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

Taxonomy, diversity, and habitat ecology of Irish tardigrades

Tardigrades (phylum Tardigrada Doyère, 1840) are obligate aquatic, microscopic animals that inhabit marine and freshwater habitats, as well as the terrestrial environment. They occur in limnoterrestrial settings including soil and in association with mosses, lichens, and leaf litter. Tardigrades are best known for their ability to survive challenging environmental conditions, as demonstrated in the terrestrial environment by their presence on all continents and in all biomes. Despite this ubiquity, tardigrades have been poorly known in Ireland. The sparse information relating to Irish tardigrades was scattered and to a great extent, taxonomically outdated. The status of the phylum in Ireland required a thorough evaluation. All previous records of Tardigrada from the Republic of Ireland and Northern Ireland were compiled and reviewed. A new record for Echiniscus quadrispinosus quadrispinosus Richters, 1902 was also added from County Clare. Resultingly, a Checklist of Irish Tardigrade Species that included 16 heterotardigrades and 35 eutardigrades was established. Most pre-existing records for Irish tardigrades originated from the work of James Murray as part of the original Clare Island Survey (1909–1911). The tardigrades of Clare Island were reinvestigated through participation in the New Survey of Clare Island. In the time between surveys the presence of many morphologically similar species complexes within Tardigrada had been reported. The importance of recognising such complexes was apparent as the original survey recorded the single taxon Macrobiotus hufelandi C.A.S. Schultze, 1834 as the most frequently occurring in samples, while the new survey found that nine morphotypes of the M. hufelandi species complex were present among its Clare Island samples. Ten other tardigrade species, including two not previously known from Ireland, Dianea sattleri (Richters, 1902) and Notahypsibius pallidoides (Pilato, Kiosya, Lisi, Inshina and Biserov, 2011), were also recorded by the New Clare Island Survey. Following these foundational studies, a new islandwide survey of Tardigrada, The All-Ireland Tardigrade Survey (AITS) was designed and launched with the objectives to characterise the Irish tardigrade fauna, evaluate its diversity, and collect habitat data using newly established standardised protocols for sample collection and processing. Five broad- scale Irish habitat types of the terrestrial environment were included: bog, built land, exposed rock, semi-natural grassland, and semi-natural woodland. A subset of AITS samples was used to demonstrate analyses for evaluating and comparing diversity between tardigrade communities including estimations of species richness, sample completeness, sample coverage, and evenness. Taxa representing multiple new records for Ireland were present within the samples comprising the selected subset. Two AITS samples from limestone pavement in Ireland’s Burren region contained specimens that were targeted for detailed taxonomic analyses involving intensive light (LM) and scanning electron microscopy (SEM) with the addition of morphometric and limited phylogenetic results. This work facilitated the description of two new species, Bryodelphax pucapetricolus DeMilio, Tumanov, Lawton, Kristensen & Hansen, 2022 and B. wallacearthuri DeMilio, Tumanov, Lawton, Kristensen & Hansen, 2022 and a revision of morphological characters important in that genus and with relevance to other heterotardigrade genera. The diagnoses of Bryodelphax Thulin, 1928 and Bryochoerus Marcus, 1936 were thereupon emended. Further recommendations, including for the revision of some related species and increased inclusion of the morphometric values of gonopore diameter and gonopore to anus distance and details of the anus morphology in the descriptions of limnoterrestrial heterotardigrade genera were made. The genus Parechiniscus Cuénot, 1926 was recorded from Ireland for the first time, as were two other heterotardigrades, Echiniscus spiniger Richters, 1904 and Testechiniscus spitsbergensis spitsbergensis (Scourfield, 1897). Detailed LM and SEM analyses of Irish Parechiniscus specimens led to a new interpretation of the configuration of the dorsal cuticular plates and the first accounts of tertiary clavae in that genus and new information on the Parechiniscus sexual system. These observations warranted a new emendation to the genus diagnosis. Leaf litter as a habitat for tardigrades was explored in Ireland and Scotland for the first time with 25 taxa encountered, including multiple new records for each country. The Irish Checklist of Species was further updated to include all newly obtained data for a total of 25 heterotardigrades and 52 eutardigrades, a 54% increase of known Irish species. The presented body of research thereby progressed the characterisation of the Irish tardigrade fauna, made contributions to the field of tardigrade taxonomy, and offered an approach to the study of tardigrade habitat ecology