Investigation of Autoregulation of ICER and Electrochemical Properties of Tryptophan

A relatively new development in the field of research-based chemistry is to study biomolecules, their interactions, and a biochemical mechanisms by examination of their physical properties and application of laboratory techniques rooted in concepts of physical chemistry. Both projects that are encompassed within this master\u27s thesis indeed fall under the umbrella of biophysical chemistry, as they apply physical chemical techniques study particular biomolecular interactions. The first of these two projects is the study of a leucine zipper protein, Inducible cAMP Early Repressor [ICER], which is a product of the cAMP Responsive Element Modulator [CREM] gene. ICER functions as a transcriptional repressor by binding to cAMP Responsive Elements [CRE\u27s] found in the promotor sequences of genes involved in cellular growth, and is abnormally expressed in certain forms of cancer in which ICER acts as a tumor repressor. It also binds to the four CRE sites on its own promotor, known as CARE-1 through CARE-4, thereby regulating its own expression. This research is based on the hypothesis that ICER may in fact autoregulate its own expression by cooperative binding to its own promotor. A technique known as Fluorescence Resonance Energy Transfer [FRET] is used to test this hypothesis and determine dissociation constants of purified ICER with double stranded DNA. Titrations were performed with purified ICER and double stranded DNA labeled with a fluorophore-quencher pair and containing one or more of the CARE sites. Observed dissociation constants were largely inconsistent and traced back to difficulties in producing purified ICER on a regular basis. Therefore, the focus shifted to reproducible purification of ICER. It includes a comparison of three purification protocols, one of which is a urea-based denaturing purification, another being a native purification, and the third a combination of the first two. Qualitative data that will illustrate this comparison includes side-by-side SDS-PAGE gel electrophoresis analysis of samples from each step of both purification procedures, among other things. The second project that makes up this thesis involves an enzyme that repairs DNA. The enzyme E. coli photolyase utilizes a light-driven electron transfer mechanism for repairing DNA damaged by UV-light exposure. The enzyme may be activated by a proton-coupled electron transfer [PCET] mechanism. PCET mechanisms are of considerable interest due to their prevalence in many physiological processes such as enzyme catalysis, as they provide an alternative reaction pathway that circumvents traditional high-energy transition states. In this case, during the electron transfer mechanism, the neutral radical form of the flavin adenine dinucleotide cofactor [FADH-] is reduced to FADH-, and an amino acid radical intermediate is formed. This amino acid, 306Trp, is of particular significance due to its role in the mechanism. Using voltammetry, it is possible to measure the reduction potential of the Trp in solution, providing insight into the mechanism involving 306Trp. The 306Trp reduction potential represents the charge recombination energy required for oxidation of that amino acid and formation of FADH-. As the mechanism of electron transfer involves a proton, its kinetics are heavily pH-dependent. Thus data was acquired over a wide range of pH values in order to quantify this relationship. Furthermore, this reaction was studied in distilled water and in D2O in order to examine solvent effects on the reduction potential of Trp. An observed inverse kinetic isotope is investigated in detail to explain the observed increase in reaction rate in D2O counterintuitive to the mass-related kinetic isotope effect. The data from this thesis confirm the hypothesis that D2O significantly affects the reduction potential and the pKa of Trp, the combined effects of which explain the observed inverse isotope effect in which the reaction occurs more quickly in D2O. All experiments were repeated with a tryptophan-like molecule, N-acetyl-Ltryptophanamide, the properties of which more accurately represent how the 306Trp residue would act in the electron-transfer mechanism as a member of a peptide chain. N-acetyl-L-tryptophanamide, unlike natural amino acids, contains no Cterminus and two amine groups. Therefore, issues of charge formation from amino acid ionization that limited the pH range for the tryptophan experiments were alleviated, and the use of N-acetyl-L-tryptophanamide allowed for greater experimental freedom and lent itself to a wider pH/pD range to be investigated

First records of marine tardigrades of the genus <i>Coronarctus</i> (Tardigrada, Heterotardigrada, Arthrotardigrada) from Mexico

Deep-water sampling in the Perdido Fold Belt, Gulf of Mexico, Mexican Economic Exclusive Zone yielded five specimens of tardigrades belonging to the genus Coronarctus Renaud-Mornant, 1974. The specimens represent the first records of the genus for Mexico. Two two-clawed larvae and two four-clawed larvae of Coronarctus mexicus Romano, Gallo, D’Addabbo, Accogli, Baguley & Montagna, 2011 and a single four-clawed larval specimen of an undescribed Coronarctus species were identified. Taxonomic analysis of the specimens contributed to the knowledge of deep-sea and Mexican marine tardigrades, two data-poor areas of study

A common genetic network underlies substance use disorders and disruptive or externalizing disorders

Here we summarize evidence obtained by our group during the last two decades, and contrasted it with a review of related data from the available literature to show that behavioral syndromes involving attention deficit/hyperactivity disorder (ADHD), externalizing disorders, and substance-use disorder (SUD) share similar signs and symptoms (i.e., have a biological basis as common syndromes), physiopathological and psychopathological mechanisms, and genetic factors. Furthermore, we will show that the same genetic variants harbored in different genes are associated with different syndromes and that non-linear interactions between genetic variants (epistasis) best explain phenotype severity, long-term outcome, and response to treatment. These data have been depicted in our studies by extended pedigrees, where ADHD, externalizing symptoms, and SUD segregate and co-segregate. Finally, we applied here a new formal network analysis using the set of significantly replicated genes that have been shown to be either associated and/or linked to ADHD, disruptive behaviors, and SUD in order to detect significantly enriched gene categories for protein and genetic interactions, pathways, co-expression, co-localization, and protein domain similarity. We found that networks related to pathways involved in axon guidance, regulation of synaptic transmission, and regulation of transmission of nerve impulse are overrepresented. In summary, we provide compiled evidence of complex networks of genotypes underlying a wide phenotype that involves SUD and externalizing disorders

Adolescent Brain Development and the Risk for Alcohol and Other Drug Problems

Dynamic changes in neurochemistry, fiber architecture, and tissue composition occur in the adolescent brain. The course of these maturational processes is being charted with greater specificity, owing to advances in neuroimaging and indicate grey matter volume reductions and protracted development of white matter in regions known to support complex cognition and behavior. Though fronto-subcortical circuitry development is notable during adolescence, asynchronous maturation of prefrontal and limbic systems may render youth more vulnerable to risky behaviors such as substance use. Indeed, binge-pattern alcohol consumption and comorbid marijuana use are common among adolescents, and are associated with neural consequences. This review summarizes the unique characteristics of adolescent brain development, particularly aspects that predispose individuals to reward seeking and risky choices during this phase of life, and discusses the influence of substance use on neuromaturation. Together, findings in this arena underscore the importance of refined research and programming efforts in adolescent health and interventional needs

TRH response pattern in adolescent schizophrenic males.

A marked elevation in human growth hormone (GH) following intravenous infusion of thyrotropin releasing hormone (TRH) was seen in three of seven adolescent male schizophrenic patients receiving neuroleptic drugs, and in one of five controls matched for age, sex, and developmental stage, receiving the same drugs. A positive family history for schizophrenia was noted in all three schizophrenic \u27responders\u27 but in only one of the remaining patients. The response patterns of thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) following TRH infusion were not significantly different in the two groups. No significant correlations were found between T3, T4, TSH, or GH response patterns and age of patient, duration of illness, medication dose or duration, weight change, or hours of sleep preceding testing