1 Legal regime of natural resources of celestial bodies Abstract The aim of this thesis is to describe the legal regime of the natural resources of celestial bodies. In recent months and years, there has been an increase in debates regarding mining on the moon and asteroids and this thesis dedicates its seven parts to the legal regime of such activities. In particular, whether such activities are possible from the point of view of international law, how are they regulated and what are the potential future developments in this area. In order to understand the legal regime governing the natural resources of celestial bodies, it is necessary to first become acquainted with the essential concepts of space law, including outer space, celestial bodies and outer space resources, whose definition is the subject of the first part of the thesis. The second part describes the development of international space law, from the first mentions in legal commentary to the creation of two important international documents in this area, the Outer Space Treaty and the Moon Agreement. Both international treaties are analysed and compared in individual chapters both in terms of their international acceptance and in terms of the regulation of activities in the field of outer space resources. Subsequently, their possible future...

<p>  </p> <p><em>Fusobacterium necrophorum</em> is a Gram-negative, anaerobic, opportunistic pathogen that causes necrotic infections in cattle leading to liver abscess, foot rot, and calf diphtheria. Particularly, liver abscess in cattle is reported at 20.7% annually, and leads to liver condemnation and an annual economic burden of about 62 million dollars to the feedlot industry. Antibiotic administration is the mainstay of treating these infections, but antibiotic resistance is unavoidable and demand for antibiotic-free, natural, and organic beef has demanded alternative therapies and preventatives. Vaccination is one of the best alternatives to prophylactic antibiotic administration. In this study, we have explored outer membrane proteins (OMPs) and outer membrane vesicles (OMVs) for potential vaccine candidates. OMPs and OMVs are vaccine targets because of their antigenic properties and host specificity. Additionally, we performed comparative genomic analysis of <em>F. necrophorum</em> species to identify additional virulence genes with vaccine potential, unique to the <em>F. necrophorum</em> and its virulent subspecies <em>necrophorum</em>. </p> <p>Protein- protein interaction investigation through binding assay and pulldown assay identified novel OMPs, namely 17kDa, 22kDa, and 66.3 kDa proteins, which were further characterized as OmpH, OmpA and Cell Surface Protein (CSP), respectively. In this study, these novel OMPs including previously characterized 43kDa OMPs were cloned, and recombinant proteins were expressed and purified. These recombinant proteins were used to generate polyclonal antibodies in rabbits, and their efficacy was studied using <em>in vitro</em> adhesion inhibition assays. The combination of two or more antibodies raised against the recombinant OMPs was significantly effective in reducing/neutralizing bacterial binding to bovine endothelial cells compared to individual antibody treatment. This suggests that a multiple subunit vaccine could be effective and provide sufficient evidence to perform <em>in vivo</em> studies. </p> <p>Similarly, we purified OMVs of <em>F. necrophorum</em> subspecies <em>necrophorum</em> 8L1 and analyzed its content using proteomics and lipidomics. Out of 342 proteins identified by tandem liquid chromatography mass spectrometry (LC-MS), OMPs and toxins were the most abundant. These included OMPs and toxins namely, 43 kDa OMP, OmpH, OmpA, CSP, FadA, leukotoxin family filamentous adhesin, N-terminal domain of hemagglutinin and other OMP transport and assembly factor protein. The presence of a subset of these proteins was further confirmed by western blot analysis. Lipidomics analysis showed that OMVs contained phospholipid, sphingolipid, and acetyl carnitine as the main lipid contents. Cytotoxicity assay on BL-3 cell line showed that these OMVs have a toxic effect on host immune cells and could impart immunomodulatory effect. All these findings suggest the vaccine potential of OMVs and demand dose-based <em>in vivo</em> study.</p> <p>In addition, we identified and characterized 5 clinical isolates of <em>F. necrophorum</em> using comparative genomics, UBCG (Up-to-date Bacterial Core Gene) based analysis enabled phylogenetic characterization of 46 <em>F. necrophorum</em> genomes into subspecies specific clades. The pangenome and recombination analysis showed the extensive disparity in accessory genes resulting in species divergence. Strikingly, we detected antimicrobial resistance gene for macrolides and tetracycline in one strain of <em>F. necrophorum</em>, a harbinger of the start of resistance and necessitating search for an alternative prophylactic method. We also noted common virulence genes, including toxins, outer membrane adhesion proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters and transporter proteins in <em>F. necrophorum</em> strains. A focused study on these genes could help identify the main genes of virulence and inform effective vaccination strategies against fusobacterial infections. </p> <p>Overall, the studies suggest adhesins and toxin and/or OMV-based subunit vaccine could be potential targets for vaccine development against fusobacterial infections.  </p>

The politics of outer space has in recent years been given attention from political elites and scientist due to increasing usage and reliance on space based assets, and due to increasing numbers of actors trying to utilize the benefits of space. Concerns have been raised if the increasing military usage of space will lead to a future weaponization of space, making some political leaders and scholars claiming the inevitability of space weaponization.  In this thesis I investigate why the United States of America this far has chosen not to weaponize space based on the strategical setting of outer space politics. The research question guiding this thesis reads: What strategic considerations explain the US decision not to weaponize outer space? In order to evaluate the strategic setting and US strategical considerations I apply Game Theory and Non-Formal Rational Choice Theory to highlight what is causing the greatest space faring nation not to weaponize space. I empirically base this study on official space policy documents and one report written by an official commission to asses US national security space management.  Based on the strategic setting of outer space politics and US strategical considerations it is found that the US has not commenced a process leading to the weaponization of space since such development would not increase its national security, but rather in several ways decrease it. I conclude that a process of space weaponization is not likely to be initiated by the US in the current strategical setting.

The temperature-programmed desorption profiles of water from three lunar analogs were measured. These experiments showed that glassy materials were hydrophobic, that water on multiphase materials occupied a continuum of adsorption sites, and that feldspar exhibited significant chemisorption of water. The competition between photodissociation and photodesorption of amorphous solid water (ASW) was investigated on three substrates: copper with a thin oxide coating, an impact melt breccia from Apollo 16, and a mare basalt from Apollo 17. The rotational temperature of desorbing H₂O did not vary significantly with substrate, but the H₂O time-of-flight spectra were broader on the lunar slabs than on copper. Additionally, the cross sections for water removal at low coverages were higher on the lunar slabs than on copper. O(³PJ) produced by 157-nm irradiation of ASW on the same three substrates was measured as a function of spin-orbit state, H₂O exposure, and irradiation time. The same Maxwell-Boltzmann components were present in each case, with translational temperatures of 10,000 K, 1800 K, 400 K, and the surface temperature, but the relative intensities of these components differed widely between substrates. Evidence for diffusion out of pores in the ASW and in the lunar slabs was observed for H2O exposures of at least 1 Langmuir. Cross sections for H2O and O(3PJ) depletion due to 157-nm irradiation of ASW were applied to icy grains in the rings of Saturn, and corresponding cross sections on the lunar substrates were used to estimate the flux of water desorbing from the Moon and the density of oxygen atoms in the lunar atmosphere.

<p>Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.</p> / Dissertation

<p>In the context of today's global mobility, information, bodies and goods are circulating across the globe, and even further into outer space. However, we face a paradox: the more we move, the more we become sedentary. The modes of transportation that enable our global mobility are working against us, insidiously dwindling our psycho-physical mobility. Globalization is thus not the world becoming bigger (or too big), but the world becoming immobile. Taking the body as the central non-place of political space, Architectures of Aliveness: Building Beyond Gravity interrogates the possibility of inhabiting circulation as a pragmatic form of resistance to the contemporary immobilization of life. In an era in which bodies and goods are ever more constantly in global circulation, architectures of aliveness ask, what would an experience of weightlessness do for us?</p><p>Biotechnology serves as the current dominant model for enlivening architecture and the mobility of its inhabitants. Architectures of aliveness invert the inquiry to look instead at outer space's modules of inhabitation. In questioning the possibility of making circulation inhabitable --as opposed to only inhabiting what is stationary--architectures of aliveness problematize architecture as a form of biomedia production in order to examine its capacity to impact psychic and bodily modalities toward an intensification of health. Problematized synchretically within life's mental and physical polarization, health is understood politically as an accretion of our capacity for action instead of essentially as an optimization of the biological body. The inquiry emerges at the intersection of biotechnology, neurosciences, outer space science and technology, and architecture. The analysis oscillates between historical and contemporary case studies toward an articulation that concentrates on contemporary phenomena while maintaining an historical perspective. The methodology combines archival research, interviews, and artistic and literary analysis. The analysis is informed by scientific research. More precisely, the objective is to construct an innovative mode of thinking about the fields of exchangeability between arts and sciences beyond a critique of instrumentality. </p><p>The outcomes suggest that architectures of aliveness are architectures that invite modes of inhabitation that deviate from habitualized everyday spatial engagements. It also finds that the feeling of aliveness emerges out of the production of analog or continuous space where the body is in relation with space as opposed to be represented in it. The analysis concludes that the impact of architecture on our sense of wellbeing is conditioned by proprioceptive experiences that are at once between vision and movement and yet at the same time in neither mode, suggesting an aesthetic of inhabitation based on our sense of weightedness and weightlessness.</p><p>These outcomes are thus transduced to the field of media studies to enchant biomediatic inquiry. Proposing a renewed definition of biomedia that interprets life as a form of aesthetic relation, architectures of aliveness also formulate a critique of the contemporary imperialism of visualization techniques. Architectures of aliveness conclude by questioning the political implications of its own method to suggest opacity and agonistic spaces as the biomediatic forms of political space.</p> / Dissertation

The Lyme disease spirochete, B. burgdorferi is atypical in that a large portion of its genome is in the form of plasmids. Many of the plasmid-carried genes form extensive paralogous gene families and encode outer-surface proteins. In this report we have assessed the humoral immune response to proteins belonging to the paralogous protein family, family 163. We have cloned and expressed BBP39, BBO40, BBQ47 and BBN39 and used these recombinant proteins to monitor the temporal nature of the antibody response to these antigens during experimental infection of mice. The immunodominant regions of each protein have also been assessed through immunoblot analyses of a series of truncations of each protein. These analyses have led to the delineation of the targets of the antibody response during infection and of the specificity of the antibody response to family 163 proteins. In addition, we quantified the expression of each gene using real time RT-PCR.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of human granulocytic anaplasmosis and bovine anaplasmosis, respectively. Both diseases can be severe, even fatal, and protective vaccines for each are lacking. We recently identified A. phagocytophilum outer membrane protein A (ApOmpA) as being critical for cellular invasion and is expressed during infection of mammalian but not tick cells. Disrupting ApOmpA-host cell interactions significantly inhibits A. phagocytophilum entry into host cells. ApOmpA and its A. marginale ortholog, AM854 (A. marginale OmpA; AmOmpA) exhibit 44% amino acid identity. The ApOmpA invasin domain is highly conserved between both proteins. In this study, we investigated the differential expression of AmOmpA in mammalian versus tick cell lines; the serological cross-reactivity between AmOmpA and ApOmpA; the potential role of AmOmpA in mediating interactions with mammalian host cells; and if inhibiting the AmOmpA-host cell interaction impairs A. marginale cellular invasion. AmOmpA is expressed throughout infection of mammalian, but not tick cells. Sera from A. marginale infected cows recognized both AmOmpA and ApOmpA. Sera from cows immunized with an A. marginale OM complex that conferred protection also recognized both proteins. Thus, ApOmpA and AmOmpA share cross-reactive B-cell epitopes. To determine if AmOmpA plays a role in promoting A. marginale infection, we assessed the abilities of recombinant AmOmpA to competitively inhibit infection of mammalian host cells. To examine the cross-reactive properties of OmpA, we showed that preincubation of host cells with GST-ApOmpA and pretreatment of A. marginale with anti-GST-ApOmpA significantly inhibit A. marginale infection of host cells; and that pretreatment of A. phagocytophilum with serum from cows immunized with an A. marginale OM complex reduces its infection of host cells. These studies advance understanding of conservation of OmpA-mediated cellular invasion between Anaplasma species and highlight the potential of OmpA as a vaccinogen that could offer protection against human and veterinary anaplasmoses.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

<p>Bacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (&#916;nlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (&#916;degP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of &#916;nlpA&#916;degP correlated with reduced OMV production with respect to the hyprevesiculator &#916;degP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the &#916;nlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.</p> / Dissertation