Throughout this thesis work, various catalysts were investigated with combinational approach to develop highly active and selective novel catalysts for direct epoxidation of propylene to PO using molecular oxygen. The promoted and un-promoted silver (Ag), copper (Cu), ruthenium (Ru), manganese (Mn) mono and multimetallic catalytic systems over different silica supports were prepared via sol-gel method and incipient wetness method. In addition to support effect, the effects of different promoters on the catalytic performances of these catalyst candidates were investigated. The study showed that commercial silica (c-SiO2) is the most effective support when compared to silica (SiO2) and silica synthesized with templete (t-SiO2). Among bimetallic catalytic systems containing Ag, Ru, Mn and Cu metals, c-SiO2 supported Cu-Ru catalyst was determined as the most active catalytic system. In addition, the most effective v catalyst and promoter in the epoxidation reaction was determined as NaCI promoted Cu-Ru catalyst supported over c-SiO2 with 35.98% selectivity&amp / 9.55% conversion (3.44% yield) at 3000C and 0.5 feed gas ratio (C3H6/O2).. In the study, the selected catalysts showed low and high PO productivity were also investigated by characterization techniques such as XRD, XPS, BET and FTIR.It was inferred from characterization tests that bimetallic systems reveal a synergistic behavior by exposing more active sites on the silica support material with respect to their monometallic counterparts. Besides, NaCl catalytic promoter has a strong interaction particularly with the Cu sites on the Cu/Ru/SiO2 catalyst surface, altering the electronic structure of Cu sites that favors to PO production.

Preclinical methods for the identification and characterization of molecules for development into new cancer drugs were investigated. Based on repurposing, i.e. the exploration of currently prescribed drugs for new indications, and as a result of a new high throughput screening (HTS) approach, the benzimidazoles benomyl and carbendazim, the alkaloid cryptolepine and the acridine acriflavine were found interesting to characterize using these methods. In mice the benzimidazoles inhibited 3H-thymidine incorporation in tissues with high cell renewal, with benomyl being more active than carbendazim.  They were rapidly absorbed with highest amounts seen in the liver, kidneys and gastro-intestinal lumen as evidenced from distribution of 14C-labeled drugs. In human tumour cell lines, the benzimidazoles showed a similar activity pattern but benomyl was more potent. This was true also in tumour cells from patients but carbendazim was relatively more active against solid tumours. Analyses of drug activity cross-resistance patterns and of drug activity – gene expression correlations in a cell line panel suggested multiple mechanisms of action for the benzimidazoles. Cryptolepine was widely distributed to tissues in vivo in the mice. It was more potent than the benzimidazoles in tumour cells, with highest activity in haematological malignancies but some patient samples of breast, colon and non small-cell lung cancer were sensitive. Cross-resistance analysis indicated cryptolepine to be a topoisomerase II inhibitor whereas drug activity – gene expression correlations suggested additional mechanisms of action. HTS on 2 000 molecules in colon cancer cell lines and normal cells identified acriflavine as a hit molecule, subsequently shown to have unprecedented activity against colorectal cancer tumour cells in patient tumour samples. Connectivity map analysis, based on drug induced gene expression perturbation patterns in a tumour cell line, indicated acriflavine to be a topoisomerase inhibitor, subsequently confirmed in a plasmid relaxation assay. In conclusion, repurposing of drugs and HTS using stringent activity criteria followed by preclinical characterization might contribute to more efficient development of new cancer drugs.

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.   In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.   The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors. / QC 20101102

<p>The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic DNA replication initiates asynchronously from hundreds to tens of thousands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time determined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replication has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress. </p><p>We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone modifications and nucleosome positioning, are an attractive candidate for DNA replication control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The <italic>S. cerevisiae</italic> ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Next, we describe the <italic>D. melanogaster</italic> replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. </p><p>Taken together our data and analyses imply that the chromatin contains sufficient information to direct the DNA replication program.</p> / Dissertation

A major driving force for development of new catalyst systems is the need for more efficient synthesis of chemical compounds essential to modern life. Catalysts having superior performance offer significant environmental and economic advantages, but their discovery is not trivial. Well-defined, homogeneous catalysts can offer unparalleled understanding of ligand effects, which proves invaluable in directing redesign strategies. This thesis work focuses on the design of ruthenium complexes for applications in dinitrogen activation and olefin metathesis. The complexes developed create new directions in small-molecule activation and asymmetric catalysis by late-metal complexes. Also examined are the dual challenges, ubiquitous in catalysis, of adequate interrogation of catalyst structure and performance. Insight into both is essential to enable correlation of ligand properties with catalyst activity and/or selectivity. Improved methods for accelerated assessment of catalyst performance are described, which expand high-throughput catalyst screening to encompass parallel acquisition of kinetic data. A final aspect focuses on direct examination of metal complexes, both as isolated species, and under catalytic conditions. Applications of charge-transfer MALDI mass spectrometry to structural elucidation in organometallic chemistry is described, and the technique is employed to gain insight into catalyst decomposition pathways under operating conditions.

Code-Division Multiple-Access (CDMA) is one of the leading digital wireless communication methods currently employed throughout the world. Third generation (3G) and future wireless CDMA systems are required to provide services to a large number of users where each user sends data burst only occasionally. The preferred approach is packet based CDMA so that many users share the same physical channel simultaneously. In CDMA, each user is assigned a pseudo-random (PN) code sequence. PN codephase synchronization between received signals and a locally generated replica by the receiver is one of the fundamental requirements for successful implementation of any CDMA technique. The customary approach is to start each CDMA packet with a synchronization preamble which consists of PN code without data modulation. Packets with preambles impose overheads for communications in CDMA systems especially for short packets such as mouse-clicks or ATM packets of a few hundred bits. Thus, it becomes desirable to perform PN codephase synchronization using the information-bearing signal without a preamble. This work uses a segmented matched filter (SMF) which is capable of acquiring PN codephase in the presence of data modulation. Hence the preamble can be eliminated, reducing the system overhead. Filter segmentation is also shown to increase the tolerance to Doppler shift and local carrier frequency offset. <p>Computer simulations in MATLAB® were carried out to determine various performance measures of the acquisition system. Substantial improvement in probability of correct codephase detection in the presence of multiple-access interference and data modulation is obtained by accumulating matched filter samples over several code cycles prior to making the codephase decision. Correct detection probabilities exceeding 99% are indicated from simulations with 25 co-users and 10 kHz carrier frequency offset or Doppler shift by accumulating five or more PN code cycles, using maximum selection detection criterion. Analysis and simulation also shows that cyclic accumulation can improve packet throughput by 50% and by as much as 100% under conditions of high offered traffic and Doppler shift for both fixed capacity and infinite capacity systems.

This thesis describes the development of microfluidic (MF) platforms for the study of biological and chemical processes. In particular the thesis is divided into two distinct parts: (i) development of a MF methodology to generate tunable cell-laden microenvironments for detailed studies of cell behavior, and (ii) the design and fabrication of MF reactors for studies of chemical reactions. First, this thesis presented the generation of biopolymer microenvironments for cell studies. In the first project we demonstrated a high-throughput MF system for generating cell-laden agarose microgels with a controllable ratio of two different types of cells. The MF co-encapsulation system was shown to be a robust method for identifying autocrine and/or paracrine dependence of specific cell subpopulations. In the second project we studied the effect of the mechanical properties on the behavior of acute myeloid leukemia (AML2) cancer cells. Cell-laden macroscopic agarose gels were prepared at varying agarose concentrations. A modest range of the elastic modulus of the agarose gels were achieved, ranging from 0.62 kPa to 20.21 kPa at room temperature. We observed a pronounced decrease in cell proliferation in stiffer gels when compared to the gels with lower elastic moduli. The second part of the thesis focuses on the development of MF platforms for studying chemical reactions. In the third project presented in this thesis, we exploited the temperature dependent solubility of CO2 in order to: (i) study the temperature mediated CO2 transfer between the gas and the various liquid phases on short time scales, and (ii) to generate bubbles with a dense layer of colloid particles (armoured bubbles). The fourth project involved the fabrication of a multi-modal MF device with integrated analytical probes. The MF device comprised a pH, temperature, and ATR-FTIR probes for in-situ analysis of chemical reactions in real-time. Furthermore, the MF reactor featured a temperature controlled feedback system capable of maintaining on-chip temperatures at flow rates up to 50 mL/hr.

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.