Thesis advisor: Jianmin Gao / Covalent drugs present a unique situation in the clinical world. Formation of a covalent bond between a drug molecule and its target protein can lead to significant increases in a number of desirable traits such as residence time, potency, and efficacy of a drug. From a kinetic perspective, the formation of a covalent bond between a drug and its target functionally eliminates the dissociation rate (koff) of the compound, ensuring that the compound will stay engaged with its target. However, development of covalent drugs has been met with caution and concern, as an irreversible covalent bond forming on the wrong target can have disastrous results, so specificity is of the utmost importance. One option for increasing specificity is by linking a covalent binding electrophile, or warhead, to a peptide. Peptide-based therapeutics have already been shown to serve as effective protein-targeting modalities with high specificity, a specificity that would greatly benefit covalent drugs. Phage display is a powerful technique for the discovery of selective peptides which utilizes the screening of vast libraries of randomized peptides to identify strong binders. This technology has been used to discover a large number of protein-targeting peptides, but also a smaller number of cyclic, covalent binding peptides that function as enzymatic inhibitors. Herein, this study aimed to explore the idea of adding covalent-binding functionality to phage libraries in novel ways and expand upon the scope of proteins that can be targeted with phage libraries containing covalent libraries. We sought to develop a mechanistic and chemical understanding of the interactions between bacteriophage and chemical warheads to best understand both the limits and the potential of this technology. In order to best understand the relationship between chemical warhead and phage particle, a model system was developed based on the M13KE pIII protein. It was found that the extracellular N-terminal domains of this protein could be expressed and purified in low yields in bacterial cells and that these domains would behave similarly in solution as in the membrane of the M13KE bacteriophage. With this protein in hand, experiments previously performed using small, cysteine containing peptides, could be performed on a full protein to mimic the phage labeling environment. This protein was used to identify efficient cysteine crosslinkers, most notably dichloroacetone (DCA) and bis-chlorooxime (BCO). The pIII protein system was then used to study the viability of bifunctional warhead molecules containing a covalent warhead and a cysteine crosslinker. Based on preliminary analyses with the pIII protein, aryl sulfonyl fluoride was chosen as a novel warhead candidate that warranted further pursuit. Kinetic NMR studies verified that aryl sulfonyl fluoride was capable of forming covalent bonds with phenols under phage labeling conditions. Labeling experiments analyzed with LC/MS seemed to indicate a degradation of the warhead. However, as the source of the degradation was not able to be determined, it was decided that various affinity assays would be used to identify if phage could be labeled with an aryl sulfonyl fluoride-DCA conjugate. Both streptavidin-bead pulldown assays and ELISA assays were used, however both assays yielded results that could not conclusively verify the integrity of the warhead. During phage labeling experiments, a phenomenon was noted that phage titers after modification showed a 2-3 order of magnitude drop in phage count. Covalent modification of phage beyond what is intended could have troubling consequences for all covalent phage libraries, and so a more in-depth approach was taken to identify and better understand phage toxicity as it relates to covalent warheads. As a model, a well-studied diazaborine-mediated warhead with a slow dissociation rate was selected and used in a range of phage toxicity screenings. Despite statistical fluctuations between trials, toxicity screenings using this warhead served to highlight a unique concern for bifunctional covalent warheads. A concentration-dependent toxicity can be seen in phage incubated with bifunctional small molecules that is not present when incubated with the monofunctional equivalents. The presence of this toxicity even towards a phage with no free thiols highlights a unique challenge of off-target labeling within phage particles that, if solved, could provide the next significant step towards developing novel covalent phage libraries. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Cyclic nucleotides such as cAMP and cGMP serve as intracellular second messengers in diverse signaling pathways that control a wide range of cellular functions. Such pathways are regulated by key cyclic nucleotide receptor proteins including protein kinase A (PKA), the exchange protein directly activated by cAMP (EPAC), the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels, and protein kinase G (PKG), and malfunction of these proteins has been linked to a number of pathologies. While it is known that cyclic nucleotide binding to these proteins leads to structural perturbations that promote their activation, the role played by dynamics in auto-inhibition and cyclic-nucleotide-dependent activation is not fully understood. Therefore, in this thesis we examined dynamics within the cyclic-nucleotide receptor proteins EPAC, HCN and PKG, and found that dynamics are critical for allosteric control of activation and/or autoinhibition of all three proteins. In particular, our findings for EPAC and HCN have highlighted dynamics as a key modulator of the entropic and enthalpic components, respectively, of the free-energy landscape for cAMP-dependent allostery, while our findings for PKG have highlighted dynamics as a key determinant of the cGMP-vs.-cAMP selectivity necessary to minimize cross-talk between signaling pathways. Ultimately, we envision that the methods outlined in this thesis will reveal key differences in the regulatory mechanisms of human cyclic nucleotide receptors that can eventually be exploited in the development of novel therapeutics to selectively target a single receptor, and thus treat physiological conditions/diseases linked to malfunction of the target receptor. / Thesis / Doctor of Philosophy (PhD) / In this thesis, we examined cyclic-nucleotide-responsive proteins that regulate key physiological processes, and whose malfunction has been linked to cardiovascular and neurological disorders. In particular, in three such proteins we examined dynamics, whose role in cyclic-nucleotide-responsive function is not fully understood. We found that cyclic-nucleotide-dependent variations in dynamics play a critical role in the function of these proteins, with the results for each protein highlighting a different role played by dynamics. Ultimately, we envision that the methods outlined in this thesis will reveal key functional differences among human cyclic-nucleotide-responsive proteins that can eventually lead to the development of novel therapeutics to treat certain diseases such as arrhythmias or epilepsy by selectively targeting a single cyclic-nucleotide-responsive protein.

No description available.

Humans navigate the world and interact with others through a complex series of communicative tools. We experience both internal and external stimuli, such as pangs of hunger or pain from an injury, and both verbal and nonverbal language. Bacteria also possess the ability to communicate, albeit in more discreet, yet no less complex ways. Bacteria rely on an incredibly diverse signaling system of triggers and responses in order to survive and to thrive. While we perceive language with our eyes and ears, bacteria employ a system of small molecules to relay both intra- and extracellular messages. They utilize this ability, known as quorum sensing to "talk" to their neighbors, express otherwise latent genetic characteristics, and to defend themselves against enemies. It has been suggested that this internal and external activity is linked, however, little is known about their interplay.  This family of molecules, the cyclic di-nucleotides, which includes c-di-GMP and c-di-AMP, are critical to regulating bacterial processes such as motility, glucose remediation, and cell wall homeostasis. Their importance has spurred numerous investigations into their mechanism of action. Although found in very low concentrations within cells, they are capable of regulating a multitude of processes due to their ability to adopt variable conformations. To date, analog design by other groups has focused on the modification of the innate phosphate moiety as well as various substitutions or deletions at the 2'-position on the ribofuranose ring. However, these analogs have not been water soluble, limiting them to in vitro investigations only. We propose that by replacing the phosphate linkage entirely we can increase water solubility and have pursued a divergent total synthesis of various cyclic di-nucleotides featuring biomimetic linkages. Herein we address the methods we explored to optimize the synthesis of our three monomers, coupling strategies employed, the novel application of a Staudinger ligation to afford our abasic macrocycles and finally our progress towards implementing a bis-glycosylation strategy to install the desired nucleobase. We are able to efficiently provide large amounts of a di-amino, azide methyl ester, and N,O-substituted furanose monomers in no more than six steps from a common intermediate. These monomers are coupled and cyclized to form our four scaffolds, amide, carbamate, squaramide, and urea. Finally, we have begun to successfully implement our Brønsted acid mediated glycosylation strategy and understand its limitations. It is our goal to develop a general method to afford a diverse array of conformationally unique and water soluble cyclic di-nucleotide analogs with which to probe these essential bacterial signaling pathways. / Chemistry

Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored. The Rv0805 gene was previously shown to code for an active phosphodiesterase from Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium. Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist. Over-expression of ML2210 in M. smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M. smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of M. smegmatis, arguing for additional cellular roles of this protein. Orthologs of Rv0805 or ML2210 are found only in slow growing mycobacteria suggesting that other cyclic nucleotide phosphodiesterases could regulate cAMP levels in fast growing mycobacteria like M. smegmatis. Since BLAST results did not retrieve an ortholog of Rv0805 or ML2210, COG1409 (COG database) containing Rv0805 was examined for the presence of other mycobacterial phosphodiesterases. Bioinformatics analysis identified Rv2795c as another PDE from M. tuberculosis. Sequence analysis of Rv2795c revealed the presence of all the motifs conserved in the class III PDEs but Rv2795c shared only 22% sequence identity with Rv0805 and 19% sequence identity with CpdA. Importantly, an ortholog of Rv2795c was identified in M. leprae. Interestingly. Rv2795c and its orthologs branched away from Rv0805, making it phylogenetically distinct and hence warranting further characterization. Recombinant, purified MSMEG_2647 (the Rv2795c ortholog from M. smegmatis) was able to hydrolyze cyclic nucleotides and other phosphodiester substrates in vitro. The Km for colorigenic substrates was higher when compared to the Km of ML2210 or Rv0805 for these substrates. However, the kinetic parameters of MSMEG_2647 for cyclic nucleotides were comparable to those of ML2210 or Rv0805. MSMEG_2647 was a metal dependent enzyme and among the panel of metals tested, Mn2+ supported the highest in vitro catalytic activity of MSMEG_2647. Zn2+ inhibited the catalytic activity of MSMEG_2647. In order to gain insight into the catalysis of MSMEG_2647, the end products of cAMP hydrolysis by MSMEG_2647 were analysed using reverse phase HPLC. The assay revealed that the end products of cyclic nucleotide hydrolysis by MSMEG_2647 were different when compared to the end products of hydrolysis of the same substrates by Rv0805 or ML2210. This suggests differences in the architecture of the active site residues of the mycobacterial MPEs. A mutational anlaysis of the active site residues in MSMEG_2647 was carried out to identify residues involved in substrate recognition and metal coordination. Although Rv0805 and MSMEG_2647 shared only a 22% sequence identity, MSMEG_2647 displayed strict conservation in the core MPE motifs. Mutation of the active residues N97 and H98 in Rv0805 had led to an abrogation of its catalytic activity. However, corresponding mutations of N76A and H77A in MSMEG_2647, did not lead to a loss in its catalytic activity. A third mutation known to be important for the catalytic activity of Rv0805 (D19) was incorporated. The corresponding residue at D19 position was mutated to an alanine. The catalytic activity of MSMEG_2647D19AN76AH77A mutant was abrogated, suggesting that while the core MPE motifs are conserved between mycobacterial PDEs, differences in the ensemble of the active site residues contributing to their catalytic activity exist. Thus, at least two biochemically diverse PDE clades are found in mycobacterial species. In order to decipher the function of MSMEG_2647, its expression was monitored during the growth of M. Smegmatis. The promoter of MSMEG_2647 displayed maximum activity during the logarithmic phase of M. smegmatis growth after which its activity declined as M. smegmatis entered the stationary phase. However in contrast to this, the transcript corresponding to msmeg_2647 mRNA was found at both logarithmic and stationary phases. The MSMEG_2647 protein was also detected at both logarithmic and stationary phases of M. smegmatis. These results suggest that additional factors may contribute to the stability of msmeg_2647 mRNA and protein levels. Localization studies of MSMEG_2647 revealed that MSMEG_2647 was present in the cytosol as well as in the cell envelope fractions. Interestingly, over-expression of MSMEG_2647 did not result in a significant increase in PDE activity in various subcellular fractions, suggesting tight regulation on the in vivo activity in various subcellular fractions, suggesting tight regulation on the in vivo activity of MSMEG_2647. In addition, over-expression of MSMEG_2647 in M. smegmatis led to only a modest decrease in cAMP levels in M. smegmatis. These results suggested additional roles of MSMEG_2647 in the biology of mycobacteria. Overexpression of MSMEG_2647 peturbed the integrity of cell wall as assessed by the use of lipophillic indicators of cell growth, crystal violet and malachite green, and a cell wall targeting antibiotic, isoniazid. Analyzing the gene neighborhood of MSMEG_2647 provided an insight into its putative function. It was observed that the stop codon of msmeg_2647 overlapped with the start codon of msmeg_2648 and stop codon of msmeg-2648 overlapped with the start codon of msmeg_2649. RT PCR was carried out at logarhtimic and stationary phases of M. smegmatis growth, which revealed that a polycistronic mRNA was being transcribed. These results confirmed that msmeg_2647, msmeg_2648 and msmeg_2649 were a part of an operon. Interestingly, these three genes as a gene cluster were confined to only those actinobacteria that produced mycolic acids. An operon often encodes products that form multiprotein complexes and operate in a common pathway. Since there were a part of an operon, a GST pull-down approach was employed to test if MSMEG_2647, MSMEG_2648 and MSMEG_2649 could physically interact. It was observed that MSMEG_2647 interacted with MSMEG_2648 and MSMEG_2649. MSMEG_2648 in turn interacted with MSMEG_2649. A role for MSMEG_2647 as a scaffold recruiting MSMEG_2648 and MSMEG_2649 is therefore proposed. In turn, a complex formation with these proteins may regulate the activity of MSMEG_2647. Attempts to generate a knock out of msmeg_2647 in M. smegmatis by homologous recombination were not successful suggesting either the gene was essential or a polar effect on msmeg_2648(an essential gene for the viability of M. smegmatis) may not allow msmeg_2647 to be deleted from the genome of M. smegmatis. In summary, this study has identified and characterized two new phosphodiesterases from mycobacteria, one from the pathogenic mycobacterium, M. leprae and the other, a PDE from M. smegmatis that is conserved in all species of mycobacteria. Several, key biochemical differences were observed using biochemical and biological approaches. It appears that the cellular roles of mycobacterial phsophodiesterases may extend beyond cAMP hydrolysis, with these proteins not only regulating cell wall properties but also acting as scaffolding proteins in the cell.

Le travail s’intéresse aux effets de l’histoire de chargement sur le comportement et la durée de vie en fatigue de deux nuances (THYSSEN et CLI)d’un acier inoxydable austénitique 304L à la température ambiante. Les essais ont été réalisés en utilisant deux catégories d’éprouvettes. Les éprouvettes de la première catégorie (vierges) ont été soumises à des essais classiques de fatigue,alors que celles de la deuxième ont subi, avant les essais de fatigue, un pré-écrouissage monotone ou cyclique en déformation imposée. Les éprouvettes vierges manifestent un adoucissement cyclique suivi d’un durcissement cyclique alors que les éprouvettes pré-écrouies ne présentent qu’un durcissement cyclique. Les résultats montrent une grande influence du pré-écrouissage qui semble bénéfique en contrainte imposée, mais néfaste en déformation imposée,même en présence d'une contrainte moyenne de compression. Ces résultats sont discutés en termes d'évolution cyclique du module d'élasticité, des écrouissages isotropes et cinématiques, et de la densité d’énergie absorbées par cycle, dans différentes configurations : avec ou sans pré-écrouissage, en contrainte ou déformation imposées... / This study deals with the effect of the loading history on the cyclic behavior and the fatigue life of two kinds (THYSSEN and CLI) of 304L stainless steel at room temperature. The experiments have been performed using two specimens’ categories. The first one (virgin) has been submitted to only classical fatigue tests while in the second category, prior to the fatigue test, the specimen is subjected to a pre-hardening process under either monotonic or cyclic strain control. Cyclic softening followed by cyclic hardening are observed for the virgin specimens while only cyclic softening is exhibited by the pre-hardened specimens. The obtained results show that fatigue life is strongly influenced by the pre-hardening: it seems beneficial under stress control but detrimental under strain control, even in the presence of a compressive mean stress. The results are discussed regarding the cyclic evolution of the elastic modulus aswell as the isotropic and kinematic parts of the strain hardening, and strain energy density per cycle, in different configurations: with or without prehardening,stress or strain control...

Three non-peripherally substituted liquid crystalline bisphthalocyanine (Pc) compounds have been studied to examine the role of central metal ions lutetium (Lu), and gadolinium (Gd) and substituent chain lengths, i.e. octyl (C8H17) and hexyl (C6H13), in determining the physical properties. For the octyl substituted Pc molecules, the head-to-tail or Jaggregates within the as-deposited spun films produced a redshift of the optical absorption Q bands in relation to their 0.01 mgml-1 solutions. Annealing at 80˚C produced a well-ordered discotic liquid crystalline (LC) mesophase causing additional redshifts irrespective of the metal ion in case of C8LuPc2 and C8GdPc2. Formation of face-to-face or H-aggregated monomers led to blueshifts of the Q bands with respect to solution spectra for C6GdPc2, both as-deposited and annealed films. Stretching and bending vibrations of pyrrole, isoindole, and metal-nitrogen bonds in Pc rings showed Raman bands at higher energy for smaller metal ion. However, no change was observed for the difference in chain lengths. As-deposited C8LuPc2 and C6GdPc2 produced comparable Ohmic conductivity, of the value 67.55 Scm-1 and 42.31 Scm-1, respectively. C8GdPc2 exhibited two orders of magnitude less conductivity than the other two due to the size effect of the central ion and side chain length. On annealing, an increase of Ohmic conductivity was noticed in the isostructural octyl substituted phthalocyanines on contrary to a reduced conductivity in hexyl substituted one. An optical band shift of the C8LuPc2 and C8GdPc2 thin films occurred on oxidation by bromine vapour. Oxidations of Pc-coated ITO were also achieved by applying potential at 0.88 V and 0.96 V electrochemically for the C8LuPc2 and C8GdPc2 compounds, respectively. To explore the applications of these compounds in biosensing, in situ interaction studies between bromine oxidised compounds and biological cofactors nicotinamide adenine dinucleotide (NADH) and L-ascorbic acid (vitamin C) were carried out using optical absorption spectroscopy. Thin films of a non-peripherally octyl substituted LC lead phthalocyanine was exposed to 99.9 % pure hydrogen sulfide gas to produce hybrid nanocomposites consisting of lead sulphide quantum dots embedded in the analogous metal free phthalocyanine matrix. Trapping of charge carriers caused hysteresis in the current-voltage characteristics of the film on interdigitated gold electrodes. The charge hopping distance was found to be 9.05 nm, more than the percolation limit and responsible for forming two well-defined conducting states with potential application as a memristor.

The work was concerned with experimental study of the turbulent flame development process of alcohol fuels, namely ethanol and butanol, in an optically accessed spark ignition research engine. The fuels were evaluated in a single cylinder engine equipped with full-bore overhead optical access operated at typical stoichiometric part-load conditions with images captured using high-speed natural light imaging techniques (or chemiluminescence). The differences in flame development between the fuels was analysed to understand better the impact of high and low alcohol content fuels on combustion. Advanced image analysis, in conjunction with Ricardo WAVE simulation, allowed for the conclusion that the faster burning exhibited by ethanol was the result of the marginally higher laminar burning velocity providing a faster laminar burn phase and accelerating the flame into the turbulent spectrum thus reducing bulk flame distortion and better in-cylinder pressure development. Such physical reactions are often over-looked in the face of chemical differences between fuels. A further study into the variation of maximum in-cylinder pressure values was conducted focussing on iso-octane and ethanol. This study identified two phenomena, namely “saw-toothing” and “creep” in which cluster of cycles feed into one another. From this it became clear that the presence of high pressure during the exhaust process had a large influence on the following cycles. This is another often overlooked phenomenon of direct cycle-to-cycle variation whereby incylinder pressures during blowdown can dictate the duration, load or stability output of the following cycle. Finally the work investigated the impact on flame development of alcohol fuels when the overlap duration was altered. While the engine produced counterintuitive figures of residual gas, ethanol was confirmed as having greater synergy with EGR by displaying less impacted combustion durations c.f. iso-octane. Care should be taken however when analysing these results due to the unique valve configuration of the engine.

International Telemetering Conference Proceedings / November 04-07, 1991 / Riviera Hotel and Convention Center, Las Vegas, Nevada / Shortened cyclic codes are not cyclic, but many cyclic shifts of various code words are still part of the shortened code set. This paper addresses the probability of false synchronization obtained through polynomial division of a serial shortened cyclic code stream in a “sliding” window correlator.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy