dans notre analyse des formes hermitiennes nous sont amenés à définir la notion de (a,b)-module indécomposable et à montrer l'analogue du théorème de Krull-Schmidt dans la théorie des modules sur un anneau commutatif. On montre par la suite l'existence de formes ou bien hermitiennes ou anti-hermitiennes sur les modules réguliers indécomposables auto-adjoints et on donne un exemple non trivial de rang 4 admettant uniquement une forme anti-hermitienne. Suit une étude des suites de Jordan-Hölder de (a,b)-modules auto-adjoints. L'intérêt se porte en particulier sur les suites de Jordan-Hölder dites elles aussi auto-adjointes et on en montre l'existence, pour tout (a,b)-module régulier auto-adjoint. En guise de conclusion on applique les résultats obtenus aux (a,b)-modules associés à une hypersurface à singularité isolée, c'est-à-dire au complété formel de son module de Brieskorn. On montre que le symétrisé de l'isomorphisme avec l'adjoint donné par R. Belgrade satisfait aux axiomes donnés par K. Saito dans la présentation de ses "higher residue pairings".

Christian tradition speaks mainly of two possible post-mortem human destinies. It holds that those human beings who, in their earthly lives, acted according to God’s will and accepted God’s love will be reconciled to Him in heaven; whereas those who have acted against God’s will and refused His love will be consigned to the everlasting torments of hell. The notion that hell is everlasting and also a place of unending suffering inevitably gives rise to the following question for theists: how could an omnipotent, all-good and allloving God allow anyone to suffer the torments of hell for eternity? The problem of hell is arguably the most severe form of the problem of evil because the evil found in hell is eternal with no possibility for redemption. Thus, the doctrine of hell gives rise to a specific moral problem caused by the apparent incompatibility between God’s goodness and love and everlasting torment in hell. There have been several attempts to shore up the doctrine of hell in the face of this problem. ‘Particularists’ argue that the doctrine is morally defensible and that some people will experience eternal torment in hell as a result of their rejection of God. Others try to evade the problem by claiming that a doctrine of hell is not in fact taught in the scriptures (at least in its traditional form), and that Christians are therefore able to reject particularism and affirm that all human beings will be saved in the end. Those who make this optimistic eschatological observation are known as 'universalists’. My thesis focuses on ‘universalists’ and, in particular, on three contemporary Christian philosophers who defend universal salvation, namely: John Hick, Thomas Talbott and Marilyn McCord Adams. All three maintain that God’s love for His human creatures is inconsistent with the claim that God does not desire to bring about their salvation. Their accounts share common roots: they are founded on an understanding of God’s nature as omnipotent love, and on an understanding of human freedom, as well as on an account of curative post-mortem punishment for sinners. All three philosophers hold that God will eventually succeed in reconciling all human beings to Himself and so no one will be damned in hell. In this thesis, I argue that Hick, Talbott and Adams fail in their attempts to make a plausible case for universalism. One of the main criticisms I consider is that there is significant tension between their universalist accounts and the value of human freedom. The necessary correlation that they assume between God’s love and the outcome of this love does not recognize the capacity for each person freely to reject the offer of salvation. Another criticism I consider is that their accounts of post-mortem punishment do not guarantee either that salvation and reconciliation with God will be the outcome of a free choice made out of love, or that all sinners will eventually be saved. In bringing these three universalist accounts into question, I examine the notions of freedom of choice and punishment as well as the relation between free choice and rationality.

This thesis explores the possibility of a modern consumption distinct from discussions of the ‘consumer,’ ‘consumption,’ ‘consumerism’ and the ‘consumer society’ and rejects the possibility of a universal or ‘human’ consumption-activity rooted in use that merely varies with space and time. This is done by exploring the roots of these terms in the philosophical anthropology of economic theory, specifically the concept of homo oeconomicus. The economic inheritance within contemporary accounts of the capitalist consumption-relation is then pursued through a review of the disciplinary approaches to the topic made by historical accounts of ‘consumer culture,’ the study of patterns of use across the social sciences, from psychology, through geography to marketing and anthropology. Finally, the contemporary sociological investigation of ‘consumption’ is critiqued and its broad reliance upon a utilitarian-derived cost/benefit model adapted to incorporate ‘sign-value’ and discussions of postmodernism are rejected. This prompts the proposal of a ‘postphenomenological’ approach to the study of modern consumption and the ‘terrain’ upon which it is available to experience. The bulk of the thesis, chapters three, four and five, are taken up with a review of the contemporary commodity-form using the phenomenological categories of space, time and causality, respectively. This allows a historical perspective to be employed in the analysis of the role of material factors in the constitution of subjective experience and its role within the emergence of modern consumption. The theory of modern consumption and the sociospatial terrain upon which it unfolds is developed through the concept of ‘affordance,’ adapted from environmental psychology and a re-definition of ‘possession’ that arises from the inter-relation of being and having. This allows the rejection of the orthodox models and theories of ‘consumption’ outlined in chapter two. The thesis concludes by advocating an engagement in a ‘playful’ modern consumption that engages with the commodity-form as the medium within which contemporary ‘experience’ is transmitted and, which, consequently, forms the of the phenomenal forms of subjective experience derived from the capitalist consumption-relation.

Martin Heidegger in 'The Origin of the Work of Art' seeks to approach the self-subsistent nature of art. The Greek Temple opens up a space within which our Being may dwell. It is the site of human civilization and religion, and of our capacity to dwell within abstractions like peace, justice, truth and representation. Art breaks open a new place and presents things in a fresh light. Language is the primary model for this activity. Paul Celan in his poetry offers a challenge to Heideggerian abstraction. Both poet and philosopher were intimately familiar with each other's work, yet there is no essay on Celan, or even a reference, in the entire Heideggerian corpus. Celan's poem 'The Straitening' conveys the breakdown of meaning that has occurred after the holocaust. In form and content it challenges any Heideggerian notion of the higher univocity achieved by great poetry. We will explore recent examples of how poets have examined the idea of cultural belonging exclusion. We present a distillation of this idea in the writings of Paul Celan, particularly his presentation of the moment of 'Shibboleth'. We explore the biblical origin of the term 'Shibboleth' in a conflict between the army of Jephtah and the Ephraimites. We look at a contemporary poem with 'shibboleth' as it theme. Seamus Heaney's 'Broagh'. A consistent theme of Maurice Blanchot's critical reflection from The Work of Fire in 1949 up to and including The Space of Literature in 1955, is the manner in which our being creatures unto death allows us to create art, and to think and write in the abstraction that is language. Life endures death and maintains itself in it. For Blanchot Rainer Maria Rilke is one of the most significant modern poets in the way in which he has presented and explored this theme. We challenge Blanchot's inadequate reading of Rilke in The Space of Literature as an instance of his own pre-conceived philosophical nihilism.

Cross-linking in soy proteins is hypothesised to have an impact on the texture of tofu. In vitro incubation showed soy proteins and its two fractions, glycinin and β-conglycinin, were cross-linked using glutaraldehyde, formaldehyde, glyceraldehyde and transglutaminase (TGA). Increasing concentration of these carbonyl compounds and TGA, and temperature of the carbonyl compounds treatment, increased the reactivity of cross-linking. Glutaraldehyde was the most reactive in forming aggregated proteins, followed by formaldehyde and glyceraldehyde. Both carbonyl moieties of glutaraldehyde are believed to be essential for the rapid cross-linking reaction. In the unfractionated soy proteins, β-conglycinin had a higher reactivity than glycinin. In in vitro incubation using TGA, soy proteins served as good substrates for TGA, in which β-conglycinin was more susceptible to TGA than glycinin in the unfractionated soy proteins. The addition of TGA, and 1 and 2 mM glutaraldehyde prior to soymilk boiling in situ resulted in a small number of cross-linked proteins, which correspond to an increase in fracture force. The addition of glutaraldehyde after soymilk boiling resulted in a slight decrease in fracture force compared to the control. At higher concentrations of glutaraldehyde (15 and 30 mM), soy proteins were mostly cross-linked, regardless of addition before or after soymilk boiling. Highly cross-linked proteins resulted in a significant decrease in the fracture force. For TGA treatment, the fracture force was increased with increasing TGA concentration from 1000 to 5000 ppm, added either before or after soymilk boiling. However, the TGA treatment showed only a small quantity of cross-linking. It is hypothesised that TGA hydrolysed glutamine of proteins to glutamate and changed the functional properties of proteins. Upon examination of the microstructure, it was found that the TGA treatment resulted in a fine-stranded network, compact structure and less porosity. These characteristics resulted in a higher fracture force. In contrast, in the glutaraldehyde treatment, the network consisted of a higher porosity, loose network and diffuse structure, which gave lower fracture force. Thus, it appears that substrate modification to the structure of the soy proteins may have a greater impact than the number of cross-links. These findings are likely to have implications for production of soy products with a wide range of textures by manipulating the soy protein properties.

The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.