GeneHuggers: In silico detection of transcriptional regulatory motifs using genomic sequence and DNA microarray data

Iacovoni, Jason Scott The Scripps Research Institute 2003 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Bioinformatics has become an integral component to post-genomic biological research. Understanding cancer biology requires the ability to both utilize high-throughput molecular methods such as gene expression profiling and the ability to interpret the large amounts of data associated with these methods. In order to use expression profiling data to understand transcriptional regulatory mechanisms, computational methods that integrate information from multiple sequence databases and methods that integrate results obtained from various sequence analysis algorithms are required. This thesis contains work related to obtaining and characterizing targets relevant to cellular transformation. The biological subject depended on the utilization of DNA microarrays for target identification. Work with such data lead to the desire to use the human genome sequence in conjunction with array results to predict transcriptional regulatory mechanisms responsible for the observed changes in gene expression. As a result, novel bioinformatics software called GeneHuggers was developed. Genomic analyses were performed with GeneHuggers and a combination of other freely available software to attempt to establish correlations between non-coding transcriptional regulatory sequence motifs and gene expression.

Theory, methods, and investigations in amyloid dynamics

Yonemoto, Isaac T The Scripps Research Institute 2009 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

This thesis is a review of the current state of experimentation in the field of amyloid dynamics. The introductory chapter outlines the basic foundations of protein chemistry and our understanding of the theory of amyloid peptide aggregation. The introduction also reviews a set of methods developed to address challenges stemming from these principles---and experiments conducted on two systems: amylin and prion which exemplify characterization of the biophysical properties of these peptides via the paradigms developed over the course of this research. Chapter 2 outlines the development of an expression system which was designed for the recombinant expression of amyloid peptides. In contrast to existent systems, this system operates by directing the peptides to bacterial inclusion bodies, which are the innate bacterial system to handle aggregating peptides and proteins. This system enables a general, high-yielding protocol for production of these peptides, and 11 peptides from 4 families of peptides were produced using this system. Chapter 3 is an experiment conducted on the aggregating peptide amylin, associated with the disease diabetes. This includes an extensive characterization of the pre-aggregated, soluble peptide and a broad profile of the kinetics of its aggregation across a very wide concentration span. The results from this study were also compared against the aggregation of biosynthetic precursors of amylin, suggesting that their aggregation is not a trigger for the pathophysiology of diabetes but rather a result of upstream defects. Chapter 4 is a research proposal outlining future directions in amyloid research that have been enabled by the development of the techniques in this thesis. Two specific aims are proposed---the first a study directly testing the hypothesis claimed in Chapter 3---that amyloid aggregation is directly influenced by the overall net charge of the peptide as well as charges found on amino acid side chains proximal to the amyloid core. The second is an in vitro evolution experiment wherein sequences of elevated amyloid propensity are enriched and mutated to generate highly amyloidogenic sequences.

Biochemical and physiological investigations of fatty acid amide hydrolase

Patricelli, Matthew Peter The Scripps Research Institute 2001 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

In chemical neurotransmission, the amplitude and duration of signals are often regulated by enzymatic inactivation of signaling substances. Fatty acid amides (FAAs) are endogenous modulators of pain perception and the sleep/wake cycle whose regulatory pathways <italic>in vivo</italic> remain an elusive subject for research. Investigations of a candidate FAA catabolic enzyme, fatty acid amide hydrolase (FAAH), are reported here. The pharmacological and biological properties of mice lacking a functional FAAH gene revealed that FAAH is the primary regulator of FAA signaling <italic>in vivo</italic> and suggest that this enzyme is an attractive pharmaceutical target for the treatment of pain and neuropyschiatric disorders. FAAH's catalytic nucleophile and base were identified as an unusual Ser/Lys catalytic dyad that has never been seen in mammals. This dyad is conserved among a large and evolutionarily diverse group of enzymes termed the amidase signature family. A residue in FAAH responsible for hydrophobic substrate contacts was identified and led to the generation of mutant enzymes with substantially altered substrate specificities. This result raised the possibility that FAAH and other amidase signature enzymes with novel and/or tailored specificities could be generated. This work validates the central importance of FAAH in regulating FAA activity <italic>in vivo </italic>, and provides a framework for understanding how FAAH and its substrates function in mammalian neuronal signaling.

Progress Toward Single-Molecule Nanopore DNA Sequencing

Chu, John Chung-Han The Scripps Research Institute 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Genetic information plays an increasingly important role in life sciences and medicine today. The ultimate goal of my Ph.D. project was to develop a nanopore-based DNA sequencing device that could drastically reduce the cost of acquiring genetic information. Chapter 1 is an overview of DNA sequencing methods, including the Sanger dideoxy-termination method and many emerging "next-generation" approaches. The potential of the nanopore DNA sequencing concept, as well as the requirements that need to be met for turning it into reality, are discussed at the end. Chapter 2 starts with an introduction to the biochemical and biophysical features of the most widely used protein (alphaHL) in the nanopore DNA sequencing field. It is followed by describing proof-of-principle experiments that suggest the feasibility of the nanopore DNA sequencing concept. More specifically, the pseudorotaxane design demonstrated that single-nucleotide variations in an intact ssDNA can be recognized, and the rotaxane design demonstrated that the natural DNA polymerase motor function can be employed to effect oligonucleotide translocation in unidirectional single-nucleotide steps. Chapter 3 begins with the discussion of a previously overlooked configuration dynamics associated with the alphaHL•DNA-PEGphos rotaxane. Such a dynamic feature of the rotaxane allows us to control and monitor oligonucleotide strand translocation in real-time. While DNA polymerase remains integral to most DNA sequencing methods, we came full circle by presenting preliminary data showing that the high spatial and temporal resolution of this rotaxane system provide the means for mechanistic studies of the DNA polymerase at the single-molecule level. Chapter 4 touches upon the importance of read-length in genome-scale sequencing, and the potential impact of the nanopore DNA sequencing method in this area of research.

Chemical Proteomic Profiling of Small Molecule-Protein Interactions

Wang, Yujia The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

A central goal in chemical biology and drug discovery is to develop small molecule probes to illuminate the functions of proteins in human diseases. The proportion of human proteome that can be targeted by chemical probes remains yet limited, underscoring the need of versatile strategies to expedite ligand discovery. We previously reported a chemical proteomic platform that utilized fully functionalized (photoreactive, clickable) fragments (FFFs) to broadly identify proteins that can interact with fragment compounds within native biological environments. Despite proven valuable in assessing the overall proteomic interaction potential of individual fragments, decerning selective small molecule-protein interactions from the non-specific background mainly driven by hydrophobic interaction remains a challenge. Careful manual review of complicated probe-vs-probe enrichment data is required for identifying authentic recognition events. In this thesis, we describe a general strategy to address this bottleneck in the form of next-generation sets of FFF probes consisting of physicochemically matched fragment pairs differing only in absolute stereochemistry. In Chapter 1, using a set of eight pairs of enantiomeric FFF probes—or “enantioprobes”—we expeditiously identified > 170 of stereochemistry-dependent small molecule-protein interactions for a diverse range of proteins including those lack chemical probes and traditionally considered as ‘undruggable’. Intriguingly, we showed that the interactions occur at functionally relevant sites on several proteins. In Chapter 2, to expand the scope and throughput of the pairwise chemical proteomic experiments, we developed a multiplexed quantitative proteomic approach capable of analyzing up to 10 separate population of cells treated under various conditions. We applied this strategy to explore the ligandable proteome in an AML cancer cell line using a set of structurally more complex enantiomeric FFF probes (elaborated enantioprobes). We showed that by elaborating simple fragment-like enantioprobes into drug-like small molecules with higher molecular weight, the stereoselective binding and chemotype selective enrichment of enantioprobes can be improved. Taken together, our findings demonstrate that incorporating chirality into FFF libraries provides a robust and streamlined method to map small molecule-protein interactions in cells. These stereoselective interactions can then form the basis for more advanced chemical probes targeting a broad range of proteins for basic and translational research.

Studies in Meroterpenoid Synthesis

Lockner, Jonathan W The Scripps Research Institute 2011 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The term "meroterpenoids" refers to natural products that contain both terpenoid and non-terpenoid components. This term encompasses a massive body of naturally occurring organic molecules, diverse in molecular constitution and biological activity. As methods for isolation and structure elucidation have grown in sophistication, so too have methods for the laboratory synthesis of many meroterpenoid targets. Described herein are various strategies and tactics examined for the pursuit of meroterpenoid (polyketide-terpenoid) natural products. Chapter 1 provides a literature review of the relevant meroterpenoid natural products that were the subject of study for this thesis research. With simplicissin as its focal point, Chapter 2 provides a review of various approaches that were examined in an aim to gain a foothold into this as-yet unassailable fungal meroterpenoid metabolite family. Chapter 3 describes the application of Minisci-type reactivity of boronic acids to practical radical cyclizations, and the successful implementation of this method for the preparation of ent-chromazonarol (and analogs thereof). This convergent approach to meroterpenoid synthesis has the potential to be applied to other classes beyond the quinone-sesquiterpenoids, including the tetraketide-sesquiterpenoids described in Chapters 1 and 2. This thesis work was carried out with the ultimate goal of devising a means for efficient synthetic access to this class of meroterpenoids. Along the way, a number of pitfalls were encountered, but valuable insight was gained which should enable further pursuit of this elusive natural product family.

Total syntheses of platensimycin and platencin

Tria, G. Scott The Scripps Research Institute 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The bacterial metabolites platensimycin and platencin are representatives of a novel class of natural products exhibiting unique architectures and potent antibacterial activities. Structurally similar, these molecules operate through inhibition of the lipid elongation/initiation enzymes FabF and FabH, found in the bacterial fatty acid synthesis pathway (FAS II). This inhibitory effect accounts for the broad-spectrum, Gram-positive antibacterial activities of both natural products against a number of key antibiotic resistant strains. The ability of platensimycin and platencin to inhibit such strains as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant Enterococcus faecium (VREF) has made them attractive targets to the synthetic organic community. Herein are presented our synthetic efforts toward both natural products, culminating in the first asymmetric total syntheses of both (--)-platensimycin and (--)-platencin. This thesis outlines the research efforts in their entirety and presents a complete discussion of the methodological means through which these practical syntheses were achieved. The work presented demonstrates the power of a number of key chemical transformations, including the hypervalent iodine-mediated de-aromatizing cyclization of an enantiopure substrate, the Cobalt-catalyzed asymmetric Diels-Alder reaction, and the one-pot reductive rearrangement of [3.2.1] bicyclic ketones to [2.2.2] bicyclic olefins.

THE LIGHT SENSING REACTION OF PHOTOACTIVE YELLOW PROTEIN

GENICK, ULRICH KARL THE SCRIPPS RESEARCH INSTITUTE 1999 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Light is the original source of energy for almost all current forms of life. Most organisms also use light to determine their daily and annual rhythms. Photoreceptors form the interface between the physical light environment and the biological signaling processes in a cell. This location at the intersection of physics and biology poses problems that are unique among sensors. The light signal is very short lived and is destroyed immediately when the receptor interacts with the signal. The response to the capture of a photon therefore has to be extremely rapid. Yet at the same time the encounter has to result in a structural signal that can be passed on to a signal transduction cascade that employs regular biochemical mechanisms. To understand how a photoreceptor is able to accomplish those two tasks we have determined the structure of three structures (ground state, early intermediate, signaling intermediate) that occur during the light cycle of Photoactive Yellow Protein (PYP) using ultra high-resolution, freeze trapping and true time-resolved crystallographic techniques. The three structures show that PYP first uses the captured photon to cause <italic>trans</italic>-to-<italic> cis</italic> isomerization of its 4-hydroxy cinnamic acid chromophore in an extremely rapid step involving only minimal atomic movements. In the second step, this change in the chromophore configuration is recognized by the protein, causing rearrangements in the active site region that propagate to the protein surface where they can be recognized by a downstream signaling partner. Based on these results we proposed a general model for protein light cycles and tested this model through the generation of site-directed mutations of key active site residues.

Insights into quorum sensing from a chemical perspective

Lowery, Colin A The Scripps Research Institute 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Quorum sensing (QS) is a mechanism of communication used by bacteria to coordinate gene expression via the exchange of diffusible chemical signals, called autoinducers. The synchronization of gene expression allows bacterial populations to engage in behaviors that are beneficial to groups of bacteria rather than individual cells, and include regulation of bioluminescence, formation of biofilms, and secretion of virulence factors. Illustrated in the latter two examples are the dire consequences on human health exerted by a coordinated group of bacteria, and as a result, QS has become an area of intense research both to understand the molecular mechanisms of this process and to mitigate the deleterious effects of a unified bacterial population. This thesis describes efforts to modulate QS through the design and synthesis of analogs of natural autoinducers, as well as the antibacterial activity of an autoinducer-derived natural product. The thesis begins with an overview of the development of autoinducer analogs, with a focus on the methodologies used and the structural relationships between the agonists or antagonists discovered and the natural signals. Chapter 2 details work towards the modulation of QS in the bioluminescent marine bacterium Vibrio harveyi. These studies were directed at finding agonists or antagonists of AI-2-based QS, a proposed interspecies signaling system, and several agonists were uncovered. Building on these findings, Chapter 3 describes the discovery of potent antagonists of the AI-2 QS of the human pathogen Salmonella typhimurium. The activity of these analogs is further elaborated in Chapter 4, where they are compared directly to the current "gold standard" of AI-2 QS inhibition and were found to be more potent antagonists of QS and less toxic towards mammalian cells. The thesis concludes with Chapter 5, which is a departure from the development of AI-2 modulators into the description of the antibacterial activity of a natural product formed from an autoinducer of the human pathogen Pseudomonas aeruginosa. In this context, it is possible to envision a scenario in which QS not only plays a role in the coordination of bacterial behavior and function, but also in the interspecies warfare that occurs in environmental settings.

Expanding the genetic lexicon

Young, Travis Scott The Scripps Research Institute 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

In vivo unnatural amino acid methodology was developed a little over a decade ago and allows the expression of proteins with amino acids beyond the canonical 20. Since its inception, unnatural amino acids have had major impacts on protein biophysical studies, broadened our understanding of immunological self-tolerance and disease etiology, and given us insights into evolution, all in ways that could not have been accomplished with a standard 20 amino acid repertoire. The breadth of unnatural amino acid applications is only surpassed by the enormity of this methodology's potential. This may be no more evident than looking at who is using unnatural amino acids. No longer is the methodology constrained to the benches of molecular biologists. Researchers from all corners of science, from virologists to physical chemists, are employing unnatural amino acids in their studies. However, the forward momentum of this technology can only be sustained with stable and efficient platforms for its use. To this end, I have focused on the expansion of unnatural amino acids to new organisms, the optimization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs in the E. coli expression system, and the repurposing of existing aminoacyl-tRNA synthetases as a rapid method for expanding the genetic lexicon. Using these new tools, I explore the evolutionary implications of an expanded code to ultimately ask the question: can unnatural amino acids provide solutions to evolutionary problems that a canonical code cannot?.