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CELF4 is a brain-specific member of the CELF RNA binding protein (RBP) family that binds a significant portion of the transcriptome with striking selectivity for the 3’UTR of neuronal and synapse-specific functional targets in the hippocampus. Celf4 knockout and haploinsufficient mice have a complex neurobehavioral phenotype similar to human patient groups identified with CELF4 mutations, specifically CELF4-inclusive deletions and translocations. We hypothesize that CELF4 operates in multiple aspects of post-transcriptional gene regulation; interacting with RNA molecules from synthesis to decay. Tissue-level ribosome profiling experiments demonstrate that loss of CELF4 results in global ribosome occupancy changes across CELF4 mRNA targets and refined our ability to interrogate the synaptic function of CELF4. Turning intra-cellularly, a snRNA-seq approach implicated the CA3 region of the hippocampus in CELF4-mediated mRNA regulation and identified synaptic targets regulated by CELF4. By leveraging both ribosome profiling footprinting and snRNA-seq differential gene expression data, we identified synaptic and epilepsy disease genes that contribute to, and drive, neurobehavioral phenotypes. In part two of this work we focus on DEE disease gene DNM1, a known target of CELF4 at the synapse. Using in vitro and in vivo approaches, we validate the regulatory relationship between mouse Dnm1 RNA and CELF4 RBP function. Lastly, we introduce a novel preclinical model of DNM1 DEE that recapitulates the seizure and behavioral phenotypes of patients suffering from dominant negative DNM1 mutations. In characterization of this model, we lay the groundwork for future investigations of cellular etiology of DNM1 pathogenic variants and therapeutic development for patient groups suffering from DEE.

<p> Bcl-2 proteins play a critical role in the regulation of apoptosis, a form of programmed cell death. Apoptosis is important during development to facilitate the elimination of supernumerary, damaged or harmful cells in multicellular organisms. Altered regulation of apoptosis is associated with many diseases such as several forms of cancer as well as autoimmune and degenerative disorders. The way in which Bcl-2 proteins regulate apoptosis is unknown and much research is focused on elucidating the molecular mechanism of their function. Bcl-2, an anti-apoptotic member of this family, is localized to the mitochondria, endoplasmic reticulum and nuclear envelope. In healthy cells, Bcl-2 adopts a typical tail-anchored topology in which the carboxyl-terminal helix (a9) is inserted into the membrane, anchoring the protein, leaving the majority of the protein in the cytosol. Previous results from our lab have shown that after the induction of apoptosis, Bcl-2 undergoes a conformational change in which the endogenous cysteine residue, C158, in the a5 helix becomes protected from a membrane impermeant cysteine specific labelling reagent, IASD (4-acetamido-4' ((iodoacetyl)amino)-stilbene-2,2'disulfonate). Modification of cysteine residues results in a change in migration ofBcl-2 in an isoelectric focusing, IEF, gel system. To investigate the nature of this conformational change, cysteine scanning mutagenesis was used to determine the topology of Bcl-2 in the late stages of apoptosis. The results from the current study showed that in rat 1 myc ERTM fibroblasts, a discontinuous sequence of residues in the a5 and a6 helices of Bcl-2 become protected from IASD labelling after the induction of apoptosis by etoposide or serum starvation. The data support a model topology in which, during apoptosis, Bcl-2 undergoes a functionally significant conformational change, going from a single spanning transmembrane protein to a polytopic membrane protein in which three helices span the membrane, a5, a6 and a9. </p> / Thesis / Master of Science (MSc)

The early region 1a oncogene of adenovirus 5 codes for proteins that can activate transcription of viral and cellular genes. This study describes the construction of three deletions and one point mutation that together span the entire coding region of the second exon of E1A. The exon-2 mutants were tested for their ability to activate transcription from the adenovirus early region 3 promoter (E3) in transient expression assays. Dl1116 (dl aa 205-221) did not affect transactivation of E3 in pKCAT-23. Sub1117 (dl exon-2 aa) and dl1115 (dl aa 188-204) were unable to activate transcription. Pm1131 (SER-219 to stop) had a reduced transactivating efficiency but was still able to stimulate transcription. These results define the 3' boundary of a transactivation domain on the E1A proteins as being between positions 188 and 204. Results obtained in our lab define the 5' boundary as being between 138-147 (Jelsma et al., 1988). The mutants that could not transactivate were tested for their ability to block wildtype E1A transactivation of the E3 promoter in assays similar to those described by Glenn and Ricciardi (1987). Dl1115 and sub1117 appeared to block transactivation by WT E1A. In transient expression assays, the fatty acid sodium butyrate was found to stimulate transcription of the CAT gene, when added to the medium of HeLa cells transfected with pKCAT-23. This suggests that sodium butyrate is transactivating the Ad 5 E3 promoter. / Thesis / Master of Science (MS)

Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from first page of PDF document. Includes bibliographical references (p. 57-64).

Des conditions de carence entrainent une mort cellulaire développementale chez le protiste Dictyostelium discoideum. Dans un système in vitro, des cellules de Dictyostelium sont mises en conditions de carence, puis l'addition des inducteurs DIF-1 ou c-di-GMP conduit à une mort cellulaire vacuolaire. DIF-1 est un polyketide produit par Dictyostelium et induisant la différenciation des cellules pré-tiges. Le dinucléotide cyclique c-di-GMP était connu comme un second messager chez les procaryotes, et comme un déclencheur de l'immunité innée dans des cellules de mammifères. Il a été montré par d'autres que des cellules de Dictyostelium peurent produire et détecter c-di-GMP.Pour analyser la signalisation par c-di-GMP chez Dictyostelium, nous avons utilisé la mutagénèse aléatoire et la mutagénèse ciblée. En utilisant des mutants inactivant stlB ou dmtA, nous avons démontré que DIF-1 endogène ou exogène est nécessaire pour la signalisation par c-di-GMP dans Dictyostelium. En conséquence, nous avons amélioré l'étape de sélection dans une mutagenèse aléatoire en utilisant c-di-GMP et un peu de DIF-1 comme inducteurs, ce qui a produit plusieurs mutants. Par ailleurs j’ai testé par mutagenèse ciblée des hypothèses basées sur les informations connues dans Dictyostelium ou d'autres types de mort cellulaire. Trois molécules ont été essayées, DDX41 comme récepteur putatif de c-di-GMP, l' uniport mitochondrial pour le Ca2+(MCU) et la Na+/K+ATPase (IonA).En résumé, au cours de ma thèse, nous avons démontré une relation entre la signalisation c-di-GMP et a signalisation DIF-1 dans Dictyostelium et identifié plusieurs nouvelles molécules de la mort cellulaire par mutagenèse aléatoire. / The protist Dictyostelium discoideum undergoes development cell death when under starvation. To investigate the molecular mechanism of Dictyostelium cell death, an in vitro system has been used. Dictyostelium cells were starved and then cell death was induced by DIF-1 or c-di-GMP. About 40h after induction, cells underwent vacuolar cell death. DIF-1 is a polyketide, produced by Dictyostelium prespore cells, which induces prestalk cell differentiation. c-di-GMP was well known not only as a second messenger produced and sensed by bacteria but also as a trigger of innate immunity in mammalian cells. Dictyostelium was recently found by another laboratory to produce and sense c-di-GMP. To analyze c-di-GMP signaling in Dictyostelium cell death, we used random mutagenesis and targeted mutagenesis. By using the knockout mutants stlB- and dmtA-, we demonstrated that endogenous or exogenous DIF-1 is required for c-di-GMP signaling in Dictyostelium. In contrast, endogenous c-di-GMP is not necessary for exogenous DIF-1 signaling. As a consequence, we improved the selection step in random mutagenesis by using c-di-GMP and a little DIF-1 as inducers, which produced several mutants. Another part of my project was to test by targeted mutagenesis some hypotheses, based on known information in Dictyostelium or other similar cell death types. Three molecules have been tested, the c-di-GMP putative receptor DDX41, the mitochondrial Ca2+ uniporter (MCU) and the Na+/K+-ATPase (IonA).In summary, during my thesis, we have demonstrated a relation between c-di-GMP signaling and DIF-1 signaling in Dictyostelium and identified several new cell death molecules by random mutagenesis.

Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions. Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation. To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes. Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities. The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets. / biokemi och organisk kemi

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 143 p. : ill. (some col.). Includes abstract. Includes bibliographical references.