Enhanced contrast in OCT imaging of tissues using birefringence, scattering and speckle signatures

The three objectives listed here are the main aim presented in each of the chapters of this thesis. The research work carried out with respect to fulfilling these objectives is one step closer towards extending the possibility of non-invasive imaging modality of OCT and PS-OCT in the field of orthopaedics and tissue engineering. - Non-invasive technique to understand the depth-dependent 3D collagen framework of articular cartilage. - Non-invasive technique to discriminate between different types of connective tissue based on angle-resolved backscattering profiles - Computer based tissue discrimination based on the speckle textural analysis of the OCT images obtained Articular cartilage was imaged using two different schemes of implementation of PS-OCT: time domain PS-OCT and swept source based continuous polarisation modulation PS-OCT system. Detailed analysis is presented for time-domain PS-OCT data obtained from bovine articular cartilage sample over multi-angle measurements and a comparative study of the phase retardance profiles obtained from experimental data is done with those obtained from a layered model of articular cartilage using extended Jones matrix calculus. This includes a noise model chosen for the time domain PS-OCT system to add noise bias to the simulated results. Optimiser algorithms are developed based on this model. This study shows the possibility of using PS-OCT imaging towards non-invasive technique to study the microstructure of articular cartilage. The technique of multi-angle imaging in PS-OCT has also been used in the study of angleresolved backscattering, with the information regarding the reflectivity profiles as obtained from a normal OCT system used for the study. The two connective tissues under study are bovine tendon sample and bovine articular cartilage sample. Articular cartilage is predominantly made of Type II collagen fibrils which are finer and more uniform in nature compared to that in tendon tissue which is predominantly Type I collagen fibrils of larger diameters and coarser packing arrangements. Single scattering model of OCT is used to obtain the angle-resolved backscattering curves and Rayleigh Gans scattering approximation based simulation is carried out to elucidate and understand the results obtained. Speckle texture analysis is carried out to extract sub-resolution based information from OCT towards computer-based classification of different types of OCT images. This has been carried out first on tissue phantoms made of agar-intralipid solutions of different concentrations. Statistical features are extracted and grouped into 3-set features to obtain scatter-plots and receiver operating characteristic curves that determine the correctness of the classification obtained of a particular group of OCT images from the total sample set. With initial success from tissue phantom based speckle textural analysis, this has been extended to study the data classification ability of normal skin from tissue engineered skin with different types of melanoma cell-lines invasion as well as discriminate different types of melanoma invasion of tissue-engineered skin from each othe

Structural insights into phosphoprotein chaperoning of nucleoprotein in measles virus

Instruct Biennial Structural Biology Conference Abstract BookletMeasles virus is an important, highly contagious, human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein, P, to prevent newly synthesized N from binding to cellular RNA. Here, we pulled down an N01-408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal, P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0 binding peptide, P1-48, to the C-terminus of an N021-408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at 2.7 Å resolution. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0 preventing both self-assembly of N0 and its binding to RNA. We compare the structure of an N0-P complex to atomic model of helical ribonucleocapsid. We thus propose a model how P may help to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of the virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development not only to treat measles but also potentially other paramyxovirus diseases.Non peer reviewe

Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-terminal Region of Phosphoprotein

ABSTRACT The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N0) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N0 assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N0 1-408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0 binding peptide, P1-48, to the C terminus of an N0 21-408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0, preventing both self-assembly of N0 and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization. IMPORTANCE Measles virus is an important, highly contagious human pathogen. The nucleoprotein (N) binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.Peer reviewe

Mapping the phylogeny and lineage history of geographically distinct BCG vaccine strains

The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in sigK, plaA, mmaA3 and eccC5 were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1 A- 33 G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non - glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1 N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera

Mapping the phylogeny and lineage history of geographically distinct BCG vaccine strains.

The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in sigK, plaA, mmaA3 and eccC5 were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature

Machine-learning based segmentation of the optic nerve head using multi-contrast Jones matrix optical coherence tomography with semi-automatic training dataset generation

A pixel-by-pixel tissue classification framework using multiple contrasts obtained by Jones matrix optical coherence tomography (JM-OCT) is demonstrated. The JM-OCT is an extension of OCT that provides OCT, OCT angiography, birefringence tomography, degree-of-polarization uniformity tomography, and attenuation coefficient tomography, simultaneously. The classification framework consists of feature engineering, k-means clustering that generates a training dataset, training of a tissue classifier using the generated training dataset, and tissue classification by the trained classifier. The feature engineering process generates synthetic features from the primary optical contrasts obtained by JM-OCT. The tissue classification is performed in the feature space of the engineered features. We applied this framework to the in vivo analysis of optic nerve heads of posterior eyes. This classified each JM-OCT pixel into prelamina, lamina cribrosa (lamina beam), and retrolamina tissues. The lamina beam segmentation results were further utilized for birefringence and attenuation coefficient analysis of lamina beam

Generation and optimization of superpixels as image processing kernels for Jones matrix optical coherence tomography

Jones matrix-based polarization sensitive optical coherence tomography (JM-OCT) simultaneously measures optical intensity, birefringence, degree of polarization uniformity, and OCT angiography. The statistics of the optical features in a local region, such as the local mean of the OCT intensity, are frequently used for image processing and the quantitative analysis of JM-OCT. Conventionally, local statistics have been computed with fixed-size rectangular kernels. However, this results in a trade-off between image sharpness and statistical accuracy. We introduce a superpixel method to JM-OCT for generating the flexible kernels of local statistics. A superpixel is a cluster of image pixels that is formed by the pixels’ spatial and signal value proximities. An algorithm for superpixel generation specialized for JM-OCT and its optimization methods are presented in this paper. The spatial proximity is in two-dimensional cross-sectional space and the signal values are the four optical features. Hence, the superpixel method is a six-dimensional clustering technique for JM-OCT pixels. The performance of the JM-OCT superpixels and its optimization methods are evaluated in detail using JM-OCT datasets of posterior eyes. The superpixels were found to well preserve tissue structures, such as layer structures, sclera, vessels, and retinal pigment epithelium. And hence, they are more suitable for local statistics kernels than conventional uniform rectangular kernels

Polarization-sensitive optical coherence tomography with a conical beam scan for the investigation of birefringence and collagen alignment in the human cervix

By measuring the phase retardance of a cervical extracellular matrix, our in-house polarization-sensitive optical coherence tomography (PS-OCT) was shown to be capable of (1) mapping the distribution of collagen fibers in the non-gravid cervix, (2) accurately determining birefringence, and (3) measuring the distinctive depolarization of the cervical tissue. A conical beam scan strategy was also employed to explore the 3D orientation of the collagen fibers in the cervix by interrogating the samples with an incident light at 45° and successive azimuthal rotations of 0-360°. Our results confirmed previous observations by X-ray diffraction, suggesting that in the non-gravid human cervix collagen fibers adjacent to the endocervical canal and in the outermost areas tend to arrange in a longitudinal fashion whereas in the middle area they are oriented circumferentially. PS-OCT can assess the microstructure of the human cervical collagen in vitro and holds the potential to help us better understand cervical remodeling prior to birth pending the development of an in vivo probe

Biochemische und Strukturelle Basis für die duale Funktionalität von Gephyrin

Neurons are specialized cells dedicated to transmit the nerve impulses throughout the human body across specialized structures called synapses. At the synaptic terminals, a crosstalk between multiple macromolecules regulates the structure and function of the presynaptic nerve endings and the postsynaptic recipient sites. Gephyrin is the central organizer at inhibitory postsynaptic specializations and plays a crucial role in the organization of these structures by anchoring GABAA receptors (GABAAR) and glycine receptors (GlyR) to the postsynaptic membrane. This 93 kDa protein features an N-terminal G domain and a C-terminal E domain and the latter interacts directly with the intracellular loop between transmembrane helices 3 and 4 of certain subunits of the GlyRs and GABAARs. Biochemical and structural analyses have already provided valuable insights into the gephyrin-GlyR interaction. Interestingly, biochemical studies on the gephyrin-GABAAR interaction demonstrated that the GABAARs also depend on the same binding site as the GlyRs for the interaction with the gephyrin, but the molecular basis for this receptor specific interaction of gephyrin was still unknown. Co-crystal structures of GephE-GABAAR α3- derived peptides with supporting biochemical data presented in this study deciphered the receptor-specific interactions of gephyrin in atomic detail. In its moonlighting function, gephyrin also catalyzes the terminal step of the evolutionarily conserved molybdenum cofactor biosynthesis. Molybdenum, an essential transition element has to be complexed with a pterin-based cofactor resulting in the formation of the molybdenum cofactor (Moco). Moco is an essential component at the active site of all molybdenum-containing enzymes with the exception of nitrogenase. Mutations in enzymes involved in this pathway lead to a rare yet severe disease called Moco deficiency, which manifest itself in severe neurodevelopmental abnormalities and early childhood death. Moco biosynthesis follows a complex multistep pathway, where in the penultimate step, the N-terminal G domain of gephyrin activates the molybdopterin to form an adenylated molybdopterin intermediate. In the terminal step, this intermediate is then transferred to the C-terminal E domain of gephyrin, which catalyzes the metal insertion and deadenylation reaction to form active Moco. Previous biochemical and structural studies provided valuable insights into the penultimate step of the Moco biosynthesis but the terminal step remained elusive. Through the course of my dissertation, I crystallized the C-terminal E domain in the apo-form as well as in complex with ADP and AMP. These structures shed lightonto the deadenylation reaction and the formation of a ternary E-domain-ADP-Mo/W complex and thus provide structural insight into the metal insertion mechanism. Moreover, the structures also provided molecular insights into a mutation leading to Moco deficiency. Finally, ternary complexes of GephE, ADP and receptor-derived peptides provided first clues regarding the integration of gephyrin’s dual functionality. In summary, during the course of the dissertation I was able to derive high resolution structural insights into the interactions between gephyrin and GABAARs, which explain the receptor-specific interaction of gephyrin and, furthermore, these studies can be extended in the future to understand GABAAR subunit-specific interactions of gephyrin. Finally, the understanding of Moco biosynthesis shed light on the molecular basis of the fatal Moco deficiency.Neurone sind spezialisierte Zellen, die über die Synapsen Nervenimpulse im menschlichen Körper übertragen. An den synaptischen Enden reguliert ein Netzwerk aus einer Vielzahl von Makromolekülen die Struktur und die Funktion der präsynaptischen Nervenenden und der postsynaptischen Kontaktstellen. Gephyrin ist der Hauptorganisator an inhibitorischen, postsynaptischen Spezialisierungen und spielt durch die Verankerung von GABAA-Rezeptoren (GABAAR) und Glycinrezeptoren (GlyR) in der postsynaptischen Membran eine zentrale Rolle für den Aufbau dieser Strukturen. Dieses 93 kDa Protein enthält eine N-terminale G-Domäne (GephG) und eine C-terminale E-Domäne (GephE), wobei letztere direkt mit der intrazellulären unstrukturierten Region zwischen Transmembranhelices 3 und 4 bestimmter Untereinheiten der GlyR und GABAAR interagiert. Biochemische und strukturelle Analysen lieferten bereits wertvolle Erkenntnisse über die Gephyrin-GlyR Interaktion. Interessanterweise zeigten Versuche zur Gephyrin-GABAAR Interaktion, dass GABAARs die gleiche Bindungsstelle auf Gephyrin benutzen wie GlyRs, wobei die molekulare Basis für diese Interaktion nicht bekannt war. In dieser Arbeit zeige ich Co-Kristallstrukturen von GephE-GABAARα3 sowie unterstützende biochemische Daten, die die atomaren Details der rezeptorspezifischen Interaktionen von Gephyrin entschlüsseln. Als zweite Funktion katalysiert Gephyrin den terminalen Schritt der evolutionär konservierten Molybdän Cofaktor Biosynthese. Dabei muss das essentielle Übergangselement Molybdän mit einem Pterin-basierten Cofaktor komplexiert werden, um den Molybdän Cofaktor (Moco) zu bilden. Moco ist essentieller Bestandteil im aktiven Zentrum aller Molybdän-enthaltenden Enzyme mit Ausnahme der Nitrogenase. Mutationen in Enzymen, die in die Molybdän Cofaktor Biosynthese involviert sind, verursachen eine Moco Defizienz, eine seltene, jedoch schwere Erkrankung, die sich durch schwere neurologische Entwicklungsstörungen und Tod im frühen Kindesalter äußert. Die Moco Biosynthese folgt einem komplexen mehrstufigen Ablauf. Im vorletzten Schritt adenyliert GephG das Molybdopterin und ein Zwischenprodukt entsteht. Im letzten Schritt wird dieses Zwischenprodukt auf GephE übertragen, das die Insertion des Metalls und die Deadenylierungsreaktion katalysiert, wodurch der aktive Moco entsteht. Frühere biochemische und strukturelle Studien brachten wertvolle Erkenntnisse über den vorletzten Schritt der Moco Biosynthese, aber die Kenntnisse über den letzten Schritt blieben vage. Während meiner Dissertation kristallisierte ich GephE in der apo-Form sowie im Komplex mit ADP oder AMP. Diese Strukturen gaben Aufschluss über die Deadenylierungsreaktion und die Formation eines ternären GephE-ADP-Mo/W Komplexes und gewährten so einen strukturellen Einblick in den Mechanismus der Metallinsertion. Darüber hinaus ermöglichten die Strukturen eine Mutation, die zu Moco Mangel führt, auf molekularer Ebene zu verstehen. Schließlich lieferten ternäre Komplexe aus GephE, ADP und von Rezeptoren abgeleiteten Peptiden ersten Aufschluss bezüglich der Verflechtung von Gephyrins dualer Funktion. Zusammenfassend konnte ich während der Dissertation hochauflösende strukturelle Einblicke in den Komplex aus GephE und GABAAR α3 Untereineinheit gewinnen, die die rezeptorspezifische Interaktion von Gephyrin erklären. Weiterhin können diese Studien in der Zukunft ausgeweitet werden, um die GABAAR-untereinheitenspezifische Interaktion mit Gephyrin zu verstehen. Schließlich erlauben die Studien zur Moco Biosynthese die tödliche Moco Defizienz auf molekularer Ebene zu verstehen