Computational techniques, mostly density functional theory (DFT), were applied to study metal-based catalytic processes for energy conversion reactions. In the first and second projects, the main focus was on activation of the light alkanes such as methane, which have thermodynamically strong and kinetically inert C–H bonds plus very low acidity/basicity. Two Mo-oxo complexes with the different redox non-innocent supporting ligands, diamide-diimine and ethylene-dithiolate, were modeled. These Mo-oxo complexes are modeled inspired by active species of a metalloenzyme, ethylbenzene dehydrogenase (EBDH). The results for the activation of the benzylic C–H bond of a series of substituted toluenes by modeled Mo-oxo complexes show there is a substantial protic character in the transition state which was further supported by the preference for [2+2] addition over HAA for most complexes. Hence, it was hypothesized that C–H activation by these EBDH mimics is controlled more by the pKa than by the bond dissociation free energy of the C–H bond being activated. The results suggest, therefore, promising pathways for designing more efficient and selective catalysts for hydrocarbon oxidation based on EBDH active site mimics. Also, it is found that the impact of supporting ligand and Brønsted/Lowry acid/base conjugate is significant on the free energy barrier of C–H bond activation. In the third project the focus was on assessing the nature of hydrogen in the transition state related to the transfer of hydrogen between a carbon and nitrogen in an experimentally studied hydroaminoalkylation process by a five-coordinate Ta complex. It was revealed that, for the studied substituents, pKa is a larger driving force in the rate-determining hydrogen transfer reaction than the BDFE, which suggest a reasonable amount of protic character in the transition state, and possible routes to the design of more active catalysts with greater substrate scope. Finally, for the last project, the focus was on hydrotris(1,2,4-triazol-1-yl)borate complex as an electrocatalyst and study the impact of metal identity down a group or across a period of the d-block on proton-coupled electron transfer (PCET), which is a key process in many electrocatalytic cycles. The studied thermodynamics and kinetics trends for a series of mid to late 3d- and 4d-transition metals show the metal and its electronic structure greatly impact the nature of the PCET processes.

Centrosome is the major microtubule organizing center in mammalian cells. The centrosome plays key roles in the formation of the mitotic spindle, cell polarity and cell locomotion. In a typical somatic cell, the centrosome is composed of a pair of centrioles that are surrounded by a mass of amorphous pericentriolar material(PCM). The centrosomal-associated protein, hNinein, has been identified as a microtubules minus end capping, centrioles position, centrosome maturation and anchoring protein. Recently data reveal ninein CCII domain is associated with centrosomal targeting signal, regulating signal and phosphorylation sites, suggesting that this protein could contribute to centrosomal targeting signal as well as regulate asymmetry centrosomes. In this report, we therefore examine whether four C-terminal of hNinein splicing isoforms, including isoform 1, isoform 2, isoform 5 and a newly finding isoform 6, represent differential characterization, expression and functional aspects. To investigate the expression level of hNinein isoform 6 in variant cancer cell lines and astrocytoma patients. Isoform 6 only exist in IMR32 cancer cell line ( neuroblastoma cell line) and 42¢Mastrocytoma patients. We speculate that isoform 6 might play a role and exist in nerve cell or neuron. Using immunofluorescence assay, we show that all three hNinein isoforms are concentrated at centrosomes in HeLa cells, but not isoform 6. Overexpression of isoform 6 in HeLa cell might influence the distribution of

As the origin of a ¡§life-and-death¡¨ signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to evaluate the cellular mechanism of this fateful phenomenon. Based on a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, this study evaluated two hypotheses. First, transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-£eB (NF-£eB) on activation of phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) cascade in the RVLM underlies brain stem death. Second, muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in the RVLM is involved in the cardiovascular responses exhibited during Mev intoxication. In Sprague-Dawley rats, our results showed that microinjection bilaterally of Mev (10 nmol) into RVLM induced a progressive augmentation in NF-£eB, PI3K, Akt or PTEN activity that paralleled the increase in NOS II or peroxynitrite level in RVLM. Loss-of-function manipulations that included pharmacological blockade, gene knockdown, or immunoneutralization of NF-£eB, PI3K or Akt in RVLM significantly potentiated and prolonged the initial increase in ¡§life-and-death¡¨ signal, reversed the cardiovascular depression, and blunted the augmented expression of NOS II or nitrotyrosine on induced by Mev. Blockade of PI3K or Akt in RVLM also significantly blunted the Mev-induced activation of NF-£eB in the RVLM. However, immunoneutralization of PTEN in RVLM significantly diminished the increase in ¡§life-and-death¡¨ signal and potentiated the increase in Akt activity. We conclude that the PI3K/Akt cascade plays a ¡§pro-death¡¨ role in our Mev intoxication model of brain stem death by upregulating NF-£eB/NOS II/peroxynitrite in the RVLM, subject to antagonism by PTEN in this process. Microinjection bilaterally of Mev (10 nmol) into the RVLM induced a significantly augmentation in PKA activity in ventrolateral medulla that was not antagonized by coadministration of a nonselevtive or selective muscarinic receptor inhibitor. However, pharmacological blockade PKA in RVLM significantly blunted the initial increase in ¡§life-and-death¡¨ signal and the accompanying augmentation of NOS I expression in the ventrolateral medulla exhibited during Mev intoxication. We conclude that a muscarinic receptor-independent activation of PKA plays a ¡§pro-life¡¨ role in our Mev intoxication model of brain stem death by up regulating NOS I/PKG in the RVLM. According to this study, we proved that Mev stimulates different mechanism, muscarinic receptor-independent/PKA and PI3K/Akt/NF-£eB, to regulate NOS I and NOS II expression respectively, and induces cardiovascular responses during ¡§pro-life¡¨ and ¡§pro-death¡¨ phases. This information should provide further insights on the cellular mechanism of central cardiovascular regulation during the progression towards brain stem death, and offer news vistas in our search for therapeutic remedies or management strategies against fatal organophosphate poisoning and brain stem death.

Oskar (Osk) is a posterior body-patterning determinant in Drosophila melanogaster and is highly concentrated at the posterior pole of the oocyte. osk mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein is made. Bruno (Bru) represses translation during osk mRNA localization by direct binding, but how Bru-mediated repression is relieved at the posterior of the oocyte is unknown. Two types of Bru protein interactions are implicated in repression of osk: Bru-Cup interaction and Bru dimerization. By mapping the Bru domains that are important for these interactions, I found that the amino-terminal domain of Bru contributes to both interactions, and deletion of this domain caused a defect in translational repression. However point mutations, within the amino-terminal domain, that disrupt both types of interaction in vitro did not interfere with translational repression in vivo. The difference may be due to other factors stabilizing the Bru-Cup interaction in vivo, as the mutant Bru still associates with Cup in vivo. My work supports the model of repression that relies on Bru interaction with Cup. I also build a new model in which Bru dimerization promotes translational activation of osk, based on my unexpected results: dimerization-defective Bru only weakly accumulated Osk::GFP fusion protein encoded by an osk::GFP reporter RNA bearing a Bru-binding region, while dimerization-competent Bru showed the opposite effect. This suggests that dimerization may contribute to switching Bru from a repressor to an activator, with dimerization controlled via a post-translational modification. Consistent with this, I found that a small fraction of Bru in ovaries is phosphorylated. PKA is a positive regulator of osk expression and phosphorylates Bru in vitro. To test if PKA regulation of osk is mediated through Bru, I examined the effect of altering PKA activity on Bru phosphorylation and Bru-mediated repression. Modulating PKA activity caused small, yet detectable changes in Bru phosphorylation and Bru-dependent translational repression using an osk::GFP reporter. However, while the studies with Bru mutants suggest that phosphorylation promotes repression by Bru, these studies argue for a role in promoting activation. Further work will be required to explain these phenomena. / text

<p><p>Syftet med detta examensarbete var att undersöka högskolestudenters erfarenheter av övergången från studier inom kemi på gymnasial nivå till högskolenivå för att se om det finns ett glapp i undervisningen. Undersökningen bestod av dels en enkätundersökning och dels svaren från tentamensfrågor av samma karaktär som enkätfrågorna. Resultatet visar att studenterna anser att det är en skillnad mellan att läsa kemi på gymnasial nivå och på högskolenivå och att den största skillnaden ligger i ett förändrat arbetssätt. Av studenternas svar på enkätfrågorna och tentamen kan slutsatsen dras att studenterna känner till termer och begrepp, men har svårt att använda dem för att förklara kemiska fenomen.</p></p>

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thesis advisor: Charles Hoffman / The study of mammalian cAMP signaling has often been confounded by the fact that ten different genes encode adenylyl cyclases (ACs) that produce cAMP from ATP and 16 different genes encode phosphodiesterases (PDEs) that hydrolyze cAMP to AMP. In this study, mammalian AC cDNAs were cloned and integrated into strains of the fission yeast Schizosaccharomyces pombe that lack their endogenous AC to determine the basal activity of all ten AC isoforms. In addition, response to the stimulatory mammalian Gsα was determined by co-expression of a mutationally-activated form of the human GNAS1 gene. AC activity was assessed using an fbp1-GFP reporter that is repressed by cAMP production and PKA activity. Results confirm that all ten isoforms have detectable basal activity, and AC1-9 definitively respond to Gsα stimulation. When matched with a sufficiently potent mammalian phosphodiesterase (PDE), strains expressing mammalian ACs make good candidates for small molecule high throughput screening (HTS) to detect AC inhibitors. A 100,000 compound screen was recently performed to detect AC and Gsα inhibitors as well as PDE activators. A promising “hit” was progesterone, which has been previously suggested to inhibit ACs in Xenopus. Initial results suggest that progesterone inhibits AC1 and the closely-related AC3. These data demonstrate the utility of using S. pombe as an effective platform for identifying inhibitors of both basal and GNAS1-stimulated AC activity. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Computational simulations of chemical systems play an ever-increasing role in many areas of biochemical research from rational drug design to probing fundamental physiological processes. Depending on the method, a vast array of properties are able to be predicted. Here we report the design and implementation of two methods for investigating diverse problems in protein biochemistry. In order to better understand protein–metal interactions—most importantly for the difficult to model transition metal ions— empirical force field parameters were developed for Pt(II), cisplatin, and other Pt(II) coordination compounds. Two force field frameworks were used: a modified version of the fixed- charge OPLS-AA and the polarizable POSSIM force field. A seven-site model was used for the Pt(II) ion. The produced parameters are compatible with the OPLS-AA and POSSIM force fields and can be used in protein–metal binding simulations in which—contrary to the common treatment of metal ions in such simulations—the position or even coordination of the ion does not have to be constrained using preexisting knowledge. It has been demonstrated that the produced models are capable of reproducing key properties of relevant Pt(II) complexes but that the POSSIM formalism yields more accurate values for energies of formation than the OPLS-AA model. This Pt(II) model was employed—along with previously developed Cu(I) parameters—to investigate the binding of platinum to the protein Atox1, a human copper chaperone implicated in the resistance mechanism of cisplatin and other platinum antitumor compounds. In collaboration with the Dmitriev and Bernholc groups, we used our models to inform and refine spectroscopic experiments as well as to serve as starting points for high-performance quantum calculations. It was shown that under physiological redox conditions, copper(I) and cisplatin can form large polymers with glutathione. These polymers were capable of transferring copper(I) to apo-Atox1 or to platinum(II) to copper-loaded Atox1. Analysis of the simultaneous binding of copper(I) and platinum(II) to Atox1 was found to occur through the formation of copper–sulfur–platinum bridges, where copper is coordinated by three sulfur atoms and platinum by four sulfur atoms. With the goal of using a simple model to be able to quickly estimate the acid disassociation constants of proteins, PKA17 has been developed and tested. PKA17 is a coarse-grain grid-based method and software tool for accurately and rapidly calculating protein pKa values given an input PDB structure file. During development, parameter fitting was carried out using a compilation of 442 Asp, Glu, His, and Lys residues that had both high-resolution PDB structures and published experimental pKa values available. Applying our PKA17 model, the calculated average unsigned error and RMSD for the residue set were found to be 0.628 and 0.831 pH units, respectively. As a benchmark for comparison, the same residue set was evaluated with the PROPKA software package which resulted in an average unsigned error of 0.761 pH units and an RMSD of 1.063 pH units. Finally, a web interface for the PKA17 software was developed and deployed (http://users.wpi.edu/~jpcvitkovic/pka_calc.html) to make PKA17 available to the wider scientific community.

[URE3] priono indukcija priklauso nuo Ras/PKA signalinio kelio aktyvumo. Galima šio kelio įtaka [PSI+] priono formavimuisi gali būti numanoma iš natyvaus Sup35 baltymo struktūros. Šio baltymo struktūroje yra nustatytos kelios menamos PKA fosforilinimo vietos. Siekiant patikrinti šią galimybę buvo atliktas trijų, Ras/PKA signalinio kelio aktyvumu besiskiriančių, izogeninių kamienų – 6-&#945;‘1-NB13-, 12-&#945;‘1-NB13-, 7-&#945;‘1-NB13 - [PSI+] priono indukcijos dažnio įvertinimas. Rezultatai parodė, kad terpėje esant turtingam azoto šaltiniui 6-&#945;‘1-NB13- kamieno ląstelėse padidinta CYR1, BCY1 ir Ras2Val19 genų raiška sumažina [PSI+] priono indukcijos dažnį. Tuo tarpu, terpėje esant neturtingam azoto šaltiniui 7-&#945;‘1-NB13 kamieno ląstelėse padidinta BCY1 geno raiška padidina [PSI+] priono indukcijos dažnį. Todėl galima daryti išvadą, kad [PSI+] priono indukcijos dažnis gali priklausyti nuo Ras/PKA signalinio kelio aktyvumo. / [URE3] prion induction depends on the activity of the Ras/PKA signal transduction pathway. Possible influence of this pathway on the formation of [PSI+] prion could be predicted from the structure of the native Sup35 protein. Several possible phosphorylation sites are known in the structure of this protein. In order to check this possibility analysis of prion induction frequency of three isogenic mutant strains – 6-&#945;‘1-NB13-, 12-&#945;‘1-NB13-, 7-&#945;‘1-NB13 – different in the activity of the Ras/PKA signal transduction pathway, was performed. Results showed that in the presence of rich nitrogen source in cells of the strain 6-&#945;‘1-NB13 the increased expression of CYR1, BCY1 and Ras2Val19 genes decreases the frequency of [PSI+] prion induction. Instead, in the presence of poor nitrogen source in cells of the strain 7-&#945;‘1-NB13 the increased expression of BCY1 gene increases the frequency of [PSI+] prion induction. So, it is possible to make a conclusion that the frequency of induction of the [PSI+] prion depends on the activity of the Ras/PKA signal transduction pathway.