Search

Riboswitches are folded structures within messenger RNA that can directly bind cell metabolites to trigger induction or suppression of essential metabolic pathways. Owing to their critical role in gene regulation, developing selective modulators of riboswitch function could lead to novel antimicrobial agents or multipurpose chemical biology tools. To find selective small molecule binders of thiamine pyrophosphate responsive riboswitches, we took a two-pronged approach. Firstly we tackled the more traditional route of screening analogues of the natural ligand in order to quickly furnish us with high-affinity ligands and structure-activity data. Secondly we applied the fragment-based approach as a fundamentally different but complementary way of achieving the same goal. By developing a suite of biophysical screening methods we were able to test a library of over one thousand low molecular weight fragments for selective binding to the E. coli thiM riboswitch. Our experiments demonstrate the validity of using fragment-based methods against RNA targets, thus extending the scope of this already valuable methodology. Our screening programme identified ligands with KD values as low as 9 nM (from the analogues) and ligand efficiencies as high as 0.69 (from the fragments). These compounds provide a solid basis for the further development of ligands that not only exhibit selective and potent binding to riboswitches but crucially also affect gene expression.

This thesis contains four essays on two topics in econometric theory in which inference involving discrete-valued indicator functions is concerned. On the fi…rst topic, I study structural dynamic discrete choice models in which the indicator represents the outcome of a discrete choice made by a forward looking economic agent as a function of utility and other structural objects. The choice is based on an explicitly postulated structural dynamic discrete Markov decision process. An attempt to infer the underlying structural parameters that generates the observed data runs into identifi…cation problems, particularly under nonparameteric and semiparameteric speci…fications. I analyse these problems in Chapter 1 and present new identifi…cation results. In Chapter 2, I propose a semiparametric estimator for the estimation of a …finite horizon structural dynamic discrete choice model. My estimator is constructed based on simultaneous estimation using the method of sieves. I provide and verify sufficient conditions for my structural model to show that the proposed sieve estimator is consistent. On the second topic, I study the use of discrete-valued indicator functions to represent restrictions of one or more inequalities in underlying econometric parameters. I exploit such representation in designing practically applicable procedures of testing econometric restrictions involving multiple inequalities. The method proposed in this thesis is to chain the several hypothesized inequalities on underlying estimable econometric parameters using discrete indicator functions. I then smooth these functions to overcome the distribution problems of discrete functions of continuous variates. Based on this idea, I propose two new methods of testing multiple inequalities restrictions. The linear normal-based chaining method is proposed in Chapter 3 and the quadratic chi-square based chaining method is provided in Chapter 4 as another alternative. Asymptotic properties of both methods are studied and Monte Carlo simulations are also carried out to assess their fi…nite sample performance.

This study explores the reasons that more and more Filipino women are seeking overseas contract work (OCW) and why so many choose to repeat the migration cycle. The experiences of overseas female Filipino workers (OFFWs) are analysed within the framework of social exclusion theory (SET). Analytical concepts, such as capital and arena, are adapted from Bourdieu in order to enrich SET with cultural and personal dimensions. OFFWs are understood as socials agents acting within overlapping social, economic, and political arenas before, during, and after OCW. Social exclusion is defined as lack of full participation in an arena in which a social agent acts. Exclusion in home arenas, for example unemployment and frustrated social aspirations, are examined, as are exclusions within the arenas of labour migration itself, for example in recruitment fees and restrictions on freedom of movement in the host country. The ways in which state policy and political and market dynamics support these exclusions is addressed. Questionnaires and interviews of current and former OFFWs reveal the considerable extent to which choices related to migration are influenced by local ideologies of age and gender, and by local social patterns. It was found that OFFWs voluntarily risk exclusion in the arenas of overseas contract work in hopes of overcoming exclusion for themselves and their families in the arenas at home in the Philippines. Short-term success and long-term failure of OCW to overcome exclusion in home arenas motivate many women to repeat their migration. The dissertation argues that persistent exclusion in the home arenas may be partly due to incommensurabilities among the various (social, economic, local, international) arenas in which OFFWs act, such that overcoming exclusion in one arena engenders exclusion in another.

The political democratisation in Taiwan which started in the 1980s had an enormous impact on educational leadership in schools in the 1990s. Democratisation was imported into school leadership as the state of reform and change reached education. Moreover, gender also became an important issue as the number of women principals increased in Taiwan. This research uses case studies of eleven junior high schools in Northern Taiwan to explore the values and vision of Taiwanese men and women principals in a time of change. It examines how they engaged in change and how their staff perceived their efforts; and also how they tried to adapt or resist democratic practices in their schools. It looks at the ways in which 'democracy' was borrowed and interpreted in the school context. The thesis reviews related theories about gender and school leadership in the West to see what insight they give in the Taiwanese context. In addition, related critiques about the influence of traditional Chinese culture and the impact of politics on school principals in Taiwan are examined. The central research finding is that although men and women principals in Taiwan share similar vision and values about educational reform, other members of school staff perceive significant gender differences in the way principals have responded to the changes of democratisation. Staff generally show a preference to work with men rather than women principals. The thesis argues that the ill-defined principalship, together with the top-down process of 'democratisation' initiated by principals in schools, has created a paradox that makes principalship in Taiwan a very challenging task. For many newly arrived women principals, who did not fit the traditional image of 'male' principals, their aspiration to work hard and to make a difference to schools often creates unexpected resistance.

This thesis consists of five parts. In chapter 1, the background to chemiluminescence, chemiluminescent compounds and their applications is given. The synthesis and chemiluminescent behaviour of substituted phenyl acridinium esters (compounds 1 and 2), heterocyclyl acridinium esters and their analogues (compounds 3-7) and phenyl 2-halo-6-methoxy acridinium esters (compounds 8-11) are detailed in chapters 2, 3 and 4 respectively. The chemiluminescence studies include chemiluminescent kinetics, chemiluminescent efficiencies, chemiluminescent wavelengths and hydrolytic stabilities of the acridinium esters. Modifications on leaving groups show obvious effects on chemiluminescent kinetics and stabilities, but no wavelength changes were observed with such modifications. However, modifications on the acridine ring do not significantly affect chemiluminescent kinetics and stabilities. Chemiluminescent efficiency was decreased by the introduction of halogen atoms into the acridine ring. Surprisingly, chemiluminescent wavelength was found to be influenced by the introduction of halo and methoxy groups to positions 2 and 6, respectively of the acridine ring. Among the newly prepared acridinium esters, compounds 2 and 3 possess better stability and good quantum efficiency, and could be developed as better chemiluminescent labels in chemiluminescent immunoassays over the model compound. Apart from the acridinium esters, three cysteine derivatives (compounds 12-14) were also prepared for chemiluminescent immunoassay. The enantiomeric purity of the product was identified by NMR. The details are presented in chapter 5.

The X-ray background (XRB) and the intergalactic medium (IGM) are two of the most interesting topics in X-ray cosmology. Historically, studies of either subject provided important information on the other. For more than 30 years, the problem of the origin of the XRB has remained unsolved, despite large advances in X-ray instrumentation. Along the way, important constraints have been placed on the state of the intergalactic medium. Based on theoretical modelling and observational constraints, I have investigated both the origin of the X-ray background and the physical state of the intergalactic medium in this thesis. The first Chapter gives an outline of the observed properties of the XRB and the manner by which X-ray studies constrain the IGM. Chapter 2 reviews previous observations of the XRB and our current understanding of the spectrum, isotropy, and the resolved component of the XRB. An analysis of ROSAT data to study the spatial structure of the XRB is presented in Chapter 3. The ROSAT PSPC data include one deep survey field and six colinear medium survey fields, from which I have obtained limits on excess fluctuations and the autocorrelation function (ACF) of the XRB in the soft X-ray energy band from 1 arcmin to 5 deg. The 2σ upper limits of these results show no strong signal of anisotropy and inhomogeneity. Incorporating the observed ACF into detailed models of evolving discrete sources, I find that the correlation length of the major source dominating the residual XRB has to be < 4h₅₀^-1 Mpc, which is less than that of normal galaxies. Spectral fitting of both resolved and unresolved components of the XRB was carried out and is shown in Chapter 4. Data from the QSF3 field observed with the ASCA SIS and the ROSAT PSPC were used for this task. The diffuse XRB has a flat spectrum, which is well fit by a single power-law extending from 0.1 to 7 keV, with a photon index ranging from 1.4 to 1.5. On the contrary, the spectrum of the resolved component appears steeper. Such a steep-spectrum component can contribute in total less than 30 per cent to the residual XRB below 3 keV and only a few per cent above 3 keV. This suggests the entire XRB is composed of two spectrally distinct populations, of which the one with a steeper spectrum has been resolved in the soft band and most of which are AGN.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.