<p>The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function.</p><p>Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. </p><p>Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect.</p><p>Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. </p><p>IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet.</p><p>It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.</p>

The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function. Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect. Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet. It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.

Neuropeptides are present in nerve fibers of the upper and lower airways. Local release of these substances may be of importance for the pathophysiology of airway disorders and may play a role in responses to different stimuli. However, little is known about the distribution of neuropeptides in the larynx. The superior laryngeal nerve is one of the vagal branches supplying the larynx. The aim of the present study was to investigate the fiber composition of this nerve and to analyse the distribution of different neuropeptides and mast cells in the larynx. The internal and the external branches of the superior laryngeal nerve had a similar number and size of the nerve fibers. Numerous unmyelinated fibers were evenly distributed in the branches. A large majority of the fibers were sensory myelinated and unmyelinated fibers; only a few of the myelinated fibers of the external branch ( 2-10 %) were motor. About a quarter of the unmyelinated fibers of the internal and the external branches had their cell bodies in the brainstem, and single myelinated and unmyelinated fibers emanated from the superior cervical ganglion. In every superior laryngeal nerve examined one to three spherical paraganglia were observed. These paraganglia contained cells which were similar to the type I and type II cells found in the carotid body and the paraganglia of the recurrent laryngeal nerve. Thin-walled sinusoidal blood vessels which were sometimes fenestrated were also present The laryngeal mucosa was supplied with nerve fibers exhibiting substance P- and calcitonin gene-related peptide-like immunoreactivity with regional differences in the distribution. The laryngeal side of the epiglottis and the ventral recess were richly supplied, and the vocal cords showed no evidence of immunoreactive nerve fibers. The distribution of connective tissue mast cells and mucosal mast cells/globular leucocytes was similar to that of nerve fibers displaying substance P- and calcitonin gene-related peptide-like immunoreactivity. These cells were found in close approximation to nerve fibers. Acetylcholinesterase-positive ganglionic cells in the larynx showed vasoactive intestinal polypeptide-, neuropeptide Y-and enkephalin-like immunoreactivity. Neuropeptide Y-like immunoreactivity was co-localized with tyrosine-hydroxylase/dopamine beta-hydroxylase-like immunoreactivity in nerve fibers in some blood vessel walls. Enkephalin-like immunoreactivity was rarely found in this location and co-localization with tyrosine- hydroxylase-like immunoreactivity was not detected. In glands and some blood vessel walls neuropeptide Y- and enkephalin-like immunoreactivity were localized in nerve fibers showing a positive acetylcholinesterase reaction and vasoactive intestinal polypeptide-like immunoreactivity. Thus, this indicates that neuropeptide Y is present in both the sympathetic and parasympathetic nervous systems, while enkephalin and vasoactive intestinal polypeptide are confined to the parasympathetic nervous system in the rat larynx. The present study shows that the superior laryngeal nerve is mainly sensory, and the study also provides a morphological basis for neuropeptide effects in laryngeal physiology/pathophysiology. / <p>S. 1-27: sammanfattning, s. 29-97: 6 uppsatser</p> / digitalisering@umu

WGS permet d'analyser et de déchiffrer l'étude de résistances de bactéries multirésistantes(MDR), en comprenant les différents mécanismes de résistance, les annuaires génétiques. Au cours de ma thèse de doctorat, j'ai réalisé: 1 revue de la littérature sur l'utilisation de nouveaux outils de diagnostic contemporains et les capacités dans la détection des foyers dans les maladies infectieuses causées par MDR. L'identification et l'analyse de résistances de bactéries multirésistantes Comme étant des Shewanellalgae, normalement de l'environnement marin, dans notre cas une souche clinique isolée du lavage bronchoalvéolaire d'un patient hospitalis avec pneumonie et Chryseobacteriumin dologenes, isolé d'une fibrose kystique du patient. Dans cette analyse, nous pouvons montrer que les bactéries environnementales telles que les S.algae peuvent être un réservoir de gènes de résistance aux antibiotiques. L'analyse exhaustive de ces bactéries a montré leur capacité à s'adapter à leurs écosystèmes, y compris l'acquisition de nouveaux éléments génétiques par transfert latéral de gènes. La détection des gènes impliqués dans la synthèse de peptides synthetasenon ribosomale et de polycétide synthétase peut avoir un rôle dans leur capacité à survivre dans des environnements hostiles tels que le tractus respiratoire des patients atteints de fibrose kystique ou leur présence chez des patients ayant subi plusieurs antibiotiques. Nous avons réalisé une analyse standardisée «insilico» afin de déterminer la résistance de ces bactéries et la présence de métabolites secondaires associés aux bactériocines et aux NRPS/PKS. L'application du NTS pour le séquençage du génome bactérien de nouvelles espèces bactériennes isolées dans le microbiome humain nous a permis de développer une plateforme capable d'analyser ces nouvelles espèces dans les 48heures. Ce travail permet de mieux comprendre la biodiversité des bactéries isolées dans le microbiome humain. / Theuse of WG Sallows to analyze and to decipherthe study of resistome of Multi Drug Resistant bacteria (MDR), understanding the different resistance mechanisms, genetic directories and their dissemination mechanisms at global level. During them y thesis I have achieved: 1. A literature review on the use of new contemporary diagnostic tools and capabilities in detecting out breaksin infectious diseases caused by MDR. 2: The identification and the analysis of resistome of multidrug resistant bacteria from clinical isolates suchasShewanellaalgae, normally marine environmental, in our case clinical strain isolated from the broncho alveolar lavage of a hospitalized patient with pneumonia and Chryseobacteriumin dologenes, isolated from a patient cysticfibrosis. In this analysis, we can show that environment albacteria suchas S.algae can be a reservoir of antibiotic resistance genes. The exhaustive analysis of these bacteria showed their ability to a dapttotheirecosystemsincludingtheacquisitionofnewgeneticelementsbylateralgenetransfer. The detection of genes in volved in the synthesis of nonribosomal peptide synthetase and polyketide synthases may have a role in their ability to survive in hostile environments suchas therespiratorytractofCFpatients or their presence inpatients having suffered multipleofantibiotic. 3:In this work,through theuse of the NTS onnew bacterial species isolated from human microbiome,we have a chieveda standardized analysis"insilico"to determine there sistome of these bacteria and the presence of secondary metabolites associated bacteriocins and the NRPS/PKS. The application of the NTS for sequenc in go bacterial genome of new bacterial species isolated in the human microbiome, allowe dus to develop a platform capable of analyzing the senew species within 48

Sortases are cell-membrane associated cysteine transpeptidases that are essential for the assembly and covalent anchoring of certain surface proteins to the cell wall in Gram-positive bacteria. Thus, they play critical roles in virulence, infection and colonization by pathogens. Sortases have been classified as type A, B, C, D, E and F based on their phylogeny and the target-protein motifs that they recognize. Sortase A (SrtA) enzymes participate in cell wall anchoring of proteins involved in bacterial adhesion, immune evasion, internalization, and phage recognition and in some cases pili formation. SrtA substrates are characterised by the presence of a C-terminal cell wall sorting signal as LPXTG motif, followed by a stretch of hydrophobic residues and a positively charged tail. Experimental and bioinformatics studies show that class A sortases are housekeeping as well as virulence determining proteins. Hence, Sortase A enzymes are considered as promising antibacterial drug targets, particularly because many organisms are developing multi-drug resistance behaviour. SrtA adopts an eight-stranded β-barrel structure and the overall fold is conserved among the sortase isoforms, with some modifications. The thesis candidate has determined the three dimensional (3D) crystal structures of wild-type and active site mutant of Sortase A from Streptococcus pneumoniae R6 strain by using X-ray diffraction method. The wild-type enzyme crystallized in P21 space group whereas active site cysteine mutant crystallized in C2 space group. In both the cases, N-terminal 81 residue deletion constructs (ΔN81) were used for crystallization. Uncommonly, both the structures showed a phenomenon of domain-swapping which resulted in the protein adopting a domain-swapped dimeric form. Two such dimers in wild-type protein and three dimers in mutant protein were observed in the asymmetric unit. To the best of our knowledge, our work reveals for the first time the occurrence of domain-swapping in sortase superfamily. Experimental techniques like size-exclusion chromatography, native-PAGE, analytical centrifugation and thiol cross-linking (carried out in our collaborator’s laboratory at National Institute of Immunology (NII), New Delhi, India) of functionally active wild-type SrtA from S. pneumoniae showed dimerization as well as domain-swapping in solution state. These results support the possibility that the protein indeed exists in a domain-swapped dimeric form and the determined structure is not the result of crystal packing artifact but is physiologically relevant as well. The work done by the thesis candidate covering crystallization of both, the active and inactive protein constructs, their structure determination using molecular replacement method, detailed structural analyses, structural comparisons with known SrtA structures and new structural findings are described in from Chapter 2 to Chapter 4. Based on the SrtA crystal structure the author of the thesis has also proposed various point mutations which are likely to disrupt domain– swapping and result in loss of dimer formation. In addition, as a part of the ongoing project in our laboratory, molecular dynamics studies of these domain-swapped dimers containing two sets of active site residues facing each other in a very compact volume have been initiated to understand substrate binding, which in future could lead to inhibitor design. Apart from the crystal structure analyses of SrtA structures, the author of the thesis has also carried out systematic crystal structure investigation of dipeptides and pentapeptides containing non-standard amino acids (ΔPhe, Aib and β-amino acids) along with computational studies. Conformationally restricted α,β-dehydrophenylalanine residue (ΔF) and α-aminoisobutyric acid (Aib) have been incorporated in highly amyloidogenic human Islet Amyloid Polypeptide (hIAPP) fragments. Amyloid deposits, observed in a vast majority of Type 2 diabetic patients, are primarily on account of misfolding and aggregation into fibrils of hIAPP, a 37 residue endocrine hormone secreted by pancreatic β-cells. It has been suggested that intermediates produced in the process of fibrillization are toxic to insulin producing β-cells. Hence, the inhibition of misfolding of hIAPP that involves structural transition from its native state (coil and/or helical and/or transient helical conformation) to β-sheet conformation, could be a possible strategy to mitigate Type 2 Diabetes Mellitus (T2DM). All the peptides discussed in this thesis were synthesized in our collaborator, Prof. V. S. Chauhan’s laboratory at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. In this work, author of the thesis has designed short peptides containing helicogenic residue, α,β-dehydrophenylalanine (ΔF) and determined their 3D crystal structures. It was found that pentapeptides, FGA∆FL and FGA∆FI act as inhibitors of hIAPP fibrillization. As revealed by crystal structure analysis, both the peptides have similar backbone conformation consisting of a ‘nest’ motif, which is an anion receptor. Molecular docking suggested that both the pentapeptides interact with the hIAPP20-27 segment, stabilizing the hIAPP in helical form by shielding the core aggregation initiation region. This reduces the possibility of oligomerization, formation of toxic intermediates and subsequently the transition to β-structure and fibrillization. Thus, the crystal structures of pentapeptide inhibitors together with computational docking studies suggest an atomic level model of the possible mode of action by which the FGAΔF(L/I) peptides manifest their fibrillization inhibition activity and this could be of value in the design of a new class of amyloid inhibitors. In another peptide design, L→U (Aib) mutation was done in core fibrillization region ANFLV i.e. hIAPP13-17. The resulting mutant peptide ANFUV as well as native fragment ANFLV was crystallized and their 3D crystal structures were determined. ANFLV crystallized in two space groups C2 and P2 adopting extended conformation. Crystal packing of ANFLV in both the crystals shows parallel beta sheet arrangement which is favoured and strengthened by hydrogen bonding between asparagine side chains of Asn-Asn pair each located in neighbouring parallel beta-strands. Hydrogen bonded Asn-Asn residue pairing in parallel beta-strands suggests its significant contribution during hIAPP fibril formation. The substitution L→U abolished its fibrillization property and the structure of ANFUV was solved by direct methods in P21 space group. The occurrence of β-bulge in ANFUV induced by Aib, as observed in crystal packing, suggests that Aib acts as a β-breaker through β-bulge inducing property in the highly amyloidogenic hIAPP segment. β-bulge forming property, an attribute of Aib as β-breaker may be responsible for the curtailment of fibrillization potential of the peptide in which the residue was incorporated. The aim of the anti-amyloid work is to design potent anti-fibrillization peptides and the work is important to design peptide based drugs to fight type II diabetes. The utilization of ΔPhe in the molecular self-assembly offers an added benefit in terms of variety and stability. Taking advantage of the conformation constraining property of ΔPhe residue, its incorporation in dipeptide molecules has been probed. The author has studied nanotube formation through molecular self-assembly, involving two classes of non¬standard amino acids i.e. ΔF and β-amino acids. FΔF in D-form, L-form and DL-mixture crystallized in different space groups forming rectangular/hexagonal channels constituting different channel dimensions. Recently, the application of FΔF nanotubes have been demonstrated in controlled drug delivery, showing the relevance of the work in health care. Another class of dipeptides containing β-amino acids (β-FF, β-FΔF, β-AΔF, β-VΔF, β¬LΔF, β-IΔF, and β-LF) was also explored for the self-assembled nanotube formation. These β-peptides were crystallized and their 3D structures were determined solely by the author of the thesis. Except the β-AΔF & β-LΔF, these peptides self-assemble and form rectangular/ hexagonal channels. Structures of ΔF and β-amino acid containing dipeptides forming ordered nanotubes through self-assembly are detailed in Chapters 8 and 9 in the thesis. Overall, the author of the thesis has crystallized and determined structures of more than twenty peptides. Experimentally, β-peptide nanotubes were observed to encapsulate drug molecules and thus might be useful as a drug delivery system. In the present thesis crystal structures of the following designed peptide sequences (including one natural sequence ANFLV) are reported in detail. Table 1 Peptide sequence Representation Length Discussed in 1. Phe-Gly-Ala-ΔPhe-Leu FGAΔFL 5 Chapter 6 2. Phe-Gly-Ala-ΔPhe-Ile FGAΔFI 5 Chapter 6 3. Ala-Asn-Phe-Leu-Val (2 forms) ANFLV_P2, ANFLV_C2 5 Chapter 7 4. Ala-Asn-Phe-Aib-Val ANFUV 5 Chapter 7 5. LPhe-ΔPhe (2 forms) LFΔF1 , LFΔF2 2 Chapter 8 6. DPhe-ΔPhe DFΔF 2 Chapter 8 7. DLPhe-ΔPhe DLFΔF 2 Chapter 8 8. LTyr-ΔPhe LYΔF 2 Chapter 8 9. LSer-ΔPhe LSΔF 2 Chapter 8 10. Boc-D,LPhe-ΔPhe Boc-DLFΔF 2 Chapter 8 11. Cbz-D,LPhe-ΔPhe Z-DLFΔF 2 Chapter 8 12. D,LMet-ΔPhe DLMΔF 2 Chapter 8 13. β-Phe-ΔPhe β-FΔF 2 Chapter 9 14. β-Phe-Phe β-FF 2 Chapter 9 15. β-Val-ΔPhe β-VΔF 2 Chapter 9 16. β-Ile-ΔPhe β-IΔF 2 Chapter 9 17. β-Leu-ΔPhe β-LΔF 2 Chapter 9 18. β-Leu-Phe β-LF 2 Chapter 9 19. β-Ala-ΔPhe β-AΔF 2 Chapter 9 20. Cyclo(Phe-ΔPhe) DKP-FΔF 2 Appendix C 21. Cyclo(Ile-ΔPhe) DKP-IΔF 2 Appendix C 22. Cyclo(Cha-Cha) DKP-ChaCha 2 Appendix C 23. Cyclo(Cha-Phe) DKP-ChaF 2 Appendix C 24. Cyclo(Cha-ΔPhe) DKP-ChaΔF 2 Appendix C 25. Cyclo(S-tritylCys-ΔPhe) DKP-CΔF 2 Appendix C Most of the dipeptides, except the N-terminal protected dipeptides, cyclic dipeptides (i.e. DKPs) and LSΔF, were found in the zwitterionic conformation and out of these, ten dipeptides resulted in tubular structures of dimensions in the nanoscale range. The thesis is organized into nine chapters and five appendices. Chapter 1 is an introduction to the work presented in the thesis, while Chapter 2, Chapter 3 and Chapter 4 describe the crystallographic work on the protein Sortase A. Chapter 5 is an introduction to the non-standard amino acids used for peptide designs and Chapter 6, Chapter 7, Chapter 8, Chapter 9 and Appendix C describe the crystallographic work on peptides. Chapter 1 starts with a general introduction to the Gram-positive bacteria containing sortase enzymes, and the bacterial cell-wall where sortase catalyzed proteins get attached for implicating their virulence during host-pathogen interactions. Pneumococcal diseases mostly affect children and their count has been observed to be higher than the combined total cases of malaria, AIDS and tuberculosis in child population worldwide. The chapter describes different virulence factors of S. pneumoniae out of which many are proteins. Among these, LPXTG containing proteins, which are the prime substrates of the sortase enzymes, are discussed in detail. Sortase enzymes, their classification and their structural studies with conserved ‘Sortase fold’ are discussed elaborately. A brief mention is made about the enzymatic activity of Sortase A to understand the transpeptidation mechanism. To appreciate the biomedical and biotechnological importance of the sortase enzyme, some potential applications of Sortase A are detailed in this chapter. A section is dedicated to describe the protein in the present study 'Sortase A from Streptococcus pneumoniae'. At the end, the scope of the present work, comprising of both protein and peptide crystallography, is presented. Chapter 2 begins with a brief account of the sequence analysis of Sortase A from S. pneumoniae and phylogenetic analysis of the sortase superfamily enzymes, followed by the details of protein purification & crystallization of two different constructs, wild-type SrtA from S. pneumoniae (Spn-∆N59SrtAWT and Spn-∆N81SrtAWT) as well as that of an active site cysteine mutant (Spn-∆N81SrtAC207A). This chapter includes X-ray intensity data collection of both types of crystals and data processing. Sortases are membrane anchored enzymes and therefore their expression as a full-length protein is a difficult task. Hence, the deletion of N-terminal transmembrane region from the enzyme is crucial for expression in its soluble form and is important for its successful crystallization. Thus, two wild-type constructs of S. pneumoniae sortase A, ∆N59SrtAWT (N-terminal 59 residue deletion) and ∆N81SrtAWT (N-terminal 81 residue deletion), and one active site mutant ∆N81SrtAC207A (N-terminal 81 residue deletion & active site Cys207 to Ala mutation) were cloned, expressed and purified. Cloning, expression and purification of the protein were done at the laboratory of our collaborator Prof. Rajendra P. Roy, Cell biology lab-II, National Institute of Immunology (NII), New Delhi, India. Crystallization of Spn-∆N59SrtAWT (~23 kDa) construct was initiated by manual screening using sparse matrix conditions from Hampton research. Initial trials were set up by following hanging-drop vapour diffusion method. Spn-∆N59SrtAWT construct crystallized in diamond, needle, rod and wedge-shaped crystal forms in more than one crystallization condition but they failed to diffract. Further trials were set up in microbatch plates that resulted in diamond-shaped crystals again, which diffracted up to a maximum of 4.0 Å resolution. Sequence comparison of the present construct was performed to modify the construct to achieve better diffraction. Thus, we made modifications in the Spn¬∆N59SrtAWT construct by deleting additional 22 residues at the N-terminal (i.e. total 81 residues deletion in the original sequence from the N-terminal) similar to SrtA from S. pyogenes. Hence, Spn-∆N81SrtAWT construct was prepared. For further crystallization experiments, we used the new construct Spn-∆N81SrtAWT. Similar to Spn-∆N59SrtAWT construct, crystallization set up for Spn-∆N81SrtAWT were done in microbatch plates at 293 K by using the Hampton conditions. During the crystallization set up, protein concentration was varied from 6-30 mg/ml. Notably, the protein crystals grown with 25 mg/ml protein concentration diffracted very well. Thus increasing the protein concentration helped to improve diffraction quality. Crystals obtained in Index-88 condition (0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0) diffracted up to 2.9 Å. Additive screen was used to improve its diffraction quality. This time many diffracting crystals were obtained and the best rod-shaped crystals grown in additive screen-79 (40% v/v (±)-1,3-butanediol) diffracted well up to 2.70 Å at home source. Thus, Spn-ΔN81SrtAWT crystallized at protein concentration of 25 mg ml-1 (in 10 mM Tris buffer, pH 7.5; 2 mM β-mercaptoethanol) with a condition containing 0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0, along with 40% v/v (±)¬1,3-butanediol as an additive agent by using microbatch-under-oil crystallization method. The chapter also includes crystallization of active site mutant Cys207Ala of ∆N81SrtAWT from S. pneumoniae (Spn-∆N81SrtAC207A). Spn-∆N81SrtAC207A mutant crystallized as a beautiful rectangular block type crystal (with a diffraction up to 2.7 Å at home source and up to 2.48 Å at synchrotron) at protein concentration of 25 mg ml-1 (in 10 mM Tris buffer, pH 7.5; 2 mM β-mercaptoethanol) with a condition containing 0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0, along with 1.0 M guanidine hydrochloride as an additive agent by using microbatch-under-oil crystallization method. Data collection was done on home-source diffraction facility for both the crystals however; mutant data in better resolution was collected by the author of the thesis at BM-14 beamline at ESRF, Grenoble, France. Thus, two crystals of SrtA, wild-type (Spn-∆N81SrtAWT) and its C207A mutant (Spn-∆N81SrtAC207A) were indexed satisfactorily in two space groups and their cell parameters are given in the following table 2. Table 2 Protein Space group a (Å) b (Å) c (Å) β (°) X-ray source Spn-∆N81SrtAWT P21 66.94 103.45 74.87 115.65 Home source Spn-∆N81SrtAC207A C2 155.57 113.33 81.34 90.80 Synchrotron The quality of both the data sets was assessed by SFCHECK and none of them showed twinning. Thus, the data sets collected were found appropriate and useful for structure determination as discussed in Chapter 3. Chapter 3 details the structure determination of Sortase A from S. pneumoniae for a wild-type construct (Spn-ΔN81SrtAWT) and for an active site cysteine mutant construct (Spn-ΔN81SrtAC207A). Sortase A from S. pyogenes was used as a search model in the molecular replacement (MR) method and a single solution for each data set was obtained through PHASER program. It resulted in four-molecules in wild-type sortase structure and six-molecules in the mutant structure in the respective crystal asymmetric unit. Iterative model building and structure refinement revealed a clear case of domain-swapping as observed in the electron density map. Finally, in the asymmetric unit of wild-type structure and in mutant protein structure two and three domain-swapped dimers were located, respectively. Simulated annealing and TLS refinement resulted in the protein structure with best refinement statistics. All these are elaborately discussed in Chapter 3. The last round of refinement of Spn-ΔN81SrtAWT converged to Rwork = 18.10% and Rfree = 23.39 % for 25152 unique reflections in the resolution range 30.7-2.7 Å whereas for Spn¬ΔN81SrtAC207A structure these parameters converged to Rwork = 18.25% and Rfree = 22.39% for 50010 unique reflections in the resolution range 47.15-2.48 Å. Chapter 4 describes the wild-type (Spn-ΔN81SrtAWT) as well as mutant (Spn¬ΔN81SrtAC207A) structures of Sortase A. The structure of Sortase A is not found in its commonly observed monomeric form but occur in a domain-swapped dimeric form. There are two dimers in Spn-ΔN81SrtAWT and three in Spn-ΔN81SrtAC207A as observed in the asymmetric unit. Each dimer contains two characteristic 8-stranded beta-barrel folds i.e. ‘sortase fold’ which is unique to the sortase superfamily. Unlike the structure of SrtA from other organisms known so far, the monomer does not form the 8-stranded beta-barrel all by itself. One monomer exchanges the β7 and β8 strands with the other monomer having β1 to β6 strands, thereby forming a complete 8-stranded β-barrel fold and such kind of two complete folds are present in each dimer. Because of the mutual swapping of strands between two monomers in a dimer, the dimer thus formed is defined as a domain-swapped dimer. This is the first time we have observed Sortase A structure in the domain-swapped dimeric form and is also the first example of domain-swapping in the sortase superfamily. Interestingly, all the catalytic residues (His141, Cys207 and Arg215) in each sortase fold in the swapped dimer lie at the secondary interface (open interface) generated by domain-swapping. Catalytic R215 (in one fold) interacts with D209 residue (in other fold of same dimer) through salt bridge interactions. Each dimer contains two pairs of such residues at the secondary interface but only one pair shows this kind of interaction. R215 (B-chain) interacts with D209 (A-chain) in AB dimer whereas R215 (D-chain) interacts with D209 (C-chain) in CD dimer. Asymmetry in the catalytic residues for their orientations and observed interactions at the secondary interface was evidenced. These active site residues were seen buried to a great extent except Arg215 which is slightly better exposed. It was difficult to find the exact substrate-binding pocket to approach the catalytic Cys207. However, biochemical and biophysical analyses (done at NII, New Delhi) provided strong evidence for the existence of the swapped-dimeric form at physiological pH as well. The enzyme exists with an equilibrium between its monomeric and dimeric forms, and the dimeric population is the most active species of the functionally active enzyme. An important role of Glu208 (in all the chains of two dimers; e.g. Chain A) was seen in the catalytic site where its side chain wobbles between His141 and H142 (both in Chain B) residues for interaction. Due to such kind of interactions the backbone conformation between C207-E208 (Chain B) shows variability, and coordinates the distance between His141 (ND1, Chain A) and Cys207 (SG, Chain B) each belonging to opposite chains in a swapped-dimer. The nature of side chain conformations of Glu208 in all the four sets of active site residues (in wild-type as well as in cysteine mutant structure) indicates that its movement presumably regulates thiolate-imidazolium acid-base pair formation which is a crucial condition for the sortase function where cysteine thiolate acts as nucleophile. Based on the crystal structure, the thesis candidate has suggested several mutants which might disrupt domain-swapping pointing to future studies on the system. Domain movement analyses by using HingeProt and DynDom servers indicate that the two-sortase folds joined with hinge loops in each dimer may show twist movement around the hinge axis. Possibly, such motion will affect the secondary interface covering active site residues and may allow increasing the exposure of the catalytic residues to perform catalysis. Presumably, such kind of domain movements may play a key role for the unique kind of regulatory mechanism for transpeptidase activity in sortase enzymes. However, more study has to be done to explore the role of these possibilities, if any, in the enzyme function and its regulation. Chapter 5 provides an introduction to non-standard amino acids, their sources and their uses in de novo peptide design; this is followed by a description of outcomes of structural investigations of modified peptides and their applications in various fields of medical and material science. Specifically, α, β-dehydrophenylalanine (ΔPhe), α-aminoisobutyric acid (Aib) and β-amino acids are discussed and their structures and conformational preferences are highlighted for their use in naturally occurring peptides or peptide fragments. Chapter 6 begins with an introduction to the human Islet Amyloid Polypeptide (hIAPP), which is an amyloidogenic protein and considered to be an important protein constituent of the amyloid plaques in pancreatic beta-cells in Type 2 diabetes patients. Therefore, fibrillization inhibition of hIAPP is considered as an important therapeutic approach to combat Type 2 Diabetes Mellitus (T2DM). In this chapter, the author of the thesis describes an approach to design peptide based inhibitors of hIAPP fibrillization using non¬standard amino acid ΔPhe (α,β-dehydrophenylalanine) residue. The first designed inhibitor has the sequence origin from hIAPP23-27 and it was developed by replacing I→ΔF (i.e. β¬favouring residue to helical conformation favouring) which resulted in FGAΔFL peptide. Fibrillization inhibition studies were done by co-incubation of hIAPP and FGAΔFL in 1:5 molar ratio and monitored by electron microscopy and thioflavin T binding assay that showed ~75% fibrillization inhibition. It suggested that the inhibitor is working effectively and thus the author determined its crystal structure by X-ray diffraction method. Peptide synthesis and experimental studies like electron microscopy and Thioflavin T binding assay were done in our collaborator’s laboratory at ICGEB, New Delhi, India. Subsequently a sequence similar peptide FGAΔFI was also designed by mutating L→I in the first inhibitor sequence. The resulting peptide FGAΔFI showed ~70% fibrillization inhibition. Following this success, crystal structures of both peptides were determined. FGAΔFL crystallized in P212121 space group whereas FGAΔFI crystallized in P21 space group. Though it was not anticipated, crystal structure analysis revealed that FGAΔFL and its analogue FGAΔFI harbour the anion receptor ‘nest’ motif. Both peptides dock with the helical form of hIAPP which may contribute to the inhibitory function of the peptides through their interaction with hIAPP in the core fibrillization region. These peptides effectively inhibit hIAPP fibrillization in vitro and it seems that these are unique examples of ‘nest-motif’ containing peptides that inhibit fibrillization. We also propose a model for fibrillization inhibition by these peptides; this has been published in Chemical Communications, a journal published by the Royal Society of Chemistry (RSC) and its reprint is enclosed within the thesis. In general, the approach described in the chapter may be applicable to target helices or helical intermediates and could be utilized in developing inhibitors useful, apart from T2DM, in other amyloid diseases including Alzheimer’s disease and Parkinson’s disease. Table 3 Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor FGAΔFL Orthorhombic, P212121 a=8.9951 (9) Åb=13.0144 (12) Åc=27.7521 (24) ÅV=3248.82 (5) Å3 Z=4 Mo Kα(λ=0.71073Å) 4703 Unique reflections 2581 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.95 % for [|Fo| > 4σ (|Fo|)] FGAΔFI Monoclinic, P21 a=8.9951 (9) Åb=13.0144 (12) Åc=27.7521 (24) Å β=92.637 (2)°V=935.59 (2) Å3 Z=2 Mo Kα(λ=0.71073Å) 4024 Unique reflections 2612 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.02 % for [|Fo| > 4σ (|Fo|)] Chapter 7 describes another important but less studied core fibrillization fragment of hIAPP (hIAPP13-17) different than the hIAPP23-27 discussed in the previous chapter. It also discusses the development of fibrillization inhibitor design from this segment. The fragment hIAPP13-17 i.e. ANFLV crystallized in two space groups; C2 with one molecule in the asymmetric unit and P2 with two molecules in the asymmetric unit. In these structures, ANFLV peptide shows fully extended conformation i.e. a β-conformation. Crystal packing shows parallel β-sheet arrangement with the involvement of dry ‘steric-zippers’. The peptide prefers cross-strand Asn-Asn residue pair by side chain hydrogen bonding and is discussed in comparison with a few crystal structures of hIAPP fragments, solved by Eisenberg’s group, containing Asn residue in their sequence. It is observed that if the Asn is located in the sequence between two terminal residues the peptide will arrange itself in parallel beta sheet. This supports a structural model of hIAPP fibril in parallel beta sheet arrangement as the hIAPP sequence contains several Asn residues. To develop an inhibitor from ANFLV, a partial success was achieved where the Leu → Aib mutant i.e. ANFUV was developed. ThT (Thioflavin T) and TEM (Transmission electron microscopy) results show that the mutant peptide does not fibrilize on its own. This strongly supports the fact that the native peptide (ANFLV) lost its inherent fibrillization characteristic with the introduction of Aib in place of Leu i.e. the resultant mutant ANFUV is a non-fibrillizing peptide. The logic behind the development was to retain ANF in the same extended conformation and then break the β-strand with β-breaker residues. The structure of ANFLV showed parallel beta-sheets along with the additional side chain-side chain hydrogen bonding in the same direction as the fibril axis. Thus, we retained the ANF region to keep the sticky segment in the design and then Leu was mutated to Aib, a known β-breaker, to alter backbone conformation. The crystal structure of the peptide ANFUV resulted in the similar ANF region in beta conformation and Aib in helical conformation. Interestingly, in this situation the conformation of Aib develops a beta-bulge observed in the crystal packing and this bulge structure probably turned the peptide to have non-fibrillizing characteristics. These results will be useful in designing peptide inhibitors by using U as a beta breaker to inhibit hIAPP fibrillization. Table 4 Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor ANFLV1 Monoclinic, C2 a=36.1350 (20) Åb=4.8050 (10) Åc=19.4190 (20) Å β=98.644 (5)°V=3333.40 (27) Å3 Z=4 Synchrotron (λ=0.77490 Å) 1982 Unique reflections 1825 [|Fo| > 4σ (|Fo|)] Direct methods: Sir92 & SHELXL97 11.71% for [|Fo| > 4σ (|Fo|)] ANFLV2 Monoclinic, P2 a=18.7940 (80) Åb=4.7970 (10) Åc=35.4160 (50) Å β=103.929 (10)°V=3099.03 (81) Å3 Z=4 Synchrotron (λ=0.77490 Å) 2651 Unique reflections 2580 [|Fo| > 4σ (|Fo|)] Direct methods: Sir92 & SHELXL97 15.39% for [|Fo| > 4σ (|Fo|)] ANFUV Monoclinic, P21 a=10.8140 (22) Åb=9.1330 (18) Åc=16.7540 (34) Å β=107.960 (30)°V=1574.07 (161) Å3 Z=2 Synchrotron (λ=0.97918 Å) 1426 Unique reflections 1398 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.45% for [|Fo| > 4σ (|Fo|)] Chapter 8 elaborates the self-assembly of α-dipeptides containing conformationally constrained achiral amino acid, α,β-dehydrophenylalanine (ΔF). The structural polymorphism in LFΔF peptide and the resulting self-assembly are discussed. Its D-isomer (DF∆F) and its racemic mixture (DLF∆F) are also discussed as these peptides self-assemble to give channel-forming assemblies. In addition to LFΔF, crystal structures of LYΔF, DLMΔF and LSΔF peptides and their self-assemblies are presented as well. Except DLMΔF xi and N-terminal protected DLFΔF (Boc-DLF∆F and Z-DLF∆F) peptides, the other dipeptides discussed in this chapter resulted in tubular structures of nanoscale dimensions through molecular self-aggregation. Table 5 Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor LFΔF1 Hexagonal, P65 a=23.1873(24) Åb=23.1873(24) Åc=5.5260(8) ÅV=2573.01(5) Å3 Z=6 Mo Kα(λ=0.71073Å) 3489 Unique reflections 2915 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 6.19% for [|Fo| > 4σ (|Fo|)] LFΔF2 Monoclinic, P21 a=5.5739(2) Åb=13.1383(4) Åc=13.5816(4) Å β=96.137(2)°V=988.90(2) Å3 Z=2 Mo Kα(λ=0.71073Å) 4865 Unique reflections 3402 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 4.35% for [|Fo| > 4σ (|Fo|)] DFΔF Orthorhombic, P21212 a=13.1690(21) Åb=25.3673(40) Åc=5.5622(9) ÅV=1858.12(5) Å3 Z=4 Mo Kα(λ=0.71073Å) 4370 Unique reflections 3426 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 4.44% for [|Fo| > 4σ (|Fo|)] DLFΔF Monoclinic, P21/c a=5.5392(14) Åb=26.0376(55) Åc=13.1839(27) Å β=90.278(16)°V=1901.46(8) Å3 Z=4 Mo Kα(λ=0.71073Å) 2051 Unique reflections 1264 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 7.08% for [|Fo| > 4σ (|Fo|)] LYΔF Hexagonal, P65 a=23.5523(4) Åb=23.5523(4) Åc=5.5183(1) ÅV=2650.96(1) Å3 Z=6 Mo Kα(λ=0.71073Å) 2746 Unique reflections 1871 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 3.91% for [|Fo| > 4σ (|Fo|)] LSΔF Monoclinic, P21 a=5.2998(20) Åb=9.6732(30) Åc=14.1827(57) Å β=95.604(27)°V=723.62(20) Å3 Z=2 Mo Kα(λ=0.71073Å) 1978 Unique reflections 1558 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 13.59% for [|Fo| > 4σ (|Fo|)] DLMΔF Monoclinic, P21/c a=9.9032(5) Åb=8.6675(4) Åc=34.0283(18) Å β=90.088(3)°V=29

No description available.

L’amyloïdose, une maladie progressive et incurable, implique une vaste panoplie de pathologies et de pathogénèses, qui est expliquée par la grande variabilité biologique et structurale des protéines responsables de la formation des dépôts d’amyloïde. L’amyline (polypeptide amyloïde des îlots pancréatiques, IAPP) est une protéine très susceptible de subir des changements de conformation impliquant les feuillets bêta et conférant aussi des propriétés physicochimiques distinctes. Cette protéine prend alors une forme fibrillaire et se dépose dans les îlots de Langerhans chez les humains atteints de diabète de type 2 ou d’insulinome. Ces dépôts d’amyloïde pancréatique (AIAPP) ont été décrits chez certaines espèces animales telles que les félins domestiques, les grands félins, le raton laveur et les primates non humains. La formation de dépôts d’amyloïde contribue à la pathogénèse du diabète de type 2, mais les mécanismes qui induisent la conversion de l’amyline (IAPP) en amyloïde (AIAPP) ne sont pas complètement compris. Les hypothèses du projet sont que certaines variations présentes dans les séquences peptidiques de l’IAPP provenant de différentes espèces animales jouent un rôle critique pour la formation de fibrilles et que plusieurs composés chimiques aromatiques/phénoliques sont capables d’abroger la formation de dépôts d’amyloïde. Le projet de recherche consiste donc à caractériser la propension des différentes isoformes animales d’IAPP à former de l’amyloïde in vitro afin d’identifier les acides aminés jouant un rôle clé dans cette transformation structurale et ultimement d’inhiber la formation d’amyloïde pancréatique. Le projet se divise en deux volets principaux. Le premier consiste à identifier les différentes séquences peptidiques de l’IAPP retrouvées chez les espèces animales. L’objectif est d’identifier les acides aminés jouant un rôle clé dans la formation d’amyloïde. Le gène de l’IAPP a été séquencé chez plus d’une quarantaine d’espèces. Le potentiel d’agrégation des séquences obtenues a été simulé à l’aide d’outils bioinformatique. Une librairie de 23 peptides a été commandée afin de procéder à des analyses physicochimiques in vitro permettant d’évaluer le potentiel amyloïdogénique (test fluorimétrique à la thioflavine T, essai de liaison au rouge Congo, dichroïsme circulaire, microscopie électronique à transmission) et cytotoxique (sur une lignée cellulaire provenant d’insulinome : INS-1). Les analyses effectuées à partir de la librairie constituée de 23 peptides ont permis d’identifier trois séquences ne formant pas d’amyloïde et qui proviennent des espèces animales suivantes : le tamarin lion doré (Leontopithecus rosalia), le grand dauphin (Tursiops truncatus) et l’alpaga (Vicugna pacos). Un site potentiellement critique est le segment 8-20 présentant le motif NFLVH qui ne forme plus d’amyloïde lorsqu’il est remplacé par le motif DFLGR ou KFLIR. Les acides aminés 29P, 14K et 18R sont également impliqués dans l’inhibition de la transformation structurale en fibrille. La dernière partie du projet consiste à inhiber la formation de l’amyloïde en utilisant des composés chimiques commercialisés (hypoglycémiants, anti-inflammatoires non stéroïdiens) ou nouvellement synthétisés dans notre laboratoire (les aryles éthyles urées). Un criblage d’une soixantaine de composés chimiques a été conduit dans cette étude. Leur efficacité a été testée sur l’IAPP humaine, qui possède un fort potentiel amyloïdogénique. Les techniques utilisées sont les mêmes que celles exploitées précédemment. L’essai de liaison croisée photo-induite ("photo-induced cross-linking of unmodified proteins", PICUP) a été réalisé afin d’étudier les formes intermédiaires (monomères, oligomères). Un total de 11 composés chimiques a démontré un potentiel à inhiber l’agrégation des fibrilles. Pour la classe des hypoglycémiants, le glyburide, le répaglinide et la troglitazone ont montré l’activité thérapeutique la plus élevée pour retarder et réduire la formation de fibrilles. Les anti-inflammatoires antiamyloïdogènes actifs incluaient le diclofenac, le méloxicam, le phénylbutazone, le sulindac et le ténoxicam. Les aryles étyles urées les plus intéressantes étaient la EU-362 et la EU-418. Tous ces composés ont conféré une protection cellulaire contre l’activité cytotoxique des fibrilles. Les molécules actives possèdent des éléments structuraux communs tels des substituants donneurs d’électrons (alcool, amine, halogène) sur un noyau benzène. En conclusion, ce projet de recherche a permis de caractériser l’IAPP chez diverses espèces animales, dont plusieurs chez lesquelles elle n’avait pas encore été décrite, de déterminer les sites jouant un rôle clé dans sa transformation en amyloïde et, ultimement, de tester le potentiel thérapeutique de nouveaux agents antiamyloïdogènes dans le diabète de type 2. Nous espérons que ce projet ouvrira ainsi la porte à de nouvelles stratégies de traitement. / Amyloidosis is a progressive and, as of now, incurable disease caused by the deposition of insoluble proteins. Amyloid research over the past decades focused on the characterization of the substantive biological variability of amyloid deposits. Amyloidosis encompasses a diversity of pathological manifestations, explained by the diversity of underlying causal proteins. In the pancreas of susceptible species, islet amyloid polypeptide (IAPP) is a precursor for an amyloid protein (AIAPP), which has a characteristic fibrillar structure and resistance to physical agents. This folded protein deposits in the islets of Langerhans of patients with type 2 diabetes or islet cell tumors (insulinoma). Amyloid deposits have also been well characterized, anatomically, in feline and non-human primate species. Amyloid fibril formation contributes to the pathogenesis of diabetes mellitus but the precise pathophysiologic factors involved in the fibrillization of IAPP as well as resultant islet injury remain to be elucidated. Further understanding of the causative factors in the fibrillogenesis of IAPP will be requisite in the development of therapeutic strategies to disrupt the amyloidosis process. This project hypothesizes that the specific variations found in IAPP peptide sequences among different animal species are critical for IAPP fibrillization. Also, some aromatic/polyphenolic compounds can abrogate fibrillization. The main objective forms the basis for development of new therapeutic tactics to impede amyloid formation and associated cellular injury. Thus this project has two specific aims. The first specific aim was to identify critical variations in IAPP amino acid sequences from different animal species and to assess their amyloidogenic potential. To accomplish this, the IAPP gene was isolated and sequenced from paraffin-embedded tissues from various animals (40 species). The aggregation potency was assessed for each sequence using in silico analysis. A library of 23 peptides was prepared from sequences that were distinctly different and their amyloidogenic potential was assessed in vitro using physicochemical analysis (thioflavin-T assay, Congo red binding assay, far-UV circular dichroism, transmission electron microscopy) and cytotoxicity assays (insulinoma cell line INS-1). Among this peptide library, three were non-amyloidogenic and corresponded to the following animal species: golden lion tamarin (Leontopithecus rosalia), commun bottlenose dolphin (Tursiops truncates) and alpaca (Vicugna pacos). Segment 8-20 of the peptide was critical for amyloid formation and the substitution of the NFLVH motif found in this region by a DFLGR or KFLIR motif impeded fibrillization. The amino acids 29P, 14K and 18R were also demonstrated to abrogate fibril formation. The second objective consisted in abrogating IAPP fibrillogenesis using conceptualized aromatic/polyphenolic structures, specifically hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl urea agents. This part of the project involved molecular screening of more than 60 compounds. Their efficacy at inhibiting amyloid formation was assessed in vitro on human IAPP, which exhibits the highest amyloidogenic potential. Techniques included the above-mentioned methods, with the addition of photo-induced cross-linking of unmodified proteins (PICUP). A total of 11 compounds showed potential in abrogating IAPP aggregation. Among the hypoglycemic agents evaluated, glyburide, repaglinide and troglitazone showed the highest potency in reducing fibril formation. The NSAIDs that displayed anti-amyloidogenic activity were diclofenac, meloxicam, phenylbutazone, sulindac and tenoxicam. EU-362 and EU-418 were the hit compounds resulting from the screening of the aryl ethyl urea (EU) class. Additionally, these anti-amyloidogenic molecules conferred a protection against fibril cytotoxicity. All of the active molecules bear a commun motif composed of benzene ring with electron donor moieties, such as alcohol, amine or halide. To conclude, this project characterized IAPP in several animal species in which it has not been previously described and improves our understanding of the amyloidogenesis process. Moreover, the therapeutic potential of hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl ureas agents as anti-amyloidogenic compounds was evaluated. It is conceivable that the additional information hereby gained on the regulation of amyloidogenesis may point towards new therapeutic strategies for diabetic patients.

Additionally, cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT) pore, failed to prevent hIAPP-induced DeltaPsim collapse, cytochrome c and AIF release and caspase-3 activation, indicating that the MPT pore was not involved in hIAPP-induced apoptosis. On the other hand, potential crosstalk between the extrinsic and intrinsic apoptotic pathways was demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. / It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by the generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little information is available about the effect of hIAPP on mitochondrial function of beta cells and protection of PC against hIAPP-induced cytotoxicity. In this thesis, I hypothesize that hIAPP may impair beta cell function with the involvement of mitochrondrial dysfunction, and this effects could be attenuated by PC. Therefore, the aim of this study was to investigate the role of mitochondria in hIAPP-induced apoptosis, the in vitro protective effects of PC and explore the underlying mechanisms. / It was found that hIAPP induced apoptosis in INS-1E cells with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (DeltaPsim). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS1-E cells was effectively restored by co-treatment with PC. / Our results showed that hIAPP inhibited the INS-1E cell growth in a dose-dependent manner. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. hIAPP induced DNA fragmentation and chromatin condensation, which were key characteristics of cell apoptosis. These changes were inhibited by PC as examined by TUNEL assay and DAPI staining. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malonaldehyde (MDA), as well as changes of activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 kinase, and these effects were effectively suppressed by PC. / Taken together, I have demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis, which was attenuated by PC through attenuating oxidative stress, modulating JNK and p38 pathways and reducing mitochondrial dysfunction. / Li, Xiaoling. / Adviser: Juliana Chung Ngor Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In dieser Arbeit wurde die Rolle selektiver, nicht-kovalenter Wechselwirkungen bei der Selbstorganisation von Diblockcopolymeren untersucht. Durch Einführung elektrostatischer, dipolarer Wechselwirkungen oder Wasserstoffbrückenbindungen sollte es gelingen, komplexe Mesostrukturen zu erzeugen und die Ordnung vom Nanometerbereich auf größere Längenskalen auszuweiten. Diese Arbeit ist im Rahmen von Biomimetik zu sehen, da sie Konzepte der synthetischen Polymer- und Kolloidchemie und Grundprinzipien der Strukturbildung in supramolekularen und biologischen Systemen verbindet. Folgende Copolymersysteme wurden untersucht: (i) Blockionomere, (ii) Blockcopolymere mit chelatisierenden Acetoacetoxyeinheiten und (iii) Polypeptid-Blockcopolymere. / In this work, the basic principles of self-organization of diblock copolymers having the in&#172;herent property of selective or specific non-covalent binding were examined. By the introduction of electrostatic, dipole&ndash;dipole, or hydrogen bonding interactions, it was hoped to add complexity to the self-assembled mesostructures and to extend the level of ordering from the nanometer to a larger length scale. This work may be seen in the framework of biomimetics, as it combines features of synthetic polymer and colloid chemistry with basic concepts of structure formation applying in supramolecular and biological systems. The copolymer systems under study were (i) block ionomers, (ii) block copolymers with acetoacetoxy chelating units, and (iii) polypeptide block copolymers.

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.