Unusual DNA secondary structures, especially G-quadruplexes and i-motifs, play important roles in gene transcriptional regulation and have been identified as novel drug targets. In this dissertation, I explored their formation in the human VEGF and RET promoters and their roles in gene transcriptional regulation. VEGF is a key regulator of angiogenesis and is up-regulated in many types of tumors. A poly-guanine/poly-cytosine (polyG/polyC) tract in its proximal promoter (-85 to -50 base pairs relative to the transcription starting site) is essential for both basal and inducible VEGF expression. I demonstrated that the guanine-rich (G-rich) and cytosine-rich (C-rich) strands in the VEGF proximal promoter are able to form G-quadruplex and i-motif structures, respectively. The major G-quadruplex formed by the VEGF G-rich sequence is an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:4:1 arrangement of three double-chain-reversal loops (two single-base loops and one loop with four bases). The complementary C-rich sequence in the same region forms an intramolecular i-motif containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration (two double-base loops and one loop with three bases). The Gquadruplexes formed by the native VEGF G-rich and its derivative sequences were also confirmed by NMR. In addition, various transcription factors including Sp1, hnRNP K, CNBP and nucleolin, which recognize different DNA structural elements including single-stranded, double-stranded or G-quadruplex/i-motif DNA in the VEGF proximal promoter, have been confirmed by EMSA, siRNA and chromatin immunoprecipitation (ChIP) assay, suggesting that the DNA in the VEGF proximal promoter region is capable of undergoing transitions between those three structures. Based on my studies, I have proposed a model to describe how various transcription factors recognize different DNA structures in the VEGF proximal promoter to regulate transcription. In the proximal promoter of another important oncogene RET, I demonstrated that the guanine-rich strand forms an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:3:1 arrangement of three double-chain-reversal loops. The complementary cytosine-rich strand forms an i-motif structure containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration. Moreover, G-quadruplex-interactive compounds TMPyP4 and telomestatin were shown to further stabilize the RET G-quadruplex structure.

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

Powerful individuals often influence the delivery of government services for their own purposes. Officials may prefer inherently to direct assistance to their own relatives and social contacts (nepotistic preferences). Alternatively, they may use government services strategically in exchange for favors (patronage) or to gain voter support (clientelism). Most existing literature examines these three phenomena separately, or does not distinguish the motivations for politicians’ influence on distribution. Causal identification has also been a problem in the empirical literature. In the first chapter of this thesis, I develop a theoretical model of interaction between three levels of actors: politicians, local patrons, and households. The model allows for politicians and patrons to influence government services for nepotistic, clientelistic, and patronage purposes. In chapters 2-4, I test the predictions of the model using two novel household survey datasets I collected along with my collaborators in rural Punjab, Pakistan. Chapter 2 tests the theoretical predictions for the interaction of politicians, patrons and voters. Chapters 3 and 4 provide quasi-experimental evidence on the causal effect of links with politicians on assistance. I find evidence that politicians exert dramatic influence on the targeting of government assistance in this setting. Consistent with the theoretical model, the most assistance goes to a small “inner circle” of their closest contacts. Politicians assist this “inner circle” based on their inherent preferences, regardless of electoral pressure. When politicians face electoral pressure, they also deliver assistance to a wider group, in particular members of the same clan. In contrast, local patrons do not appear to have significant independent influence over the targeting of the government assistance programs I study, but they do provide other types of assistance to households. Their behavior is more consistent with the idea that they are motivated by inherent preferences for assisting their contacts. The results have implications for the interpretation of empirical literature on nepotism, clientelism, and patronage. They can also inform the policies of donor agencies and civil society organizations who aim to engage or pressure governments to reduce corruption and improve public spending.

Master of Science / Department of Chemistry / Santosh Aryal / Nanomedicine-based therapeutics have exhibited clear benefits when compared to unmodified drugs, which include improved pharmacokinetics, drug retention, targeting efficiency, and minimizes toxicity. Every year thousands of bone cancer cases are diagnosed in the United States. Moreover, development of bone metastasis occurs in over 80% to 90% of various cancers that metastasize and signals the entry of the disease into an incurable phase. Cancer in bones can cause pain, fractures, hypercalcemia, and compression of the spinal cord, due to deposits that can erode into the bone using bone-absorbing cells. Bisphosphonates are drugs that reduce the activity of bone-absorbing cells and targets overexpressed calcium. They are characterized pharmacologically to inhibit bone resorption, skeletal distribution, and renal elimination. In addition, they can target bone microenvironment and bind strongly with calcium. The goal of this thesis is to engineer targeted nanomedicine drug with the ability to spatiotemporally control therapeutics delivery to the bone. Herein we synthesized biopolymers with functional end group moieties as alendronate (a molecular member of bisphosphate), which can target overexpressed calcium ions at the vicinity of the bone lesion where bone resorption takes place. In order to achieve our goal, a ring opening polymerization of cyclic L-lactide initiated by ALE in the presence of catalytic amount of stannous octoate was conducted in an inert environment. Thus, formed polymers are characterized for their chemistry and physicochemical properties using various analytical tools. These polymers were characterized by nuclear magnetic resonance (¹H-NMR) and Fourier Transfer Infrared Spectrometer (FT-IR), which shows monomer conversion and the presence of amide and phosphate moiety. Thereafter we engineered bone-homing polymeric nanoparticles of 80nm diameter by nanoprecipitation for controlled delivery of Dox, a first line anticancer drug used in clinics. The in-vitro results show that the nanoparticles have the ability to accumulate and internalized into the bone cancer cells, deliver drugs efficiently, and are least toxic. Therefore, innovative and efficient bisphosphonate functionalized Poly-l-lactide polymers were synthesized to target bone microenvironment.

Ce travail est consacré à l’évaluation des performances de ciblage et à l’analyse d’impact de trois projets. La première étude porte sur l’accès à l’eau courante en République du Congo ; et l’auto-ciblage pratiqué à travers une tarification spécifique ne bénéficie qu’aux ménages connectés qui sont plutôt non pauvres. L’auto-ciblage fonctionne mieux pour le projet des travaux publics au Libéria ; la proportion de pauvres qui participent à ces travaux est élevée. En fait ce projet n’a aucune barrière à l’entrée, ce qui explique en partie ce bon résultat. Le ciblage du programme de modernisation agricole en RDC n’est pas très efficace non plus, les bénéficiaires n’étant souvent pas pauvres. L’impact en termes de réduction de la pauvreté est important dans les deux projets concernés par ce type d’analyse (modernisation agricole en RDC et travaux publics au Libéria), tout au moins pour les populations bénéficiaires. Le cas du projet de modernisation agricole en RDC montre néanmoins qu’il est important de prendre en compte d’autres facteurs pour obtenir un impact important, comme lever les contraintes liées au marché du crédit et à l’accès aux infrastructures / This work analyses the targeting performance and the impact evaluation of three projects. The first paper is about the water subsidies in the Republic of Congo. The study shows that the self-targeting scheme, using an Inverse Block Tariff structure has poor performance, indeed only those households who are connected can benefit from it, and they are usually non-poor. The self-targeting mechanism used in the Public Work program in Liberia works better since the proportion of poor involved in the program is high. This project has no entry barrier which partly explains the good result. The targeting performance of the Modernization of Agriculture project in the DRC is also poor, most of the beneficiaries being non-poor. The impact in terms of poverty reduction is important in both projects involved in this type of analysis (Modernization of Agriculture in the DRC and Public Work in Liberia), at least for the beneficiaries. The case of the DRC project, however, shows that it is important to lift some other constraints like access to credit and infrastructures in order to improve the impact of the program.

In this thesis I examine the effect of introduction of inflation targeting as a monetary policy regime on the difference between actual inflation and perceived inflation. Perceived inflation is used in the analysis in contrast with previous research, because inflation perceptions are extracted from consumer surveys conducted in individual European Union countries on the whole population sample and thereby enable me to examine the effect that the introduction of inflation targeting has across the whole population. A panel data set of 19 European Union members and 1 candidate, including 7 inflation targeters, is used in the analysis, with monthly information from the period beginning in January 1990 and ending in December 2012. Based on the analysis using fixed-effects model with specific dummy variables to capture the difference-in-differences element, I find that inflation targeters experience lower differences between actual and perceived inflation and that the difference between actual and perceived inflation decreases after the introduction of inflation targeting. Furthermore, various groups divided according to socio-economic characteristics of the consumer survey respondents tend to be affected in a different way by the introduction of inflation targeting, although to a limited extent. JEL...

After the recent financial crisis of 2007, a connection between monetary policy and financial stability has started to be thoroughly investigated. One of the particular areas of this research field deals with the role of various financial variables in the monetary policy rules. The main purpose of this research is to find whether direct incorporation of the financial variables in the monetary policy rule can bring macroeconomic benefits in terms of lower volatility of inflation and output. So far, the main emphasis of the research has been placed on the investigation of the augmented Taylor rules in the context of a closed economy. This thesis sheds light on the performance of the augmented Taylor rules in a small open economy. For this purpose, a New Keynesian DSGE model with two types of financial frictions is constructed. The model is calibrated for the Czech Republic. The thesis provides four conclusions. First, incorporation of the financial variables (asset prices and the volume of credit) in the monetary policy rule is beneficial for macroeconomic stabilization in terms of lower implied volatilities of inflation and output. Second, the usefulness of the augmented monetary policy rule is the most apparent in case of the shock originating abroad. Third, there is a strong link between the financial and the...

As mitocôndrias são os principais locais de geração de espécies reativas de oxigênio (ERO), que são produzidos como subprodutos da cadeia de transporte de elétrons. O ferro livre e as ERO podem se envolver em processos potencialmente nocivos, sendo que a desregulação do metabolismo do ferro nessa organela tem sido associada a várias doenças, como a Ataxia de Friedreich (FA). O transporte seletivo de quelantes de ferro a esta organela é proposto como um meio de melhorar sintomas de FA. A desferrioxamina (DFO) é um sideróforo bacteriano com grande afinidade ao ferro, mas baixa penetração celular. Já os peptídeos altamente catiônicos TAT49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) e SS20 (Phe-(D)-Arg-Phe-Lys) são conhecidos por permear as membranas citossólicas e mitocondriais. Nós preparamos conjugados de DFO com peptídeos que penetram as mitocôndrias e estudamos suas características de ligação ao ferro in vitro. Eles foram preparados e conjugados em fase sólida com DFO (produzindo quatro mtDFO), que em seguida foram purificados e caracterizados por meio de LC/MS e análise de aminoácidos. Os mtDFO de alta pureza exibiram capacidade de ligação de ferro idêntica à do quelante livre DFO. Os mtDFO também foram capazes de suprimir a oxidação catalisada pelo sistema ferro-ascorbato. A fim de avaliar a localização intracelular dos mtDFO, estes foram marcados com TAMRA (mtDFO-TAMRA). Frente a uma linhagem de carcinoma de ovario, os mtDFO foram em geral pouco tóxicos e altamente localizados nas mitocôndrias. Não foram observados níveis expressivos de danos a DNA, indução de apoptose, geração de ERO na mitocôndria, arraste de ciclo celular ou de apoptose. Resultados preliminares da aplicação dos mtDFO a cardiomiócitos murínicos com baixo nível de frataxina (modelo de FA) indicam um restabelecimento de aproximadamente 60% na morfologia mitocondrial, o que pode ser interpretado como uma melhora nas funções dessa organela. Estes resultados indicam que os mtDFO produzidos podem ser uma parte importante no arsenal terapêutico para FA. / Mitochondria are the main site for the generation of reactive oxygen species (ROS) as sub products of electron transport chain. Free iron and ROS may interact to generate potentially harmful species, and iron homeostasis in this organelle has been linked to several diseases, such as Friedreich Ataxia (FA). Selective targeting of iron chelators to mitochondria has been proposed as a means of alleviating FA symptoms. Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, however low cell penetration. Highly charged peptides TAT49-57 Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) and SS20 (Phe-(D)-Arg-Phe-Lys) are known as both cell- and mitochondria-permeant. We prepared conjugates of DFO with mitochondria-penetrating peptides and studied their iron binding characteristics in vitro. They were prepared in solid phase and conjugated to DFO (generating four mtDFO), which were then purified and characterized by LC/MS and Amino acid analysis. MtDFO exhibited iron binding ability identical to free DFO. The mtDFO of high purity were also able to quench the oxidation catalyzed by the iron-ascorbate system. Cell localization studies were performed by tagging mtDFO with TAMRA. In A2780 cells, mtDFO displayed in general low toxicity and high levels of mitochondrial location. No expressive levels of DNA damage, apoptosis, mitochondrial ERO generation or cell cycle arresting were observed. Preliminary results of the application of mtDFO on mouse cardiomiocytes with low levels of frataxin (animal model of FA) indicate a recovery of ca. 60% of mitochondrial morphology. This is interpreted as an improvement of the functions of the organelle. These results indicate that mtDFO may be important allies in the therapy of FA.

As mitocôndrias são os principais locais de geração de espécies reativas de oxigênio (ERO), que são produzidos como subprodutos da cadeia de transporte de elétrons. O ferro livre e as ERO podem se envolver em processos potencialmente nocivos, sendo que a desregulação do metabolismo do ferro nessa organela tem sido associada a várias doenças, como a Ataxia de Friedreich (FA). O transporte seletivo de quelantes de ferro a esta organela é proposto como um meio de melhorar sintomas de FA. A desferrioxamina (DFO) é um sideróforo bacteriano com grande afinidade ao ferro, mas baixa penetração celular. Já os peptídeos altamente catiônicos TAT49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) e SS20 (Phe-(D)-Arg-Phe-Lys) são conhecidos por permear as membranas citossólicas e mitocondriais. Nós preparamos conjugados de DFO com peptídeos que penetram as mitocôndrias e estudamos suas características de ligação ao ferro in vitro. Eles foram preparados e conjugados em fase sólida com DFO (produzindo quatro mtDFO), que em seguida foram purificados e caracterizados por meio de LC/MS e análise de aminoácidos. Os mtDFO de alta pureza exibiram capacidade de ligação de ferro idêntica à do quelante livre DFO. Os mtDFO também foram capazes de suprimir a oxidação catalisada pelo sistema ferro-ascorbato. A fim de avaliar a localização intracelular dos mtDFO, estes foram marcados com TAMRA (mtDFO-TAMRA). Frente a uma linhagem de carcinoma de ovario, os mtDFO foram em geral pouco tóxicos e altamente localizados nas mitocôndrias. Não foram observados níveis expressivos de danos a DNA, indução de apoptose, geração de ERO na mitocôndria, arraste de ciclo celular ou de apoptose. Resultados preliminares da aplicação dos mtDFO a cardiomiócitos murínicos com baixo nível de frataxina (modelo de FA) indicam um restabelecimento de aproximadamente 60% na morfologia mitocondrial, o que pode ser interpretado como uma melhora nas funções dessa organela. Estes resultados indicam que os mtDFO produzidos podem ser uma parte importante no arsenal terapêutico para FA. / Mitochondria are the main site for the generation of reactive oxygen species (ROS) as sub products of electron transport chain. Free iron and ROS may interact to generate potentially harmful species, and iron homeostasis in this organelle has been linked to several diseases, such as Friedreich Ataxia (FA). Selective targeting of iron chelators to mitochondria has been proposed as a means of alleviating FA symptoms. Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, however low cell penetration. Highly charged peptides TAT49-57 Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) and SS20 (Phe-(D)-Arg-Phe-Lys) are known as both cell- and mitochondria-permeant. We prepared conjugates of DFO with mitochondria-penetrating peptides and studied their iron binding characteristics in vitro. They were prepared in solid phase and conjugated to DFO (generating four mtDFO), which were then purified and characterized by LC/MS and Amino acid analysis. MtDFO exhibited iron binding ability identical to free DFO. The mtDFO of high purity were also able to quench the oxidation catalyzed by the iron-ascorbate system. Cell localization studies were performed by tagging mtDFO with TAMRA. In A2780 cells, mtDFO displayed in general low toxicity and high levels of mitochondrial location. No expressive levels of DNA damage, apoptosis, mitochondrial ERO generation or cell cycle arresting were observed. Preliminary results of the application of mtDFO on mouse cardiomiocytes with low levels of frataxin (animal model of FA) indicate a recovery of ca. 60% of mitochondrial morphology. This is interpreted as an improvement of the functions of the organelle. These results indicate that mtDFO may be important allies in the therapy of FA.

This thesis focuses on designing and synthesis of novel molecular imaging probes based on non-covalent funtionalisation of pure single-walled carbon nanotubes (SWNTs). Several synthetic strategies for the supramolecular chemistry functionalisation of SWNTs, cytotoxicity measurements and cellular imaging of supramolecularly functionalised carbon nanotube probes are discussed. Chapter one is a literature review as the thesis Introduction. This describes aspects of the physical and chemical properties, structural importance and synthesis methods of single-walled carbon nanotubes (SWNTs), also opens the discussion of the different functionalisation methods to enhance the solubility and biocompatibility of SWNTs for biomedical applications. Several approaches for the design of functionalisation SWNTs for molecular imaging reported in the current literature are highlighted. Techniques and facilities for accessing cell imaging ability and behavior of these synthesized molecular imaging probes, including confocal laser scanning microscopy (CLSM) and fluorescence-lifetime imaging microscopy (FLIM) are described briefly. Chapter two explores the synthesis of specifically designed naphthalene diimide (NDI). These molecules are known to form 3 dimensional (3D) helical organic nanotubes through hydrogen bonding. In this work an iodine-tagged NDI was allowed to self-assemble onto the surface of SWNTs. The cavities of the NDI organic nanotubes can accommodate SWNTs strands in their hydrophobic interior as observed high-resolution transmission electron microscopy (HR TEM). A new hybrid material, NDI@SWNT, was prepared and characterised as dispersed in organic solvents and aqueous media and in the solid state by HR TEM, tapping mode atomic force microscopy (TM AFM), scanning electron microscopy (SEM), circular dichroism, Raman and fluorescence spectroscopies (steady-state single and two-photon techniques). These measurements indicate that amino acid-functionalised NDI interacts strongly with SWNTs in dispersions and forms a donor-acceptor complex denoted NDI@SWNT. The interaction of this nanohybrid with cancer cells was explored using fluorescence microscopies. Chapter three describes the synthesis of series of molecular imaging agents based on two cancer targeting peptides (bombesin and RGDfK). Two types of NDIpeptide conjugates (Iodine-tagged NDI-Bombesin and Tryptophan-NDI-RGDfK) were designed and synthesized through EDC-coupling method. New compounds synthesized were characterised by mass spectroscopy and also HPLC. Fluorescence lifetime imaging microcopy and confocal laser scanning microscopy were utilised for investigating cellular behaviors (stability, fluorescence intensity and localization) of these molecular imaging probes. Chapter four describes the synthesis of amphiphilic conjugated thiophenes (dodecathiophene, denoted as T12). In this system, the thiophene backbone structure was chosen as a biocompatible coating for carbon nanotubes as simple molecular mechanics modeling suggested that it would be perfectly fitted to the curvature of SWNTs. T12 showed very good capability for debundling of SWNTs and forming corresponding solution dispersions of T12@SWNTs describes the potential of T12@SWNTs as a stable fluorescent bioimaging nano-probe for tracking cancer cells. Chapter five describes the successful filling of SWNTs with Cu2+ by radiochemistry methods (using 'hot' 64Cu ions anchored onto NaOAc) and also by a 'cold' optimised procedure for excess Cu(OAc)2. Filling with other metal ions was also tested, for example KReO4 and Zr(OAc)4. The filling experiments with Zr(OAc)4 in solution did not prove successful at normal pH but results were promising when pH was adjusted to ca. 2 by adding H2SO4. Any significant leakage of metal ions from open SWNTs was avoided by a simultaneous encapsulation of C70 molecules at the ends of SWNTs. Functionalisation of SWNTs by the supramolecular wrapping the surface of SWNTs in aqueous media with a naturally occurring glucan (β-1,3-1,4-Dglucan, denoted here as β-D-glucan) was also explored. Several boronic acid fluorophores were successfully synthesized and tested for the labeling of β-D-glucan @SWNTs by molecular recognition between boronic acids and this polysaccharide. Their cellular translocation behaviour and fluorescence properties were investigated by confocal fluorescence imaging and fluorescence lifetime imaging. Both methods show that localisation in sub-cellular (MCF-7 cells) regions and that the glucan coating significantly enhances the cell membrane translocation of SWNTs. Chapter six reports an efficient and economical strategy of supramolecular complex formulation of thermally reduced graphene. Naphthalene diimide (NDI) was used to form a stable and energy transfer complex which showed efficient quenching and significant red-shift of fluorescence of NDI when adsorbed onto graphene surfaces. The effect of thermally reducing annealing procedure to convert graphene oxides in graphene-nanoflake like materials was investigated. A new hybrid material (denoted here as NDI@TRG) synthesized was characterised by transmission electron microscopy (TEM), Raman spectroscopy, thermogravimetric analysis (TGA) and fluorescence microscopy in the dispersed phase Chapter seven contains full experimental details for the work described in this thesis. The Appendix contains details of the crystallographic data and supplementary information on cell imaging photos and fluorescence lifetime point decay data for SWNT nanocomposites.