Single-particle cryogenic electron microscopy (cryo-EM) has become a powerful mainstay tool in high resolution structural biology thanks to advances in hardware, software and sample preparation technology. In my thesis, I utilized this technique to unravel the function of various challenging biological macromolecules. My first focus was bacterial ribosomal biogenesis: understanding how bacteria assemble their ribosomes. Ribosomes are the factories of the cell, responsible for manufacturing all proteins. Ribosomes themselves are huge, with the bacterial version made of 52 proteins and 4566 RNA nucleotides. How these components assemble has long been a mystery. Early groundbreaking work sketched out a biogenesis pathway using purified components in vitro – but under non-physiological conditions. We sought to understand how the bacterial ribosome – specifically the large subunit 50S – is built inside the cell. To achieve this, we engineered a conditional knock-out bacterial strain that lacked one specific ribosomal protein (L17). This caused the cells to accumulate incomplete intermediates along the 50S biogenesis pathway. These intermediates were purified and examined with mass spectrometry and single-particle cryo-EM. Two major hurdles arose in this project: firstly, the biogenesis intermediates exhibited a preferred orientation when vitrified for cryo-EM analysis. This means that instead of showing many different views required for reconstruction of the 3D structure, the intermediates only adopted one view on the cryo-EM grid. To overcome this problem, we engineered a method to induce additional views on the microscope by tilting the stage. Using another test protein that also exhibited preferred orientation (hemagglutinin), we optimized and characterized this new tilt methodology and showed it was generally applicable to overcoming preferred orientation, regardless of type of specimen. We also created a software tool, called 3DFSC (3dfsc.salk.edu), for other microscopists to calculate the degree of directional anisotropy in their structures due to preferred orientation. Using this tilt strategy finally enabled the structural elucidation of our 50S intermediates. The second challenge in the project was the large amount of heterogeneity present in the sample. Through hierarchical 3D classification schemes using the latest software tools, we obtained 14 different 50S intermediate structures, all from imaging a single cryo-EM grid. By analyzing the missing components of each intermediate, and corroborating these observations with mass spectrometry data, we outlined the first in vivo 50S assembly pathway, and showed that ribosome assembly occurs step-wise and in parallel pathways. My second focus was on pushing the resolution limits of single-particle cryo-EM using adeno-associated virus (AAV) serotype 2 homogeneous virus-like particles (VLPs) that lack DNA. Exploiting several technical advances to improve resolution, including use of gold grids, per-particle CTF refinement, and correction for Ewald sphere curvature, we managed to obtain a 1.86 Å resolution reconstruction of the AAV2L336C variant VLP, the highest resolution icosahedral virus reconstruction solved by single-particle cryo-EM to date. Using our structure, we were able to show improvements using Ewald sphere curvature correction and shed light on the mechanistic basis as to why the L336C mutation resulted in defects in genome packaging and infectivity compared to the WT viral particles. My third focus was the understanding of small membrane proteins involved in infectious diseases. Membrane proteins are a challenge to work with due to the need for them to be extracted from the lipid bilayer for studies as compared to soluble proteins. Infectious diseases have a huge burden on society, with the top three infectious agents accounting for 2.7 million deaths in 2016. The third most deadly infectious disease is malaria, a mosquito-borne parasite which kills 450,000 people annually. One drug used early on for treating malaria was chloroquine but its usefulness waned due to development of resistance. Chloroquine resistance is mediated by the chloroquine resistance transporter (PfCRT). Although small (49 kDa) for single-particle cryo-EM, we solved its structure by using fragment antibody technology to add mass and help with image alignment and 3D reconstruction. The 3.2 Å structure resembles other drug metabolite transporters, and the chloroquine resistance mutations map to a ring around the central cavity, suggesting this central pore as the drug binding site. Tuberculosis (TB) is the top killer, above malaria and HIV/AIDS, being responsible for 1.3 million deaths. In TB, a common antibiotic target is the bacterium’s cell wall synthesis machinery. One family of such enzymes is the arabinosyltransferases, which synthesize the critical arabinose sugars. Using single-particle cryo-EM, we solved two high resolution structures of one such essential enzyme, AftD. Due to the low yield of the protein, a picoliter automated sample dispensing robot was crucial to allow for initial cryo-EM analysis. We then performed mutagenesis studies in M. smegmatis, a TB model organism, which uncovered the critical amino acid residues in the active site and determined that a bound acyl-carrier-protein was likely involved in allosteric inhibition of AftD’s active site. Another member of the family, EmbB, is the target of a widely used frontline TB drug called ethambutol. We have solved the high resolution structures of the apo and putative drug-bound states of EmbB, allowing us to map out, for the first time, both the active site and drug-resistance mutations of this crucial enzyme. The atomic structures of the functional pockets of Mycobacterial AftD and malarial PfCRT will hopefully enable structure-based drug design to improve existing drugs or potentially even develop new treatments against these infectious maladies. In conclusion, the continual and breathtaking improvements in single-particle cryo-EM methodology has been instrumental in allowing the elucidation of the aforementioned biological macromolecules from ribosome biogenesis intermediates, to AAV2 vehicle, Plasmodium drug resistance transporter to mycobacterial glycosyltransferases – structures of which help explain biological function.

<p>Different prosthetic groups containing a trialkylstannyl- and an electrophilic group have been synthesised and labelled with the accelerator produced ⁷⁶Br (T_1/2=16 h) through oxidative bromination. The labelled prosthetic groups were conjugated to amino-containing macromolecules such as proteins and 5´-modified oligonucleotides.</p><p><i>N</i>-Succinimidyl 4-[⁷⁶Br]bromobenzoate <b>14 </b>was synthesised in 65 % radio-chemical yield and was conjugated to 5´-hexylamino-modified phosphodiester and phosphorothioate oligonucleotides in 12-19 % isolated radiochemical yield. The stability of the ⁷⁶Br-oligonucleotide-conjugates <i>in vivo</i> in rats was investigated. No degradation from the 5´-end, resulting in labelled, low molecular weight compounds was detected. Compound <b>14</b> has also been used for labelling of different proteins in 23-61% radiochemical yield.</p><p><i>N</i>-Succinimidyl-5-[⁷⁶Br]bromo-3-pyridinecarboxylate <b>17</b> and methyl-4-[⁷⁶Br]bromo-benzimidate <b>15 </b>were synthesised from the corresponding trimethylstannyl-compound in 25% and 40 % yield respectively. Compounds <b>14 </b>and <b>17</b> were conjugated to ε-Boc-octreotide in 55 and 50% isolated radiochemical yield respectively after microwave heating. Compound <b>15</b> did not react with octreotide under the conditions investigated. The two ⁷⁶Br-labelled octreotide derivatives showed different lipophilicity and different binding-properties to tissue from meningiomas.</p><p>Hyaluronic acid, a polysaccharide, was modified with tyramine and labelled by oxidative bromination using ⁷⁶Br in 10% radiochemical yield.</p><p>The generator produced ⁶⁸Ga (T_1/2=68 min) was used to label octreotide and oligonucleotides modified with the metal chelating group 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). ⁶⁸Ga-DOTA-octreotide was isolated in 65% radiochemical yield and a phosphorothioated ⁶⁸Ga-DOTA-oligonucleotide was isolated in 35% radio-chemical yield after 30 min synthesis time.</p><p>Compound<b> 14 </b>was reacted with 3-aminomethylbenzylamine to give compound <b>18</b>. The specific radioactivity<b> </b>of<b> 18 </b>was determined to be 36 GBq/µmol by measuring the ratio between the mass-peaks for the ⁷⁶Br and ⁷⁹Br-compounds using packed-capillary LC-MS.</p>

Nowadays, polymer chemists undertake considerable efforts to achieve the preparation of new macromolecules and a perfect control over the macromolecular engineering, i.e. the mass parameters but also over the chain and end-group compositions, topology, etc… is definitively expected. In addition, more complex architectures, such as brush (co)polymers, jellyfish-like topologies…, are required to improve or drastically modify the physicochemical properties of the materials. As a direct consequence of the development of such complex molecular objects, sophisticated techniques are required for the in-depth characterization of the macromolecules, since the exact compositions and structures should be fully and unambiguously identified. Given the fact that the usual characterization tools such as Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC) are extensively used, their abilities have been intensively developed to account for the increasing complexity and diversity of the targeted molecules. Nevertheless, all the usual techniques are averaging methodologies since they only provide pieces of information about the polymer mixture instead of affording data on the individual macromolecules. Since few decades, mass spectrometry (MS) has become as used as NMR and GPC for polymer characterization. In the context of large molecules analysis, MS undoubtedly underwent an impressive craze with the development of two modern ionization procedures, namely Electrospray Ionization (ESI) and Matrix-assisted Laser Desorption/Ionization (MALDI). Those ionization procedures permit the vaporization of macromolecules allowing the intact polymers to be analyzed without a too extensive level of degradation. ESI and MALDI are often considered as soft ionization methods since they offer the possibility to observe ions corresponding to the intact molecules. After their production in the ion source, ions corresponding to the polymer molecules can be mass analyzed by the mass spectrometer and important parameters such as the molecular weight distribution (Mn and Mw), polydispersity index (PDI), the nature of the monomer units and the end-groups can be derived from the measure of the mass-to-charge ratios of the produced ions. In the first part of the present thesis, we studied the MS behavior of different classes of polymers when submitted to ESI and MALDI ionizations. The investigations were devoted to the validation of MS as a truly reliable methodology for fragile polymers such as aliphatic polyesters for instance. In this context, a preliminary MS investigation on semi-telechelic polyethers revealed the importance of the source parameters for the characterization of polymers presenting fragile moieties. We also demonstrated the huge importance of the matrix molecule selection for the MALDI analyses of polymers. In particular, we introduced a new matrix for the MALDI measurements of electroconjugated polymers such as polythiophenes. After the study of the influence of the source parameters on the MS data, a complete study by single stage MS and double stage MS (MS/MS) on newly synthesized polylactides (PLA) was performed. The PLA samples were prepared following original procedures using carbene as catalyst. Finally, to achieve the MS study of PLA ions, we used ion mobility-mass spectrometry (IM-MS) experiments to obtained information on the tridimensional structure of the gas phase PLA ions. In particular, we put a special emphasis on the influence of the charge and size of the polymer chains on their gas-phase conformations. The conclusions derived from the MS/MS and IM-MS results were fully supported by theoretical calculations. In the second part of the thesis, the acquired MS experience was applied to the fine characterization of macromolecules presenting complex architectures obtained by two different polymerization procedures: (i) cobalt-mediated radical polymerization of inter alia acrylonitrile and vinyl acetate and (ii) ring-opening polymerization (ROP) of lactones using non-organometallic catalysts. In particular, mass spectrometry was used to tune the experimental conditions for the ROP of â-lactones using different phosphazenes as catalysts. As an ultimate conclusion, this work points to the very efficient synergy between polymer synthesis, mass spectrometry and theoretical calculations. We believe that this thesis paves the way for innumerable possibilities in the future.

Different prosthetic groups containing a trialkylstannyl- and an electrophilic group have been synthesised and labelled with the accelerator produced 76Br (T1/2=16 h) through oxidative bromination. The labelled prosthetic groups were conjugated to amino-containing macromolecules such as proteins and 5´-modified oligonucleotides. N-Succinimidyl 4-[76Br]bromobenzoate <b>14 </b>was synthesised in 65 % radio-chemical yield and was conjugated to 5´-hexylamino-modified phosphodiester and phosphorothioate oligonucleotides in 12-19 % isolated radiochemical yield. The stability of the 76Br-oligonucleotide-conjugates in vivo in rats was investigated. No degradation from the 5´-end, resulting in labelled, low molecular weight compounds was detected. Compound <b>14</b> has also been used for labelling of different proteins in 23-61% radiochemical yield. N-Succinimidyl-5-[76Br]bromo-3-pyridinecarboxylate <b>17</b> and methyl-4-[76Br]bromo-benzimidate <b>15 </b>were synthesised from the corresponding trimethylstannyl-compound in 25% and 40 % yield respectively. Compounds <b>14 </b>and <b>17</b> were conjugated to ε-Boc-octreotide in 55 and 50% isolated radiochemical yield respectively after microwave heating. Compound <b>15</b> did not react with octreotide under the conditions investigated. The two 76Br-labelled octreotide derivatives showed different lipophilicity and different binding-properties to tissue from meningiomas. Hyaluronic acid, a polysaccharide, was modified with tyramine and labelled by oxidative bromination using 76Br in 10% radiochemical yield. The generator produced 68Ga (T1/2=68 min) was used to label octreotide and oligonucleotides modified with the metal chelating group 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). 68Ga-DOTA-octreotide was isolated in 65% radiochemical yield and a phosphorothioated 68Ga-DOTA-oligonucleotide was isolated in 35% radio-chemical yield after 30 min synthesis time. Compound<b> 14 </b>was reacted with 3-aminomethylbenzylamine to give compound <b>18</b>. The specific radioactivity<b> </b>of<b> 18 </b>was determined to be 36 GBq/µmol by measuring the ratio between the mass-peaks for the 76Br and 79Br-compounds using packed-capillary LC-MS.

The synthesis and optoelectronic properties of biphenyl based conjugated copolymers with varying acceptor units in the polymer backbone were investigated. The well known Donor-Acceptor Theory was used to establish the synthetic pathway for the structural modifications. Solubility issues regarding biphenyl polymer was solved by copolymerizing with soluble units. For this purpose / poly 4-(biphenyl-4-yl)- 4&rsquo / -tert butylspiro[benzo[d]imidazole-2,1&rsquo / -cyclohexane] (P1), poly 4-(biphenyl-4-yl)- 2- dodecyl-2H-benzo[d][1,2,3]triazole (P2) and poly(4-(5-(biphenyl-4-yl)-4-hexylthiophen- 2-yl)-2-dodecyl-7-(4-hexylthiophen-2-yl)-2H-benzo[d][1,2,3]triazole (P3) were synthetized using Suzuki coupling process. Electrochemical properties of these polymers were examined by cyclic voltammetry, spectroelectrochemistry and kinetic studies. Polymers P2 and P3 showed both p- and n-doping behaviors and multicolored electrochromic states. Optical studies revealed that emission color of biphenyl is tuned from blue to orange and the polymers are good candidates for light emitting diode applications. OLED application of P3 was established and outputs of the device were increased by energy transfer studies. The preliminary investigation indicated that P3 possesses promising efficiencies.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of tissues. In this approach, three dimensional (3D) scaffolds are of great importance. Scaffolds function both as supports for cell growth and depot for sustained release of required active agents (e.g. enzymes, genes, antibiotics, growth factors). Scaffolds should possess certain properties in accordance with usage conditions. Wet-spinning is a simple technique that has been widely used for the fabrication of porous scaffolds for tissue engineering applications. Natural polymers can effectively be used in scaffold fabrication due to their biocharacteristics. Among natural polymers, chitosan and alginate are two of the most studied ones in tissue engineering and drug delivery fields because of being biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. In this study, two kinds of porous scaffolds were produced as chitosan and alginate coated chitosan fibrous scaffolds by wet-spinning technique In order to investigate the delivery characteristics of the scaffolds, loading of gentamicin as a model antibiotic and bovine serum albumin (BSA) as a model protein was carried out in different loading models. Resultant scaffolds were characterized in terms of their structural formation, biodegradation, biomineralization, water uptake and retention ability and mechanical properties. Additionally, release kinetics of gentamicin and BSA were examined. Efficiency of gentamicin on Escherichia coli (E.coli) was examined. Characterization of scaffolds revealed their adequacy to be used in bone tissue engineering applications and capability to be employed as bioactive agent delivery systems.

In the present work, zinc phthalocyanine and tetra nitro substituted zinc phthalocyanine were sytnhesized by using phthalic anhydride and nitro phthalic acid precursor respectively under microwave irradiation. Molecular structures of these pigments were confirmed by FT-IR and UV-visible spectroscopy analyses. Furthermore, these pigments were deposited on mica-titania pigment substrate in dimethyl formamide solvent to obtain the combination pigment. FT-IR analysis was conducted to analyze the deposition of pigments onto the mica-titania surfaces. Fluorescence spectroscopy analysis was performed to observe zinc phthalocyanine, tetra nitro zinc phthalocyanine pigments, and their combination pigment with mica- titania pigments. The surface morphologies of zinc phthalocyanines on the mica titania pigments were investigated by scanning electron microscopy. Optimum deposition temperature and deposition time were determined by v depositing varying amounts of zinc phthalocyanines. The paint samples of these combination pigments were prepared by alkyd based paint formulation. L*a*b* values of the paint samples were determined by color measuring spectrophotometry. The optimum deposition temperature was determined as 120 oC and the optimum deposition time was determined as half an hour and effective deposition was obtained at 120 &deg / C. It is observed that, with the increasing amount of ZnPc, the interaction between ZnPc particles increases and they desorp the surface of mica-titania pigment. The best result was obtained with 0.08 g ZnPc at 120 oC.

In order to study their electrochemical and optical properties, two donor-acceptordonor (D-A-D) type monomers / 4&#039 / -(tert-butyl)-4,7-bis(4-hexylthiophen-2- yl)spiro[benzo[d]imidazole-2,1&#039 / -cyclohexane] (BIHT) and 4&#039 / -(tert-butyl)-4,7-bis(2,3- dihydrothieno[3,4-b][1,4]dioxin-5-yl)spiro[benzo[d]imidazole-2,1&#039 / -cyclohexane] (BIED), were electrochemically polymerized. These properties were investigated by cyclic voltammetry and UV&ndash / Vis-NIR Spectroscopy techniques. Effects of different donor groups / 3-hexylthiophene and 3, 4-ethylenedioxythiophene (EDOT), on new benzimidazole group, 4&#039 / -(tert-butyl)spiro[benzo[d]imidazole-2,1&#039 / -cyclohexane], were investigated. These pendant groups cause slight differences in optical band gaps, which are 1.19 eV for PBIHT, 1.15eV for PBIED. PBIHT and PBIED have both nand p- doping. Both of them have broad absorption in the visible region. In NIR region, both polymers revealed 46 % transmittance with high switching times. PBIED and PBIHT showed multichromic properties. PBIED is green at neutral state. For the most oxidized and reduced states, it is blue and red, respectively. PBIHT is blue at neutral state, transmissive-gray in oxidized state and transmissive greenishgray in reduced state.

In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.

THE CHARACTERIZATION OF SOME METHACRYLATE AND ACRYLATE HOMOPOLYMERS, COPOLYMERS AND FIBERS VIA DIRECT PYROLYSIS MASS SPECTROSCOPY &Ouml / zlem G&uuml / ndogdu, Suriye Ph.D., Department of Polymer Science and Technology Supervisor: Prof. Dr. Jale Hacaloglu December 2012, 177 pages Poly(methyl methacrylate) possesses many desirable properties and is used in various areas. However, the relatively low glass transition temperature limits its applications in textile and optical-electronic industries. Monomers containing isobornyl, benzyl and butyl groups as the side chain are chosen to copolymerize with MMA to increase Tg and to obtain fibers with PMMA. In this work, thermal degradation characteristics, degradation products and mechanisms of methacrylate homopolymers, poly(methyl methacrylate), poly(butyl methacrylate), poly(isobornyl methacrylate) and poly(benzyl methacrylate), acrylate homopolymers, poly(n-butyl acrylate), poly(t-butyl acrylate), poly(isobornyl acrylate), two, three and four component copolymers of MMA and fibers are analyzed via direct pyrolysis mass spectrometry. The effects of substituents on the main and side chains, the components present in the copolymers and fiber formation on thermal stability, degradation characteristics and degradation mechanisms are investigated. According to the results obtained, the depolymerization mechanism yielding mainly the monomer is the main thermal decomposition route for the methacrylate polymers, acrylate polymers degradation occurs by H-transfer reactions from the main chain to the carbonyl groups. However, when the alkoxy group involves