Phase Locked Loop and Modulo Games, The Dogma Loops; and “Finding Ibrida”

This dissertation consists of two independent musical compositions and an article detailing the process of the design and assembly of an electric guitar with particular emphasis on the carefully curated suite of embedded effects.

The first piece, 'Phase Locked Loop and Modulo Games' is scored for electric guitar and a single echo of equal volume less than a beat away. One could think of the piece as a 15 minute canon at the unison at the dotted eighth note (or at times the quarter or triplet-quarter), however the compositional motivation is more about weaving a composite texture between the guitar and its echo that is, while in theory extremely contrapuntal, in actuality is simply a single [superhuman] melodic line.

The second piece, 'The Dogma Loops' picks up a few compositional threads left by ‘Phase Locked Loop’ and weaves them into an entirely new tapestry. 'Phase Locked Loop' is motivated by the creation of a complex musical composite that is for the most part electronically transparent. 'The Dogma Loops' questions that same notion of composite electronic complexity by essentially asking a question: "what are the inputs to an interactive electronic system that create the most complex outputs via the simplest musical means possible?"

'The Dogma Loops' is scored for Electric Guitar (doubling on Ukulele), Violin and Violoncello. All of the principal instruments require an electronic pickup (except the Uke). The work is in three sections played attacca; [Automation Games], [Point of Origin] and [Cloning Vectors].

The third and final component of the document is the article 'Finding Ibrida.' This article details the process of the design and assembly of an electric guitar with integrated effects, while also providing the deeper context (conceptual and technical) which motivated the efforts and informed the challenges to hybridize the various technologies (tubes, transistors, digital effects and a microcontroller subsystem). The project was motivated by a desire for rigorous technical and hands-on engagement with analog signal processing as applied to the electric guitar. ‘Finding Ibrida’ explores sound, some myths and lore of guitar tech and the history of electric guitar distortion and its culture of sonic exploration.

Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. RESULTS: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. CONCLUSIONS: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Molecular cloning and expression of the cDNA for the hamster alpha 1-adrenergic receptor.

The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.

An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

Cloning of the cDNA for the human beta 1-adrenergic receptor.

Screening of a human placenta lambda gt11 library has led to the isolation of the cDNA for the human beta 1-adrenergic receptor (beta 1AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human beta 2-adrenergic receptor (beta 2AR). The 2.4-kilobase cDNA for the human beta 1AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian beta AR but only 54% homologous with the human beta 2AR. This suggests that the avian gene encoding beta AR and the human gene encoding beta 1AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with beta 1AR binding. This pattern is quite distinct from the pattern obtained when the beta 2AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical beta 1AR specificity. This contrasts with the typical beta 2 subtype specificity observed when the human beta 2AR cDNA is expressed in this system. Mammalian beta 1AR and beta 2AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.