Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic model developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G2-phase cell prior to division is inherently twice the cell volume of a G1-phase cell after cell division. By the same logic, it is also apparent that a G2-phase cell possesses twice the number of ribosomes of a G1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G1 and G2/M phase cell proportions remained unchanged, whereas S phase cells progress to G2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G2/M phase in both infected and non-infected cultures were caused through medium exchange.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.

Die Malaria ist heute noch immer die bedeutendste parasitäre Infektionskrankheit des Menschen. Hiervon sind in Endemiegebieten neben Kleinkindern insbesondere schwangere Frauen betroffen. P. falciparum weist eine hohe genetische Diversität auf. So sind in Endemiegebieten Infektionen mit P. falciparum in der Regel polyklonal. Man spricht in diesen Fällen von der Multiplizität der Infektion. Bei Schwangeren sequestrieren mit P. falciparum infizierte Erythrozyten, die spezifische Oberflächenproteine exprimieren, in der Plazenta. Hierdurch bedingt können pathologische Schwangerschafts-verläufe klinische Manifestationsformen der Malaria darstellen. Um die Diversität von P. falciparum und die Multiplizität der Infektion bei schwangeren Frauen zu erforschen, wurden in einer Querschnittsstudie im holoendemischen Malariagebiet von Agogo in Ghana über den Zeitraum von einem Jahr 474 Gebärende mit einer nachgewiesenen plazentaren Infektion von P. falciparum untersucht. Hierzu wurden die Gene, die für das "Merozoiten-Oberflächen-Protein-1" (msp-1) und "Merozoiten-Oberflächen-Protein-2" (msp-2) kodieren, aus peripher und plazentar gewonnen Isolaten typisiert. Plazentar gewonnene Isolate waren im Vergleich zu peripher gewonnenen mit einer signifikant höheren Prävalenz an polyklonalen Infektionen und einer höheren Multiplizität der Infektion assoziiert. Die höchste Multiplizität der Infektion wurde bei Erstgebärenden und jüngeren Patientinnen beobachtet. Mit zunehmendem Alter und einer höheren Anzahl an vorangegangenen Schwangerschaften fielen signifikant sowohl die Multiplizität der Infektion als auch die Parasitendichte. Zudem wurde eine hohe Korrelation zwischen der Multiplizität der Infektion und der Parasitendichte nachgewiesen. Weder das Alter noch die Parität beeinflussten diese Korrelation. Der Einfluss von Alter und Parität auf die Multiplizität der Infektion konnte somit nicht unabhängig von der Parasitendichte nachgewiesen werden. Multivariate Analysen zeigten aber, dass es unabhängig von der Parasitendichte bei plazentaren Infektionen mit zwei und mehr als zwei Klonen im Vergleich zu monoklonalen plazentaren Infektionen mit einer höheren Wahrscheinlichkeit zu einer Frühgeburt kam. Dies betraf insbesondere Erstgebärende und Frauen mit submikroskopischen plazentaren Infektionen. Ob bei polyklonalen Infektionen eine Sequestration von P. falciparum in der Plazenta durch alle oder nur durch einen Teil der zahlreichen Genotypen geschieht, die an einer Infektion bei Schwangeren beteiligt sind, ist nicht entgültig geklärt. Es wurden aus zusammengehörenden plazentar und peripher gewonnenen P.-falciparum-Isolaten die Verteilungsmuster der Genotypen verglichen. Zwar korrelierte die Multiplizität der Infektion plazentarer und peripherer Isolate, die Genotypenmuster der Plazenta und der Peripherie waren jedoch deutlich unterschiedlich. Nur in 12% der Fälle konnte eine Genotypisierung eines peripher gewonnenen Isolates das klonale Gesamtbild der Infektion nachweisen. In 67% der Fälle waren neben identischen Genotypen wenigstens in einem der beiden Isolate unterschiedliche Genotypen nachweisbar. Einzelne spezifische Genotypen traten in der gesamten Untersuchungsgruppe öfter in der Plazenta als in der peripheren Blutprobe auf. Bei Frauen, die mit den Genotypen der Allelfamilie FC27 infiziert waren, lagen signifikant häufiger klinischen Manifestationen der Malaria vor. So konnte in multivariaten Analysen eine Assoziation zwischen FC27 und einer Frühgeburtlichkeit nachgewiesen werden. Darüber hinaus war FC27 zumindest in univariater Analyse mit einer Anämie und einem verminderten Geburtsgewicht assoziiert. Dies konnte insbesondere für Primiparae und für Gebärende mit submikroskopischen plazentaren Infektionen beobachtet werden. / Malaria is still one of the most considerable parasite infections of the human being. Pregnant women are at an increased risk in endemic areas. P. falciparum shows a high genetic diversity. In endemic areas infections with P. falciparum are very often polyclonal. They are described as multiple Infections or as the multiplicity of infection. In pregnant women P.-falciparum-infected-erythrocytes which exprimate specific surface proteins sequester in the placental tissue. Often this is the course of preterm delivery, low birth weight and anaemia. To investigate the diversity of P. falciparum and the multiplicity of infection in pregnant women a cross-sectional study was conducted in the holoendemic area of Agogo in Ghana. In this study over a period of one year 474 labouring women infected with placental P.-falciparum where investigated. To examine the diversity and the multiplicity of infection merozoite surface protein-1 (msp1) block 2 and merozoite surface protein-2 (msp2) genotypes were determined in Isolates from peripheral and placental blood samples. The study showed that in comparision to isolates of peripheral blood samples isolates of placental blood samples where associated with a significant higher prevalence of polyclonal infections and a higher multiplicity of infection. The highest multiplicity of infection was found among primiparae and young women. With age and parity multiplicity of infection as well as parasite density decreased. In addition a high correlation between the multiplicity of infection and parasite density could be demonstrated. Age and parity did not influence this correlation. Thus the influence of age independent from parity on the multiplicity of infection could not be proved. However, multivariate analyses showed, that independently from parasite density placental Infections with two or more clones were in comparison to monoclonal Infections associated with a higher probability of preterm delivery. This was the case especially in primiparae and in women with submicroscopical placental Infection. Presently it is not clear, whether all or only a subset of co-infecting genotypes sequester in the placental tissue. To address this issue the genotype distribution of matched placental and peripheral P. falciparum isolates where investigated. While the multiplicity of infection of placental and peripheral isolates correlated the genotype pattern of the placenta and the periphery differed extensively. Only 12% genotyping of a peripheral Isolate showed the entire picture of the infection. In 67% of the cases despite finding identical genotypes differing genotypes in at least one of the two Isolates were detectable. Specific genotypes appeared more often in the placental than in the peripheral Isolate. In women, who were infected with genotypes of the allelic family FC27 clinical manifestation of malaria were observed more often. In multivariate analysis an association between FC27 and a preterm delivery was established. Beside this at least in univariate analyses FC27 was associated with low birth weight and anaemia. This was the case especially for primiparae and labouring women with submicroscopic placental infections.