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Ribozymes are catalytic RNA molecules capable of performing functions normally attributed to proteins. The hairpin ribozyme is derived from the (-) strand of the satellite RNA of Tobacco Ringspot Virus, where it functions in processing rolling circle transcription intermediates. The hairpin ribozyme catalyzes the breaking of a phosphodiester bond to form a 2'-3' cyclic phosphate and a 5' OH on the product termini. RNA substrates are recognized through Watson Crick base pairs. In theory, ribozymes are able to cleave any RNA that forms the correct secondary structure. Therefore, ribozymes have been designed to recognize specific targets through base pair interactions with the substrate recognition sequence of the ribozyme. This feature of catalytic RNAs gives them endless potential as antisense reagents. While tremendous effort has gone into elucidating the kinetic mechanism of ribozymes in vitro, very few studies have addressed ribozyme function in the intracellular environment. Previous studies have had varying success. And while in some cases ribozymes have clearly reduced gene expression, the experiments were not quantitative and did not provide any information regarding the kinetic pathway of catalysis in vivo. Improved understanding of intracellular cleavage reactions requires the development of a system that can directly measure cleavage rates in vivo. Utilizing a self-cleaving ribozyme cassette inserted into the yeast PGK1 gene we have developed a system to detect ribozyme cleavage products and directly measure the cleavage rates of the hairpin ribozyme in yeast. Furthermore, we have performed controls confirming detected cleavage activity is reflective of intracellular catalysis. As ribozyme activity requires the formation of a catalytically active structure, cleavage can act as a monitor for the assembly of a functional molecule. We have used this system to address the effect of helix stability on intracellular hairpin ribozyme activity. The results of these experiments have important implications for the design of antisense ribozymes. Furthermore, catalysis by small RNAs in vivo serves as a model system for more complex RNA catalyzed reactions that are implicated in mRNA processing and translation.

Analyses of 11 new erbB transducing viruses had previously correlated differences in disease potential with deletions or truncations in sequences encoding the carboxy-terminal domain of the chicken EGF receptor. erbB sequences of two of these viruses, one inducing only erythroblastosis (AEV-5005) and the other inducing only angiosarcoma (AAV-5005), were molecularly cloned. Sequence analysis confirmed the presence of an internal deletion in AAV-5005. The deletion was found to be inframe and to have removed 177 nucleotides. The deleted sequence had encoded a region between the kinase domain and the autophosphorylation sites of the EGF receptor. To establish that the deletion was responsible for the change in disease potential two recombinant viruses were constructed. One recombinant virus (srE) contained erbB sequences from AEV-5005. The other recombinant virus (srE/A) was identical to srE except that a restriction fragment from AAV-5005 which contained the deletion was substituted for the homologous fragment of AEV-5005. The srE virus induced only erythroblastosis, while the srE/A virus induced angiosarcoma as well as erythroblastosis. This demonstrated that the deletion was sufficient to induce angiosarcoma. Metabolic labelling did not reveal any difference in the expression or processing of these proteins and both became phosphorylated in an in vitro kinase assay. The biochemical basis for the difference in disease potential of these two related proteins remains to be determined.

Sickle cell disease is caused by the intracellular polymerization of human hemoglobin containing a mutation of a glutamic acid to a valine residue at the sixth position of the β chain. This substitution of a hydrophobic residue for a hydrophilic residue greatly decreases the solubility of the hemoglobin tetramer, promoting the formation of ordered fibers in deoxygenated red blood cells. These fibers are composed of double strands of hemoglobin tetramers, which effectively bury the valine side chain in a hydrophobic pocket, eliminating its unfavorable interaction with solvent. Inhibition of fiber formation would greatly alleviate the symptoms of sickle cell disease; therefore, the elucidation of the structure of the fiber is critical to developing treatments for the disease. In this thesis, an analysis of the crystal structures of the double strand component of the fibers formed by HbS and other site-directed mutants displaying altered sickling properties is undertaken. Structure of HbS at high resolution: The structure of HbS was solved at 2.05 Å resolution. This high-resolution analysis produced significant improvements in the previous 3.0 Å model. In particular, the accurate positioning of side chains, and the placement of more than 500 solvent molecules was achieved. Some side chains in the physiologically relevant contacts were moved by as much as 3.5 Å away from their locations in the lower resolution model. The structure also demonstrates that well ordered water molecules are located in both the axial and lateral contacts, some of which may be exploited in the design of sickling inhibitors. Structures of Hbβ6L and Hbβ6W: The crystal structures of two human hemoglobins with mutations at the β6 position that display altered sickling behavior were solved and refined at high resolution. Both mutants were crystallized under conditions similar to those used with HbS. Hbβ6L showed a greater tendency to polymerize, and displayed reduced delay times in aggregation assays. The refined structure was very similar to that of HbS, with changes confined mostly to the lateral contacts. However, the packing of Leu β6 in the linear crystalline double strands is sub-optimal. This has important ramifications for the differences between the crystalline double strands and those within the physiological HbS fiber. Hbβ6W, on the other hand, showed a reduced tendency to polymerize and shorter delay times in polymerization assays. The protein crystallized in a different space group from the other two mutants (HbS and Hbβ6L) under the same crystallization conditions, and formed fundamentally different double strands in the crystals. This structure demonstrates conserved interactions at the axial contacts, but very different interactions at the lateral contacts. The alternate double strand formed by this protein in the crystal may be useful as a model for tetramer interactions within the physiological fiber, or as a structural model for heterogeneous nucleation. The structures of each of these mutants help explain their altered polymerization behavior.

The two non-allelic preproinsulin genes of the mouse have been cloned and their nucleotide sequences determined. The mouse preproinsulin I gene, like its rat counterpart, has only one intron. Homology between the two mouse genes extends in the 5' direction to about position -500. Homology 3' of the coding sequence terminates shortly after the polyadenylation signal with a dA rich region found in gene I. The coding sequences of the two genes have been compared. The deduced amino acid sequences of the mature hormones are identical to the published protein sequences and to the corresponding sequences of rat insulins I and II. The prepeptides of mouse insulin I and II differ at six positions. However, they maintain hydrophobic cores that are required for transport of the nacent peptide across microsomal membranes. The B-peptide of mouse insulin I differs from insulin II at two positions: at position B9 a proline has replaced a serine, and at position B29 a lysine has replaced a methionine, compared to the sequence of insulin II. The A-peptides of the two hormones are identical. The C-peptide of mouse proinsulin I has a deletion eliminating amino acids C17 (Gly) and C18 (Ala) compared to the sequence of proinsulin II. The presence of this deletion in mature RNA was confirmed through an S1 nuclease assay. The transcriptional start sites for the preproinsulin genes were determined with S1 and Mung Bean nuclease assays, and with a primer extension assay. The data indicate that transcription of the mouse preproinsulin genes starts 6 bp 5' of the site reported for the rat II gene. Single-stranded DNA probes were used to determine the structure of the 3' ends of the preproinsulin mRNAs. Hybridization conditions were used which only allowed each probe to detect its cognate mRNA. Digestion of the resulting DNA-RNA duplex molecules with S1 nuclease followed by gel electrophoresis demonstrated that transcription of mouse preproinsulin I mRNA terminates 18 bases after the polyadenylation signal. Transcription of preproinsulin II mRNA terminates 43 bases after the polyadenylation signal, thus extending 25 bases past the last point of homology between the two genes. The 3' end-specific probes were used in experiments designed to determine the ratio of preproinsulin I and II mRNA in pancreatic extracts of normal, fasted and fasted and refed mice. In all cases the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. These results were extended to include an analysis of preproinsulin mRNA from freshly isolated islets and islets incubated for 48 hours in the presence of 2.8 mM or 16.7 mM glucose. With both high and low glucose concentrations, the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. The mouse islets were also incubated with 3H-leucine and the ratio of insulin I and II determined after fractionation by HPLC. Unlike the mRNA results, the level of insulin II, within the islets and secreted into the media, exceeded insulin I by about 2:1 under all conditions. The available preproinsulin prepeptide amino acid sequences have been compared. The sequence of mouse I prepeptide differs from most other insulin prepeptides at amino acid position 4. At that position a tryptophan residue that is conserved in most insulin prepeptides has been replaced by a leucine in the mouse I prepeptide. This change causes a shift in the hydropathy profile in that region of the mouse insulin I prepeptide making it more hydrophobic. Every other insulin prepeptide is relatively hydrophilic at that position. This difference is postulated to interfere with signal recognition particle mediated regulation of translation and/or transport of nacent mouse preproinsulin I to microsomal membranes, and nay account for the discordant mRNA-peptide ratios. The structure of the insulin genes in a number of myomorph rodents has been examined. The data indicate that only members of the sub-family Murinae have two insulin genes.

G-proteins are heterotrimeric complexes composed of α, β, and τ subunits and are involved in coupling receptor and effector functions during signal transduction across plasma membranes. G-proteins Gs and Gi are stimulatory and inhibitory to the catalytic subunit of adenylyl cyclase, respectively. A chimeric G-protein α subunit cDNA was constructed from the complete 5' untranslated region of Gαs52 (the 52 kD α subunit of Gs), the first 356 codons of the rat Gαs52, and the last 36 codons and 428 bp of the 3' untranslated region of the rat Gai2 (the α subunit of Gi2) cDNA. Expression of the chimeric G-protein alpha subunit (Gαs/i(38)) causes a constitutive increase in adenylyl cyclase activity in three different fibroblast cell lines. In turn, the elevated cyclase activity leads to higher levels of basal cyclic AMP and protein kinase A activity. The effect of Gαs/i(38) on cyclase does not seem to be through an inhibiton of Gαi function, but instead appears to be the consequence of direct action on the catalytic subunit, resulting in both a decreased time required for maximal cyclase activation and a greater maximal activation as well. Such alterations are not noted in cells expressing exogenous, wild-type Gαs. This data is based primarily on reconstitution assays using cholate extracts from fibroblast Gαs/i(38) clones and membranes derived from the S49 murine T-cell lymphoma (cyc- variant). Endogenous G-protein steady-state changes were detected by immunoblot analysis, but do not appear to account for the observed phenotypic alterations in Gαs/i(38) expressing clones. Furthermore, the validity of the above findings is unequivocally demonstrated through the use of amethopterin-mediated amplification of the chimeric Gαs/i(38) gene transcript and the consequent activation of adenylyl cyclase.

The B family of DNA-dependent DNA polymerases (pols) are uniquely sensitive to inhibition by N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (BuPdGTP). The affinity of members of the B family for BuPdGTP varies greatly, as does the ability of certain members to use the modified nucleotide as a substrate. For example, eukaryotic pol α has high affinity for the nucleotide, but incorporates it into DNA poorly, while T4 pol has lower affinity, but incorporates the nucleotide well. This thesis addresses two questions: 1) What are the amino acid residue(s) that impart sensitivity to BuPdGTP? and 2) is incorporation of BuPdGTP required for inhibition? To answer the first question, molecular modeling was used with the crystal structure of RB69 pol [Wang et al., 1997], an enzyme closely related to T4 pol [Wang et al., 1995]. This modeling identified a structural pocket adjacent to the polymerase active site that could serve as the butylphenyl "receptor". Based upon this modeling, a chimeric T4 pol containing the residues corresponding to the butylphenyl receptor from human pol α was designed and engineered for expression. The chimera was hypothesized to have a pol α-like phenotype with respect to its response to BuPdGTP (higher sensitivity/ lost ability to incorporate). The chimera was found to be unstable during purification, leaving the hypothesis unresolved. To answer the second question, non-substrate derivatives of BuPdGTP were in which the α,β anhydride oxygen of the triphosphate were replaced with either a CH2 or NH. The ability of the latter derivatives to inhibit polymerase activity and to serve as substrates was measured on T4 pol, RB69 pol and human pol α. Both derivatives retained high potency, but were not substrates under the conditions tested. These compounds were potent, selective inhibitors of B family pol that should be useful in the formation of a stable complex of enzyme:DNA:inhibitor for crystallization trials.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2006. / Includes bibliographical references (p. 211-218). Also available electronically via ProQuest Digital Dissertations.

Se encuestó a los médicos titulados por tesis de las siete escuelas de Medicina de Lima en el 2011 de las siete escuelas de Medicina de Lima para conocer las características del proceso de titulación por tesis, sus motivaciones y sus percepciones sobre dicho proceso. Se incluyó 98 tesistas (87% del total), 99% realizó tesis observacionales y el 35% realizó la tesis de manera individual (solo un tesista) en grupo de tres. La principal motivación fue que era bueno para su currículo (94%). En aquella universidad donde la tesis es obligatoria se empezó antes la elección del tema y el asesor. La percepción de mayor y menor dificultad en el proceso fue la realización de los trámites administrativos (53%) y seleccionar a su asesor (11%), respectivamente. Se deben revisar los procesos y tiempos administrativos para que no dificulten la realización de tesis, puesto que la nueva Ley Universitaria obliga a la elaboración de tesis para titularse. / We surveyed physicians who obtained their medical degree with a thesis in 2011 from the seven medical schools in Lima to know the characteristics of the degree by thesis process, as well as participants’ motivations and perceptions of that process. We included 98 students who did a thesis (87% of total); 99% conducted observational thesis, 30% did so in groups of three. The main motivation was that it was good for their curriculum vitae (94%). At the university where the thesis is compulsory, the process began with the choice of topic and adviser. Perceived “greatest” and “least” difficulty in the process was the completion of administrative procedures (53%) and selection of their advisor (11%), respectively. Administrative timeliness and processes should be reviewed so as not to impede the completion of thesis, since the new University Act requires the completion of a thesis to graduate.

Selenium is toxic at high doses, yet metabolically essential in trace amounts, and therefore provides an excellent illustration of the rule of paracelsus that "the dose alone determines the poison". The only mammalian selenoprotein of known function is glutathione peroxidase (GPx). This enzyme is expressed ubiquitously, and is responsible for detoxifying peroxides and hydroperoxides which, if left unchecked, may damage important biomolecules such as DNA and membrane fatty-acids. GPx is a homotetramer; each subunit contains one mole atom of selenium incorporated as a selenocysteine residue in the active site of the enzyme. Using oligonucleotides generated against the known bovine GPx amino-acid sequence, cDNA clones were isolated corresponding to the human GPx mRNA. Sequence analysis indicated that the selenocysteine in the active site of the enzyme was incorporated at an opal terminator (UGA) codon. Therefore the glutathione peroxidase mRNA constitutes the first example of natural suppression of a terminator codon in human cells. Regulation of human GPx gene expression by selenium was examined. Selenium replete HL-60 cells possessed approximately 30-fold more enzymatic GPx activity than selenium deficient cells. However steady-state GPx mRNA levels and rate of transcription of the GPx gene differed by less than 1.5-fold. Cycloheximide abolished the increase in enzymatic activity observed upon selenium replenishment. Cellular immunoreactive GPx protein levels correlated with enzymatic activity, indicating that the human GPx gene is regulated post-transcriptionally by selenium. The mechanism of this post-transcriptional regulation was investigated. A selenium labelled tRNA species was identified which exhibited features in common with a previously characterized tRNAUGA. This data suggested that selenium may be incorporated into GPx via a co-translational mechanism using a selenocysteinyl tRNA intermediate. Selenium did not alter cytoplasmic levels of the tRNAUGA, indicating that accumulation of cytoplasmic suppressor tRNA was not the point of regulation of GPx by selenium. A model is proposed for the co-translational insertion of selenocysteine into GPx mediated by a charged tRNA species present in selenium replete but absent from selenium deficient cells. Models are also proposed to explain the discrimination between the selenocysteine UGA codon and authentic UGA terminator codons. The regulation of the GPx gene was examined during mono-myelocytic differentiation of HL-60 cells in vitro and also during interferon-gamma activation of human peripheral blood macrophages and PMN. During phagocyte cell differentiation or activation, the ability to generate peroxide developed, however the peroxide-destroying capacities of GPx did not increase concomitantly. Complex regulatory patterns involving both transcriptional and translational controls were observed. The association of GPx gene expression with chronic granulomatous disease was explored. No correlation was found with either the autosomal or X-linked forms of the disease, a finding contradictory to previously published material.