Cadherins are a family of cell-surface proteins mediating adhesion that are important in development and maintenance of tissues. The family is defined by the repeating cadherin domain (EC) in their extracellular region, but they are diverse in terms of protein size, architecture and cellular function. The best-understood subfamily is the type I classical cadherins, which are found in vertebrates and have five EC domains. Among the five different type I classical cadherins, the binding interactions are highly specific in their homo- and heterophilic binding affinities, though their sequences are very similar. As previously shown, E- and N-cadherins, two prototypic members of the subfamily, differ in their homophilic K_D by about an order of magnitude, while their heterophilic affinity is intermediate. To examine the source of the binding affinity differences among type I cadherins, we used crystal structures, analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and electron paramagnetic resonance (EPR) studies. Phylogenetic analysis and binding affinity behavior show that the type I cadherins can be further divided into two subgroups, with E- and N-cadherin representing each. In addition to the affinity differences in their wild-type binding through the strand-swapped interface, a second interface also shows an affinity difference between E- and N-cadherin. This X-dimer interface, which is a weakly binding kinetic intermediate in E-cadherin, has a much stronger affinity in N-cadherin: nearly as strong as N-cadherin wild-type binding. In the swapped and X-dimer interactions of E- and N-cadherin, differences in hydrophobic surface area can mostly account for the affinity difference. However, several mutants of N-cadherin have a K_D an order of magnitude stronger even than the wild-type N-cadherin. In these mutants, the source of the strong affinity seems to be entropic stabilization through an equilibrium between multiple conformations with similar energies. We thus have a molecular-level understanding of vertebrate classical cadherins, with a detailed understanding of their adhesive mechanism and their binding affinity determinants. However, the adhesive mechanisms of cadherins from invertebrates, which are structurally divergent yet function in similar roles, remain unknown. We present crystal structures of the predicted N-terminal region of Drosophila N-cadherin (DN-cadherin). Of the 16 total predicted EC domains, we have crystallized the EC1-3 and EC1-4 segments. While the linker regions for the EC1-EC2 and EC3-EC4 pairs display binding of three Ca^2+ ions similar to that in vertebrate cadherins, domains EC2 and EC3 are joined in a bent orientation by a novel, previously uncharacterized Ca^2+-free linker. Based on sequence analysis of the further ECs of DN-cadherin, we predict another such Ca^2+-free linker between EC7 and EC8. Biophysical analysis demonstrates that a construct containing the first nine predicted EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins. Intriguingly, this segment contains both the crystallized and predicted Ca^2+-free linkers, suggesting a complex binding interface. Sequence analysis of the cadherin family reveals that similar Ca^2+-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins. In cases of long cadherins, there are frequently multiple Ca^2+-free linkers in a single protein chain. It thus appears that a combination of calcium-binding and calcium-free linkers can allow cadherins to form three-dimensional arrangements that are more complex than the extended, calcium-rigidified structures in classical cadherins. Discovery of the Ca^2+-free linker, together with the differing numbers and arrangements of ECs and other domain types, implies that the cadherin superfamily is more structurally diverse than previously thought. Because little is known about the function and even less about the structure of the majority of the superfamily, studying the linear architecture (i.e. the precise sequence of ECs and the characteristics of the interdomain linkers) at the scale of the superfamily would give significant new insights on the structure and function of less-understood cadherins. With this motivation, we have constructed a cadherin database with relevant information on two different scales: the protein and the domain. On the whole protein level, we represent the architecture of each cadherin by recording the arrangement of ECs, different linker types, and other (non-EC) domain types in the protein. On the individual EC level, based on the sequence, we record the domain characteristics that give rise to the different structural features at the protein level. We have annotated over 9,600 proteins from 560 organisms, containing over 69,000 ECs; and built an online interface to search and access this information. Our aim is to provide a tool for understanding the protein architecture, function, and relationships among cadherins, a structurally diverse protein family. Together, these studies examine the relationships between sequence, structure and function of cadherins at different scales. In the classical cadherin study, small changes of one or two residues can dramatically alter the dimer conformations and thus lead to large differences in binding affinity between highly related cadherins, or between wild-type and mutant proteins. These seemingly small mutations can result in even higher binding affinity with the effect of entropic stabilization by multiple conformations. In DN-cadherin, the absence of certain calcium-binding motifs in adjacent ECs leads to a new linker type and a new interdomain orientation. This, in turn, has great implications in the global shape, and possibly the binding mechanism of the protein. The cadherin database aims to provide information at different structural levels in order to allow users to draw connections between primary sequence, domain structure and protein architecture, to ultimately learn about protein function.

Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171

*Please refer to dissertation for diagrams. / In addition, a precursor of tetraphenylene-based monodentate ligand ( S,S)-114 was also prepared, and the structures of five compounds, namely 89, 124, 125, 133 and 137 were examined by X-ray crystallographic analysis. These structural determinations were relevant in establishing regiochemistry and absolute stereochemistry.* / In the synthesis of enantiopure (S,R,S)-48 and (R,S,R)-48, two routes were successfully employed. One way was to follow the same pathway for the synthesis of racemic 48 by using enantiopure (S,S)- and (R,R)-1,8,9,16-teramethoxytetraphenylenes [(S,S)-89 and (R,R)- 89] as staring materials. Another way was by direct resolution of racemic 48 via derivatization into its two diastereomeric hexakis-(S)-camphorsulfonates 135 and 136. / In the synthesis of racemic 48, 3-nitrophenol (115) was employed as the starting material which upon a series of standard reactions provided 2,2'-diiodo-1,1'-biphenyl (119). Through sequential lithium-iodine exchange and Cu(II)-mediated oxidative cyclocoupling, 119 was converted to 1,8,9,16-tetramethoxytetraphenylene (89) . The key intermediate 1,8-dihydroxy-9,16-dimethoxytetraphenylene (87) was obtained by partial demethylation of 89. This intermediate was transformed to 1,4,5,8-tetrahydroxy-9,16-dimethoxytetraphenylene (126) by a quinone-hydroquinone strategy. Demethylation of 126 furnished the target compound 48. / This thesis describes the synthesis of 1,4,5,8,9,16-hexahydroxytetraphenylene (48)* in its racemic and enantiopure (S,R,S) and ( R,S,R) forms. Some essential background and previous works in this area are presented in the first chapter. / Wu Anhui. / "August 2006." / Adviser: Henry N. C. Wong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1649. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 80-85). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

No description available.

Cellular development and function necessitate precise patterns of gene expression. Control of gene expression is in part orchestrated by a class of remote regulatory elements, termed enhancers, which are brought into contact with promoters via DNA looping. Enhancers typically contain clusters of transcription factor binding sites, and TF recruitment to them is thought to play a key role in transcriptional control. In this thesis I have addressed two issues regarding gene regulation by enhancers. First, with recent genome-wide enhancer mapping, it is becoming increasingly apparent that genes are commonly regulated by multiple enhancers in the same cell type. How a gene’s regulatory information is encoded across multiple enhancers, however, is still not fully understood. Second, numerous recent studies have found that enhancers are enriched for expression-modulating and disease-associated genetic variants. However, understanding and predicting the effects of enhancer variants remains a major challenge. I focussed on a human lymphoblastoid cell line (LCL), GM12878, for which ChIP-Seq data are available for 52 different TFs from the ENCODE project. Significantly, Promoter Capture Hi-C data for the same LCL are available, making it possible to link enhancers to target genes globally. In the first part of the thesis, I investigated how gene regulatory information is encoded across enhancers. Specifically, I asked whether a gene tends to use multiple enhancers to bring the same or distinct regulatory information. I found that there was a general trend towards a “shadow” enhancer architecture, whereby similar combinations of TFs were recruited to multiple enhancers. However, numerous examples of “integrating” enhancers were observed, where the same gene showed large variation in TF binding across enhancers. Distinct groups of TFs were associated with these contrasting models of TF enhancer binding. To investigate the functional effects of variation at enhancers, I additionally took advantage of a panel of LCLs derived from 359 individuals, which have been genotyped by the 1000 Genomes Project, and for which RNA-Seq data are publically available. I used TF binding models to computationally predict variants impacting TF binding, and tested the association of these variants with the expression of the target genes they contact based on Promoter Capture Hi-C. Compared to the standard eQTL calling approach, this offers increased sensitivity as only variants physically contacting the promoter and predicted to impact TF binding are tested. Using this approach, I discovered a set of predicted TF-binding affinity variants at distal regions that associate with gene expression. Interestingly, a large proportion of these binding variants fall at the promoters of other genes. This finding suggests that some promoters may be able to act in an enhancer-like manner via long-range interactions, consistent with very recent findings from alternative approaches.

No description available.

Biocompatible polymers are used exhaustively within the biomedical arena, demonstrating a mechanical and chemical diversity that few other materials possess. As polymer technologies evolves to cater for new medical demands, even the most niche biomedical application becomes an achievable reality. However, the discovery of new polymers is hindered by the complexity and intricacy in which the biological milieu interacts with a new substrate, reducing the ability to predict the appropriateness of a certain polymer for a specific application. This drawback can be countered by the high-throughput evaluation of large numbers of chemically diverse polymer candidates. In this thesis, the use of polymer microarrays is invoked to address two separate medically-relevant issues: the control of inflammation, and the improvement of cancer screening. In addition, I provide details of how polymer microarray techniques and technology can be employed to expand the repertoire of biomaterials research. Mitochondrial DNA (mtDNA) is an alarm molecule that contributes to the cytokine storm observed during severe tissue injury. An application where control of this systemic inflammation is achieved through scavenging of mtDNA by a polymer was proposed. Primary screening highlighted that 166 out of the 380 polymers evaluated bound to blood cells, making them unsuitable for a blood-based application. The remaining 214 blood-compatible polymers were cross-examined for mtDNA binding. Through polymer microarray and subsequent scale-up of promising candidates, a poly(methoxyethyl methacrylate-co-di(ethylamino)ethyl acrylate-co-methoxyethyl acrylate) was found to have a remarkable ability to scavenge mtDNA. Removal of cell-free mtDNA using this polymer is proposed to remove a key trigger of systemic inflammation. Cervical cancer screening includes the cytological evaluation of patient material for developed or developing abnormalities. An application was sought that would enrich for cancerous/pre-cancerous cells and improve upon current standards for detection. Four cancerous cervical cell lines (HeLa, CaSki, SiHa, and C33a) and four precancerous cell lines (W12E, W12G, W12GPX, and W12GPXY) were interrogated to identify polymers with consistent binding that may improve routine cytological evaluation. A short-list of 24 polymers was assembled, and cells from liquid based cytology samples from healthy patient were spiked with DiI-labelled cancerous/precancerous cells and the short-listed polymers were re-evaluated for preferential binding. An enrichment of abnormal cervical cells was observed with three polymers, which could form the foundation for improved screening resources. Inkjet printing can be a useful tool in developing patterned substrates, such as polymer microarrays. A piezoelectric drop-on-demand printer was used to explore the methods in which these can be fabricated. A wettability assay using picolitre volumes was developed and used to characterise O2 plasma treatment of glass slides. Additionally, the printing of a cell-binding polymer using this approach enabled the decoration of cells with precise spatial resolution.

Caput Mortuum is a visual representation of my own spiritual quest for enlightenment using alchemy. Ancient alchemists sought perfection in all things and visualized a personal spiritual hierarchy that resided within all physical matter. The lowest tier of this scale represents the dull and lifeless material while the highest could touch the Divine. Reshaping the material world revealed the ordinary item's latent potential, aiding in its own transformation as well as the alchemist's into more perfect beings. Inspired by this idea, I seek to bring ultimate perfection to every piece I create. By manipulating and altering books and book forms I replicate the physical work alchemists performed, each time changing myself with the book, elevating our spiritual beings in order to bring perfection from within.

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and the mechanistic pathway of covalent binding to nucleophilic thiols. The BQ binding by Cys34 on human serum albumin was studied, and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudo-first order rate constants (k) of methyl-, t-butyl-, and chlorine-substituted BQD reactions with NBT, whereas electron pair resonance (EPR) studies were performed to investigate the possible free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine substituted (activated) BQD to be more reactive toward NBT, than the methyl and t-butyl-substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA of NBT onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQD stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to allergenic potency. Hammett and Taft (HT) constants were then used to estimate the influence of these substituents on chemical reactivity. HT values demonstrated chlorine substituted BQD to be more reactive than methyl substituted BQD. BQ and BQD dermal allergenicity, as evaluated in the murine local lymph node assay, (LLNA) was consistent with that predicted by reactivity and HT parameters. These results demonstrate the effect of substituents on BQ reactivity and dermal allergic sensitization, and suggest the potential utility of chemical reactivity data and HT values for electrophilic allergen identification and potency ranking.

Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses? In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.