The role of highly conserved amino acids within the carboxy-terminal hydrophobic domain of herpes simplex virus I (HSV-I) glycoprotein gB was studied by introducing point mutations using the method of site directed mutagenesis. A segment of this hydrophobic domain of glycoprotein gB contains a nuclear envelope (NE) targeting signal and the effect of these point mutations on targeting to the nuclear envelope was determined. A complementation assay was employed to determine the effect these mutations have on HSV-I infectivity .The point mutations created within the transmembrane domain of glycoprotein gB had no effect on nuclear envelope targeting and localization. However, single point mutations introduced into the first and second hydrophobic domains of glycoprotein gB, G₇₄₃R and F₇₇₀S, affected the targeting and localization of full-length glycoprotein gB at the nuclear envelope. When the transmembrane domain ofHSV-I glycoprotein gB containing the following point mutations A₇₉₀Q, A₇₉₁S, A₇₈₆S, A₇₈₆Y and A₇₉₀S, was introduced into a chimeric protein consisting of the cytoplasmic domain and ectodomain of a plasma membrane protein, vesicular stomatitis virus glycoprotein G, NE targeting and localization were affected. These point mutations may affect the targeting of glycoprotein gB by altering the structure of the targeting signal within the protein. It can be hypothesized that the presence of the cytoplasmic domain. ectodomain domain, and the first and second transmembrane domains within full-length glycoprotein gB can compensate for the effect these point mutations have on nuclear envelope targeting. since the same point mutations had no effect on the targeting · and localization of full-length glycoprotein gB. Complementation assays showed that the glycoprotein gB mutants, A₇₈₆S, A₇₈₆Y, A₇₈₆N, A₇₉₀Q, A₇₉₁S, F₇₇₀S, or G₇₄₃R, were unable to complement a gB-null virus even though these mutant proteins are localized at the nuclear envelope. These proteins may not have been incorporated into the viral capsid due to misfolding or due to the fact that sequences required for interaction with other viral proteins were lost. Another possibility is that the mutant proteins were incorporated into the HSV virion but were not biologically active. / Thesis / Master of Science (MS)

The potential of human adenovirus (Ad) to serve as a vector for expression of heterologous genes was evaluated. An experimental gene, consisting of sequences coding for the glycoprotein of vesicular stomatitis virus (VSV) attached to the promoter and polyadenylation signal of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, was inserted into early region 3 of adenovirus, in both orientations. The TK promoter was functional in both orientations. The TK promoter was functional in both orientations and responded to trans-activation by HSV infection. Abundant expression of VSV G however depended on the presence of a second transcript. This transcript was present only in the recombinant carrying the insertion in the orientation parallel to the E3 promoter (AdG12) and was initiated upstream of the insertion, within Ad sequences. The potential of Ad recombinants to serve as vaccine vectors was investigated using the recombinant AdG12. Antibody against VSV G was induced in cows, pigs, and dogs in response to infection with AdG12. Protection of mice, immunized with AdG12, against a lethal challenge with VSV demonstrated the biological effectiveness of this immune response. / Thesis / Master of Science (MS)

The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein. / Thesis / Master of Science (MS)

Spider orb-webs have evolved to intercept prey, absorb and dissipate the kinetic energy from prey impact, and retain prey until a spider can subdue their catch. Orb-web structure and function engages scientists from many disciplines, including engineering, behavior, materials science, ecology, and evolution. This dissertation examines the sticky capture spiral component of an orb-web. This composite material is made of supporting fibers covered in sticky glue droplets. These threads are both adhesive and extensible, and their performance is influenced by ambient conditions. The questions I addressed are framed in an ecological context, although they also add to our understanding of materials science. The results of the first study showed that temperature increased the viscosity of glycoproteins within Argiope aurantia droplets, mediating the effect of daily humidity changes, an important environmental effect on the glue's performance. The second study demonstrates that capture spiral droplets of spiders that build webs in habitats ranging from full sun to shade and nocturnal species (Argiope aurantia, Leucauge venusta, Neoscona crucifera, Verrucosa areenata, Micrathena gracilis) is resistant to degradation after a day's worth of UVB exposure. Conversely, after the equivalent of two days of UVB exposure the glue degrades in webs built by M. gracilis that build webs in the shade and N. crucifera, a nocturnal species. The less harsh UVA has little affect on capture spiral glue function, both for species that build webs in full sun and those that build webs at night. The third study documented web asymmetry in Argiope trifasciata orb-webs and identified differences in droplet characteristics across the webs. These spiders differently allocated resources, with the bottom region of the web having twice the droplet volume as the top, and half the ratio of aqueous to glycoprotein material as the inner droplets. Additionally, during foraging times, the bottom of the web experiences higher humidity than the top, which has the potential to increase droplet toughness in this region. This study expands the understanding of web asymmetry by examining the differences in glue characteristics as an additional level of flexibility for web fine-tuning. / Ph. D.

Francisella tularensis is a highly virulent zoonotic pathogen that is the causative agent of tularemia in humans. Two subspecies of F. tularensis are the most virulent in humans: tularensis (type A) and holarctica (type B), with less than 10 organisms via aerosol of a type A strain having the ability to cause fatal infection. Over the last decade much research has been done on the pathogenesis of this unique intracellular bacterium and many different virulence factors have been identified. The goal of this dissertation has been to identify and characterize the capsule-like complex (CLC) surface antigen of F. tularensis, and to determine its role in virulence and immunoprotection in a mouse model. In addition, I have investigated the role of CLC in biofilm formation. The CLC appears as a negatively staining material surrounding F. tularensis cells during transmission electron microscopy (TEM). I found that the CLC in the type B live vaccine strain (LVS) could be significantly diminished by deleting two glycosyl transferase genes (LVSΔ1423-22) in the putative polysaccharide locus, FTL_1432-FTL_1421. In addition, I determined that the CLC was not a typical polysaccharide capsule, but was in fact composed of over 50 proteins and glycoproteins including known virulence determinants, such as GroEL, DnaK, and ClpB. Upon further evaluation of the CLC, I determined that it was composed of an increase in production of outer membrane vesicles and tubules (OMV/T). These OMV/T appeared to be self-aggregating into what I visualized through TEM as the CLC. LVSΔ1423-22 was attenuated in the mouse model, and BALB/c mice immunized with CLC and adjuvant were protected against challenge with LVS. In addition to virulence, the CLC appears to play a role in biofilm formation and development. F. tularensis type B strains lacking the surface antigens CLC or CLC and O-antigen, develop a 2-7-fold more robust biofilm than the parent strains. The biofilm matrix contains a glucan-like EPS, proteins, and extracellular DNA, and further characterization may lead to determining if the biofilm acts as an environmental survival mechanism for F. tularensis. In summary, the CLC appears to be a novel surface antigen composed of upregulated OMV/T that is present in type A and B F. tularensis. Deficiency in CLC contributes to increased biofilm formation that could contribute to the survival of F. tularensis in a wide range of environmental niches. Furthermore, the CLC contributes to virulence of type B strains and elicits a protective immune response to type B challenge. A CLC-deficient type A strain could be a candidate for a new live vaccine strain, and therefore further investigation of such a mutant is warranted. / Ph. D.

Orb weavers intercept insects using non-hardening bioadhesive droplets, supported by two flagelliform fibers. Droplets contain an adhesive glycoprotein core and aqueous layer that confers hygroscopicity. The first study investigates the durability of these droplets to cycling, or repeatedly adhering, extending, and pulling off. Droplets of four species proved resilient, cycling 40 times. Cycling, coupled with droplet humidity responsiveness, qualifies them as smart materials. However, thread adhesion is complex, relying on an integrated performance of multiple droplets and the flagelliform fibers. As insects struggle, the flagelliform fibers bow and the droplets extend, forming a suspension bridge configuration whose biomechanics sum the adhesion of droplets and dissipate the energy of struggling insects. Given this performance, the second study predicts that the material properties of both thread components have evolved in a complementary way. Comparative phylogenetics of 14 study species revealed that their elastic moduli are correlated, with glycoproteins being six times more elastic than flagelliform fibers. Spider mass affects the amount of each material, but not their properties. Since glycoprotein performance changes with humidity, we hypothesized that orb weavers generate greater adhesion at their foraging humidity. After delimiting low and high humidity species groups (eight and six species, respectively), bridge force was determined as total contributing droplet adhesion at three humidities. Only three spiders generated greater adhesion outside of their foraging humidity. The distribution of force along a suspension bridge differed from a previously reported pattern. We also characterize the sheet configuration, which generates force similar to suspension bridges. / Master of Science / In nature, adhesives are used for a variety of functions. Many animals use adhesives use adhesives when climbing. Examples include toe pads of geckos, tarsal pads of ants, and tube feet of and sea urchins. Here, adhesion is repeatedly generated and released as the animal moves. However, some animals depend on permanent adhesives to anchor to surfaces. Marine mussels and barnacles, whose adult forms are sessile, use adhesives to resist the powerful action of waves and currents. Adhesion also plays a critical role in prey capture, where it prevents prey from escaping. The sticky droplets of a sundew plants and the adhesive capture threads of spider orb webs trap flies. Biologists and engineers study these bioadiehsives in search of inspiration and principles that will guide the development of new materials, including adhesives that function underwater, harden rapidly, or remaining pliable after adhering. This study investigated the material properties of capture threads spun by orb weaving spiders, which rely on non-hardening sticky droplets, supported by two protein fibers to capture insects. Inside each droplet is an adhesive core allows droplets to adhere to an insect and to extend as it struggles to escape. Surrounding this core is an aqueous layer that attracts atmospheric water, causing droplets to track changes in ambient humidity. A study of the cycling (or reusability) of four species' droplets repeatedly adhered a droplet to a surface and extending it to pull-off. These droplets were very resilient, cycling 40 times. Cycling, coupled with droplet humidity responsiveness, qualifies them as smart materials. However, prey capture is more complex, relying on the integration of multiple droplets and their supporting flagelliform fibers. As insects struggle, these fibers bow and the droplets extend, forming a suspension bridge configuration whose biomechanics sum the adhesion of droplets to resist an insect escape. The threads of 14 species were examined to test the hypothesis that material properties of both thread components have evolved in a complementary way to optimize adhesive performance. This revealed that the elasticities of the two capture thread components were correlated, with support fiber elasticity being greater. Capture threads generated the greatest adhesion at humidities during times that a spider feeds, although the distribution of this force across a suspension bridge showed different patterns among the species. The functional integration of a capture thread's components and its ability to respond to environmental humidity gives it exciting biomimicry potential.

Fear and anxiety are fundamental emotional states that are critical for survival. These states are characterized by a variety of coordinated responses, including behavioral and autonomic changes, that need to be properly integrated. For the past decades, most studies have separated the behavioral and autonomic elements, generating a gap in understanding their integrative nature. In this thesis, a framework analysis is presented that allows for the integration of cardiac, behavioral, and neuronal readouts in freely moving mice during different emotional states. Furthermore, a growing body of evidence demonstrates that a vital component of these states is the physiological report of bodily states, or interoception, which allows for quick adaptation to changing situations. A set of distinctive interoceptive pathways has been described from the periphery to the brainstem; however, the circuits that process and integrate cardiac interoceptive signals in higher orders are poorly understood. The midbrain periaqueductal gray (PAG) is a region crucially involved in defensive states through its modulation of both, cardiac and behavioral components. Preliminary studies demonstrate an anatomical connection between the major cardiac interoception brainstem area, the nucleus of the solitary tract, and the PAG; however, the functional characterization and the specific neuronal substrates responsible for interoception in this area have not been described. An interesting particularity of the PAG is that the ventro-lateral subcolumn is the highest order of the neuraxis where inhibitory neurons that express the glycine can be found. In the lower brainstem and spinal cord, glycinergic inhibitory neurons have demonstrated a role in processing sensory and autonomic signals from the periphery, raising the question of whether the PAG glycinergic neurons could be involved in integrating cardiac interoceptive signals as part of a defensive state. In this thesis, using virally mediated trans-synaptic retrograde tracing, I showed that glycinergic PAG neurons receive inputs from cardiac regulatory areas in the brainstem and project massively to forebrain and midbrain regions. By employing advanced techniques such as deep brain calcium imaging with a miniaturized microscope and optogenetics, this study provides compelling evidence for the involvement of glycinergic PAG neurons in controlling heart rate and maintaining cardiac macrostate dynamics within physiological levels. The results of the optogenetic manipulation further revealed that a change in the heart rate macrostate caused by the glycinergic PAG neurons leads to anxiety-like behaviors, providing further evidence for the role of these neurons in regulating defensive states. Overall, by unraveling the neural circuitry underlying interoception in the PAG, our study paves the way to better understand fear and anxiety disorders. / Furcht und Angst sind grundlegende emotionale Zustände, die für das Überleben entscheidend sind. Diese Zustände sind durch eine Vielzahl von koordinierten Reaktionen gekennzeichnet, darunter auch Verhaltensänderungen und autonome Veränderungen, die richtig integriert werden müssen. In den letzten Jahrzehnten haben die meisten Studien die verhaltensbezogenen und autonomen Elemente getrennt, was zu einer Lücke im Verständnis ihrer integrativen Natur führte. In dieser Arbeit wird eine Rahmenanalyse vorgestellt, die die Integration von kardialen, verhaltensbezogenen und neuronalen Messwerten bei sich frei bewegenden Mäusen während verschiedener emotionaler Zustände ermöglicht. Darüber hinaus gibt es immer mehr Belege dafür, dass eine wichtige Komponente dieser Zustände die physiologische Rückmeldung von Körperzuständen an das Gehirn, die sog. Interozeption ist, die eine schnelle Anpassung an sich ändernde Situationen ermöglicht. Es wurde eine Reihe von ausgeprägten interozeptiven Bahnen von der Peripherie bis zum Hirnstamm beschrieben. Jedoch sind die Schaltkreise, die kardiale interozeptive Signale in höherer Ordnung verarbeiten und integrieren, kaum bekannt. Das periaquäduktale Grau des Mittelhirns (PAG) ist eine Region, die sich entscheidend an Verteidigungszuständen beteiligt ist und deren Modulation sowohl kardiale als auch verhaltensbezogene Signale beeinflusst. Vorläufige Studien zeigen eine anatomische Verbindung zwischen dem wichtigsten kardialen Interozeptionsbereich des Hirnstamms, dem Nucleus tractus solitarius, und dem PAG; die funktionelle Charakterisierung und die spezifischen neuronalen Substrate, die für die Interozeption in diesem Bereich verantwortlich sind, sind bislang jedoch nicht beschrieben worden. Eine interessante Besonderheit des PAG ist, dass die ventro-laterale Subkolumne die höchste Ordnung der Hirnachse mit hemmenden glyzinergen Zellkörpern aufweist. Im Hirnstamm und im Rückenmark haben diese hemmenden Neuronen eine Rolle bei der Verarbeitung sensorischer und autonomer Signale aus der Peripherie gezeigt, was die Frage aufwirft, ob die glyzinergen Neuronen des PAG an der Integration kardialer interozeptiver Signale als Teil eines Verteidigungszustandes beteiligt sein könnten. In dieser Arbeit habe ich mit Hilfe von viral vermitteltem trans-synaptischem retrograden Tracing gezeigt, dass glyzinerge PAG-Neuronen Inputs von kardialen Regulationsbereichen im Hirnstamm erhalten und massiv in Vorder- und Mittelhirnregionen projizieren. Durch den Einsatz fortschrittlicher Techniken, wie z. B. Tiefenhirn- Kalziumbildgebung mit einem miniaturisierten Mikroskop und Optogenetik, liefert diese Studie überzeugende Beweise für die Beteiligung glyzinerger PAG-Neuronen an der Kontrolle der Herzfrequenz und der Aufrechterhaltung der kardialen Makrostate-Dynamik innerhalb physiologischer Grenzen. Die Ergebnisse der optogenetischen Manipulationen zeigten außerdem, dass eine durch die glyzinergen PAG-Neuronen verursachte Veränderung des Makrostates der Herzfrequenz zu angstähnlichem Verhalten führt, was einen weiteren Beweis für die Rolle dieser Neuronen bei Furcht und Angst darstellt. Insgesamt ebnet unsere Studie durch die Entschlüsselung der neuronalen Schaltkreise, die der Interozeption im PAG zugrunde liegen, den Weg zu einem besseren Verständnis von Angst und Angststörungen.

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The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.

The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.