Persister cells are able to survive in the presence of high concentrations of antibiotic, and re-grow once the antibiotic has been removed. Unlike conventional antibiotic resistance, the antibiotic tolerance of persister cells is due to phenotypic switching, and is non-inherited. There is growing evidence for a role of persisters in various persistent bacterial diseases. Burkholderia pseudomallei is a pathogen which causes melioidosis, which often persists in the host despite antibiotic treatment. As persister cells may contribute to persistent melioidosis, this study investigated persisters in B. thailandensis, as a model for B. pseudomallei. Treatment of B. thailandensis with ceftazidime, ciprofloxacin, imipenem or trimethoprim demonstrated persister cells which survived antibiotic treatment. Persister frequencies were increased in the absence of oxygen, and higher in stationary phase cultures compared with growing cultures. Drug concentration did not affect persister frequencies, and inherited antibiotic resistance was not detected. Different persister fractions were detected using treatment with multiple antibiotics, indicating heterogeneous susceptibility to antibiotics. In order to increase understanding of the molecular basis of B. thailandensis persister cells, a transposon mutagenesis-based sequencing approach was used on persister cultures. This indicated some issues with genome coverage and mutant diversity. Genes were identified from mutants present before and/or after ciprofloxacin treatment. In order to try to eradicate persister cells from a culture, two anti-persister strategies were tested. Itaconate appeared to stimulate growth of B. thailandensis, increasing susceptibility to the antibiotic ceftazidime. However, the overall effect of the combination was no greater than ceftazidime alone in the conditions tested. Metronidazole was effective against a persister culture under anaerobic conditions, suggesting it may be useful in treating anaerobic persisters. Treatment of B. pseudomallei infected mice with metronidazole and ceftazidime did not improve survival over ceftazidime treatment alone.

<p>Recent technological advances enable us to examine bacterial population dynamics with high temporal resolution with capacity for collecting high throughput data. Precise quantification of bacterial population dynamics can help us to further extend our understanding of how bacteria respond to environmental conditions. Such analysis provides critical information for improving antibiotic treatment protocols and for predictable engineering cellular behavior with synthetic gene circuits. </p><p>A fundamental question in bacterial population dynamics is how fast bacteria are killed in response to antibiotics. Due to their mode of action, β-lactams are more effective against fast-growing bacteria than against slow-growing bacteria. Indeed, it has been recognized that the rate of lysis by β-lactam antibiotics depends on the growth rate of the bacteria, based on previous works. However, past studies examined the growth rate modulation of lysis only during balanced growth and for very limited combinations of bacteria and drugs. Although there is evidence that growth plays key role in determining bacterial response to antibiotics, more comprehensive understanding on how wide range of growth rates affect antibiotic dose response had been overlooked. Instead, bacterial growth has been largely described to be in either growing or non-growing states. </p><p>To examine the general applicability of this growth rate dependence of antibiotic response, I examined how growth rate influences the lysis rate induced by beta-lactams. I found that there is a robust correlation between growth and lysis rates beyond what had been demonstrated in the previous work. Even during unbalanced growth, and regardless of how growth rate was modulated, the robust correlation between growth and lysis rates in bacterial populations were observed. Also, my data suggested a striking versatility of this correlation in different bacterial specie-drug pairs. Thus, my quantification greatly expands previous work by further examining the dependence of lysis rate on growth rate, and extends our understanding of the phenomenon associated with β-lactam antibiotic treatment, and of possible consequences arising from variable lysis rate. My strategy on modulating growth rates and measuring corresponding lysis rates demonstrates a simple and robust method for examining this phenomenon. These results have direct implications in two aspects.</p><p> First, my quantification method allows greater degree of freedom in modulating growth states of bacteria. Indeed, I was able to examine a wide range of growth rates in bacteria that allowed analyses of robust correlation in growth and lysis rates. The simple correlation reported from my work suggests the underlying reasoning for slow or fast lysis of bacterial population that can lead to designing optimal protocols depending on the growth rates of bacterial population. Due to frequent observation of slow-growing cells under conditions such as biofilm of pathogenic bacteria that complicates clinical symptoms and treatments in patients, they have been an important aspect of study for antibiotic tolerance. A quantitative understanding of the robust correlation between growth and lysis rates is critical for designing effective treatment protocols using β-lactams. </p><p> Second, the robust correlation serves as a foundation for predicting dynamics of synthetic gene circuits engineered for practical applications. In my work, I developed a prototype microbial swarmbot, which employs spatial arrangement to control growth dynamics of engineered bacteria. I demonstrated an engineered safeguard strategy to prevent unintended bacterial proliferation with this platform technology. In this work, I adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside microbial swarmbots will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. In this work, using antibiotics to control growth dynamics of the engineered populations was critical, and optimization of the growth dynamics depended on their environmental conditions that modulated their growth rates.</p><p>Together, my investigation on quantifying and analyzing bacterial growth dynamics demonstrated that understanding of bacterial population dynamics is crucial in addressing antibiotic tolerance in bacteria as well as in using them for engineered functions. By further examining the dependence of lysis rate on growth rate, we extended our understanding of the phenomenon associated with β-lactam antibiotic treatment, and of possible consequences arising from variable lysis rate. This information is important in designing a modular and readily generalizable platform technology as well. Therefore, my work demonstrates quantitative approach towards understanding of bacterial populations, and lays the foundation for engineering integrated and programmable control of hybrid biological-material systems for diverse applications.</p> / Dissertation

<p>Normal healthy people are not susceptible to tuberculosis (TB) but immunocompromised and HIV positive patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and amikacin) for TB patients is 6-9 months for normal patients but if <i>Mycobacterium tuberculosis</i> (Mtb) becomes multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in 2018, there were about half a million new cases among which 78% were multidrug resistant TB. This antibiotic resistance is due in part to its ability to survive in the macrophage in our body by entering a non-replicating persistent state. Mtb also contains efflux pumps that increase antibiotic tolerance by pumping out the drugs. Therefore, if the efflux pump activity can be blocked by using efflux pump inhibitors, then it might increase antibiotic susceptibility of the pathogen. In our study, we used <i>Mycobacterium smegmatis</i> (Msm) as a model organism for Mtb and subjected it to a combination of three stresses (low oxygen, low pH and low nutrients) that mimic the physiological stresses in the human body and report that these conditions produced a non-replicating state in Msm. This is the first report of the use of this combination of stresses to produce a non-replicating state in Msm. Our results show that non-replicating Msm became completely tolerant to isoniazid and displayed increased tolerance to rifampin and clarithromycin by nearly 2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to construct a gene knockout mutant lacking two potential ATP-binding cassette transporters to study their functions as drug exporters. However, we were unable to obtain homologous recombination mutants. Further studies on efflux pump inhibitors could potentially enable greater understanding of antibiotic tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the development of novel therapies that shorten treatment time for tuberculosis.</p>

Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

Staphylococcus aureus (S. aureus) est un pathogène humain majeur dont l’impact sur la santé publique est majoré par les phénomènes de résistance et de tolérance aux antibiotiques. Au cours de cette thèse, nous avons identifié deux nouveaux systèmes toxine-antitoxine (STA) de type I appartenant au core génome et exprimés par S. aureus nommés sprG2/SprF2, sprG3/SprF3. Ces nouveaux STA sont homologues au STA sprG1/SprF1 localisé dans un îlot de pathogénie. Nous avons montré que des interactions croisées influençant le niveau des ARN sprG et SprF pouvaient avoir lieu entre les STA homologues sprG/SprF, mais que celles-ci n’avaient pas d’impact sur la neutralisation spécifique de chaque toxine SprG par son antitoxine SprF. Nous avons démontré que les peptides encodés par sprG2 et sprG3 sont bactériostatiques, contrairement aux peptides encodés par sprG1 qui sont bactéricides. Nous avons démontré que l’expression des ARN sprG et SprF pouvait varier en réponse à des stress environnementaux comme un stress hyperosmotique ou un stress oxydatif. Pour le STA sprG1/SprF1, nous avons démontré que l’antitoxine SprF1 pourrait participer à l’entrée en persistance de S. aureus, en atténuant la traduction globale via son association aux ribosomes. Ainsi, SprF1 est le premier exemple d’une antitoxine ARN avec une double fonction de neutralisation de la toxine sprG1 via son extrémité 3’ et de fixation aux ribosomes pour atténuer la traduction de S. aureus via son extrémité 5’. Ensemble, ces travaux de thèse suggèrent que les STA sprG/SprF seraient impliqués dans l’adaptation de S. aureus au stress antibiotique ou dans l’échappement au système immunitaire. / Staphylococcus aureus (S. aureus) is a human pathogen that causes nosocomial and community-associated infections. The antibiotic resistance and tolerance of S. aureus increase its impact on public health. During my PhD thesis, we identified two novel type I toxin-antitoxin systems (TAS) located in the core genome and expressed in S. aureus named sprG2/SprF2 and sprG3/SprF3. These TAS are homologues of the sprG1/SprF1 TAS, encoded in a pathogenicity island. We showed that cross-interactions affecting sprG and SprF RNA level can occur between sprG/SprF homologous TAS, but without any impact on the specific neutralization of a sprG toxin by its SprF antitoxin. We demonstrated that overexpression of sprG2- and sprG3-encoded peptide induce bacteriostasis, as opposed to the sprG1-encoded peptides that induced S. aureus death. We showed that sprG and SprF RNA levels can be modulated by environmental triggers such as hyperosmotic and oxidative stresses. Concerning sprG1/SprF1, we demonstrated in S. aureus strain N315 that the SprF1 antitoxin could be involved in persister cells formation, by a translation attenuation mechanism via its association with the ribosomes. SprF1 is the first example of an untranslated RNA antitoxin with a dual function that neutralize sprG1 toxin and that bind to ribosome to attenuate S. aureus translation. Altogether, these thesis experiments suggest an involvement of the sprG/SprF TAS in S. aureus adaptation to antibiotic stress or in the escape of the immune system.

Together with the current antibiotic resistance crisis, bacterial persistence appears to play an increasingly important role in the frequent failure of antibiotic treatments. Persister cells are rare bacteria that transiently become drug tolerant, allowing them to survive lethal concentrations of bactericidal antibiotics. Upon antibiotic removal, persister cells are able to resume growth and give rise to a new bacterial population as sensitive to the antibiotic as the original population. Interest in persister cells seriously increased in the past few years as these phenotypic variants were shown to be involved in the recalcitrance of chronic infections, such as tuberculosis and pneumonia and in the well-known biofilm tolerance to antibiotics. Persistence has therefore been extensively studied throughout the last decade, which led to the discovery of large variety of different molecular mechanisms involved in persisters formation. However, the specific physiology of bacterial persisters remains elusive up to now, mainly because of the transient nature and the low frequencies of persister cells in growing bacterial cultures. This work aims to gain a better understanding of the physiology of Escherichia coli persisters by combining population analyses with single-cell observations.In the first part of this thesis, we developed an experimental method allowing for measuring persistence with increased reproducibility. The method was further refined, which allowed us to observe four distinct phases in the ofloxacin time-kill curve, suggesting the existence of a tolerance continuum at the population level at treatment time. Characterization of these four phases notably revealed that the growth rate and the intrinsic antibiotic susceptibility of the strain define the number of surviving cells at the onset of the persistence phase, while persister cells survival mainly relies on active stress responses (SOS and stringent responses in particular).We next investigated the molecular mechanisms underlying the well-known correlation between persistence and the growth rate. Interestingly, we showed that the growth rate determines the number of survival cells at the onset of the persistent phase, whereas it does not affect the death rate of persister cells during antibiotic treatment. Furthermore, slow growth was shown to influence survival to ofloxacin independently of the replication rate, thereby suggesting that target inactivation solely cannot explain this correlation. However, our preliminary data indicate that ppGpp induction upon ofloxacin exposure substantially increases in slow growing bacterial populations, supporting a model in which slow growth would allow bacteria to respond faster to the antibiotic treatment, thereby generating more persisters than fast growing bacterial populations.Finally, both population and single-cell analyses were performed to assess the influence of the SOS response on persistence to ofloxacin. Firstly, population analyses revealed that the SOS response is required for survival of both sensitive and persister cells, but only during recovery, after ofloxacin removal, presumably allowing cells to induce SOS-dependent DNA repair pathways, required to deal with the accumulated ofloxacin-induced DNA lesions. The SOS response therefore appears as a good target for anti-persisters strategies, as shown by the 100-fold decrease in persistence upon co-treatment of a bacterial population with an SOS-inhibitor and ofloxacin. Secondly, single-cell analyses revealed that persister cells sustain similar DNA damages than sensitive cells upon ofloxacin treatment and induce SulA- and SOS-independent filamentation upon antibiotic removal, probably reflecting the presence of remaining cleaved complexes, formed during ofloxacin exposure. Importantly, we showed filamentation to occur in persister cells upon ampicillin treatment as well, thereby suggesting these filaments to be part of a more general survival pathway, which molecular basis remains unknown. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The discovery of antibiotics is considered as one of the most significant scientific achievements of the 20th century – lives of millions of people and animals have been saved. Thenceforth, substantial amounts of administered antibiotics and their metabo-lites have been excreted into waste stream via urine and faeces. In this dissertation, primary focus is the qualitative balance of 14 antibiotics and one metabolite in urban water management and in urban waters, respectively. In particular, antibiotics pre-scribed to human beings are drained in the urban sewer system and finally enter the environment: (i) Continuously via the effluent of the wastewater treatment plant after a partially effective removal or degradation or (ii) Intermittent via combined sewer overflow structures due to capacity limitations of the urban drainage system. The fate and the potential effects and risks of these substances on ecosystems and hu-man health are of major concern – their direct toxic effect to all trophic levels as well as the global spread of antibiotic resistance genes are challenging. Hence, an assessment of microbial community activity due to antibiotic exposure is presented. In particular, systematic work has been carried out to study the presence and character-istics of 14 antibiotics in urban waters. In detail, investigations were conducted to gain scientific knowledge with respect to adsorption, desorption, abiotic, biotic and photolyt-ic degradation as well as activity-inhibition of microorganism communities in sewage and of natural freshwater biofilm communities, respectively, due to inevitable urban drainage overflows. In order to provide information to assist potential management strategies, which miti-gate surface water pollution and minimize the adverse impacts of antibiotics on activity of microorganism communities, the following specific topics were addressed: ⑴ The occurrence of 14 antibiotics and one metabolite were determined in sewag-es at three sampling sites in the city of Dresden, Germany. ⑵ The adsorption affinities of 14 antibiotics and one metabolite to size dependent sewer sediments were determined in experimental investigations, three sam-pling campaigns and subsequently an antibiotic-specific adsorption coefficient, normalized to organic content, was quantified. ⑶ The desorption affinity and -dynamics of 14 antibiotics and one metabolite were quantified in size dependent sewer sediments in experimental investigation and with statistical analysis. ⑷ The abiotic, biotic and photolytic degradation affinity of 14 antibiotics and one metabolite were quantified based on batch experiments with three different sewages at 7°C and 22°C, with artificial irradiation and different dilution ratios of the sewage at 30°C and subsequently a model framework decrypted ranges of abiotic, biotic and photolytic degradation coefficients. ⑸ The occurrence of three antibiotics, namely ciprofloxacin, clarithromycin and doxycycline was determined in sewage sampled during dry weather conditions in a small catchment of Dresden, which spills intermittently combined sewage (a mixture of sewage and storm water) to an adjacent brook in the case of capacity limitations of the urban drainage system during periods of intense rainfall and subsequently the three antibiotics were determined in the adjacent brook water. ⑹ Then, the activity-inhibition of microorganism community in sewage of this small catchment was quantified due to an exposition with three different antibiotics and three different antibiotic concentrations. ⑺ Last but not least, the activity-inhibition of natural freshwater biofilm communities in the adjacent brook was quantified via exposure to three antibiotics, which were individually dosed in three different concentrations, and also in mixture. ⑻ Finally, a two-dimensional hierarchical cluster analysis with dendrogram and heat map based on before mentioned activity inhibition of natural freshwater biofilm communities were conducted to identify hot spots of antibiotic tolerant and resistant bacterial subpopulations due to inevitable urban drainage system overflows.:List of Figures IV List of Tables VIII Symbols and Abbreviations XII List of Publications on the Ph.D. topic XIX 1 General Introduction 2 1.1 Background 2 1.2 Aims and Objectives 3 1.3 Innovation and Contribution to the Knowledge 4 1.4 Outline of this Thesis 4 1.5 References 6 2 Adsorption and Desorption Affinity of 14 Antibiotics and One Metabolite for particulate components in urban drainage systems 10 2.1 Introduction 11 2.2 Materials and Methods 12 2.2.1 Study area 12 2.2.2 Sewer sediment and sewage sample collection 12 2.2.3 Sediment fractionation 13 2.2.4 Antibiotic determination in sewage and sediment 13 2.3 Results and Discussion 18 2.3.1 Antibiotics in composite sewage samples 18 2.3.2 Antibiotics adsorbed to sewer sediments 19 2.3.3 Organic-bound antibiotic load as a linear function of liquid concentration 20 2.3.4 Adsorption dynamics and adsorption coefficient determined by bath experiments 20 2.3.5 Mineral composition of sewer sediment SED#1B 23 2.3.6 Initial characteristics of sediment SED#1B 23 2.3.7 Desorption dynamics and desorption coefficient of SED#1B 24 2.4 Conclusions 25 2.5 References 26 3 Abiotic, Biotic and Photolytic Degradation Coefficients of 14 Antibiotics and One Metabolite 32 3.1 Introduction 34 3.2 Materials and Methods 35 3.2.1 Study area and sample collection 35 3.2.2 Experimental set up 35 3.2.3 Modelling framework 38 3.2.4 Procedure of model calibration 40 3.3 Results and Discussion 43 3.3.1 Primary metabolic parameter 43 3.3.2 Secondary metabolic parameter 44 3.4 Conclusions 50 3.5 References 50 4 Activity-Inhibition of Microorganisms due to an Exposition with different Antibiotics and Concentrations 56 4.1 Assessing Antibiotic Resistance of Microorganisms in Sanitary Sewage 56 4.1.1 Introduction 57 4.1.2 Material and Methods 58 4.1.2.1 Sampling Site and Antibiotic Agents 58 4.1.2.2 Analyzing Antibiotics 60 4.1.2.3 Respiration Rate 60 4.1.3 Results and Discussion 60 4.1.3.1 Concentration Range of Antibiotics and Typical Sewage Parameters 60 4.1.3.2 Oxygen Uptake Rate 62 4.1.4 Summary and Conclusions 63 4.1.5 References 64 4.2 Hot Spots of Antibiotic Tolerant and Resistant Bacterial Subpopulations in Natural Freshwater Biofilm Communities due to Inevitable Urban Drainage System Overflows 66 4.2.1 Introduction 68 4.2.2 Material and Methods 69 4.2.3 Results and Discussion 72 4.2.4 Conclusions 76 4.2.5 References 76 5 Summery and General Coclusions 82 5.1 Adsorption and Desorption Affinity 82 5.2 Abiotic, Biotic and Photolytic Degradation 83 5.3 Activity-Inhibition of Microorganism Communities due to Antibiotic Exposure 84 5.4 Enhancement of the Stockholm County Council (2014) assessment of antibiotics 84 5.5 References 87 6 Proposed Directions of Future Research 90 7 Appendixes 94 7.1 Chapters 94 7.2 Figures 95 7.3 Tables 115 7.4 References 139