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Generation of early human neuroepithelial progenitors from primary cells for biomedical applications
(2018)
Patient-specific induced pluripotent stem cells (iPSCs) emerged as a promising cell source for disease modeling and drug screening as well as a virtually unlimited source for restorative therapy. The thesis deals with three major topics to help realizing biomedical applications with neural stem cells.
To enable the generation of transgene-free iPSCs, alternatives to retroviral reprogramming were developed. Hence, the adaptation and evaluation of reprogramming using excisable lentiviral constructs, Sendai virus (SeV) and synthetic mRNA-based methods was assessed in the first part of this thesis. hiPSCs exhibit the pluripotency markers OCT4, SSEA-4, TRA1-60 which were confirmed by immunofluorescence and flow cytometry. Besides, the potential to differentiate in cell types of all three germ layers was detected, confirming pluripotent identity of proliferating colonies resulting from various reprogramming strategies. However, major differences such as high efficiency with SeV in contrast to a relatively low efficiency with mRNA in regard to passage number and the phenotype of starting fibroblasts were observed. Furthermore, a prolonged clone- and passage-dependent residual presence of viral RNA genes was identified in SeV-iPSCs for up to 23 passages using RT-PCR underlining the importance of careful monitoring of clone selection. In contrast, viral-free reprogramming by synthetic mRNA represents a fully non-integrative approach but requires further refinement to be efficiently applicable to all fibroblasts.
The second part of this thesis deals with the establishment of a rapid monolayer approach to differentiate neural progenitor cells from iPSCs. To achieve this, a two-step protocol was developed allowing first the formation of a stable, primitive NPC line within 7 days which was expanded for 2-3 passages. In a second step, a subsequent adaptation to conditions yielding neural rosette-like NPCs followed. Both neural lines were demonstrated to be expandable, cryopreservable and negative for the pluripotency marker OCT4. Furthermore, a neural precursor identity including SOX1, SOX2, PAX6, Nestin was confirmed by immunofluorescence and quantitative RT-PCR. Moreover, the differentiation resulted in TUJ1-positive neurons and GFAP-positive astrocytes. Nonetheless, the outcome of glial differentiation from primitive NSCs remained low, whereas FGF/EGF-NPCs were efficiently differentiated into GFAP-positive astrocytes which were implicated in a cellular model of the blood brain barrier.
The third and major objective of this study was to generate human early neural progenitor cells from fetal brain tissue with a wide neural differentiation capacity. Therefore, a defined medium composition including small molecules and growth factors capable of modulation of crucial signaling pathways orchestrating early human development such as SHH and FGF was assessed. Indeed, specific culture conditions containing TGFβ inhibitor SB431542, SHH agonist Purmorphamine, GSK3β inhibitor CHIR99021 and basic FGF, but no EGF enabled robust formation of early neuroepithelial progenitor (eNEP) colonies displaying a homogeneous morphology and a high proliferation rate. Moreover, primary eNEPs exhibit a relatively high clonogenicity of more than 23 % and can be monoclonally expanded for more than 45 passages carrying a normal karyotype. Characterization by immunofluorescence, flow cytometry and quantitative RT-PCR revealed a distinct NPC profile including SOX1, PAX6, Nestin and SOX2 and Prominin. Furthermore, primary eNEPs show NOTCH and HES5 activation in combination with non-polarized morphology, indicative of an early neuroepithelial identity. Microarray analysis unraveled SOX11, BRN2 and other HES-genes as characteristic upregulated genes. Interestingly, eNEPs were detected to display ventral midbrain/hindbrain regional identity. The validation of yielded cell types upon differentiation indicates a strong neurogenic potential with more than 90 % of TUJ1-positive neurons. Moreover, astrocytes marked by GFAP and putative myelin structures indicating oligodendrocytes were identified. Electrophysiological recordings revealed functionally active neurons and immunofluorescence indicate GABAergic, glutamatergic, dopaminergic and serotonergic subtypes. Additionally, putative physiological synapse formation was observed by the presence of Synapsin and PSD-95 as well as by ultrastructural examination. Notably, rare neurons stained positive for the peripheral neuronal marker Peripherin suggesting the potential of eNEPS to give rise to cells of neural tube and neural crest origin. By the application of specific differentiation protocols an increase of TH-positive neurons or neural crest-derivatives such as putative A- and C-sensory neurons and mesenchymal cells was identified. Taken together, primary eNEPs might help to elucidate mechanisms of early human neurodevelopment and will serve as a novel source for cell replacement and further biomedical applications.
Luminescent organotransition metal complexes are of much current interest. As the large spin-orbit coupling of 2nd and 3rd row transition metals usually leads to rapid intersystem crossing from S1 to T1, which enables phosphorescence, there is a special interest in using triplet-emitting materials in organic or organometallic light emitting diodes (OLEDs). Marder et al. have found that, reductive coupling of both para-R-substituted diarylbutadiynes and diaryldodecatetraynes on Rh(PMe3)4X leads to quantitative yields of bis(arylethynyl)-rhodacyclopentadienes with complete regiospecificity (R = BMes2, H, Me, OMe, SMe, CF3, CN, CO2Me, NMe2, NO2, C≡C-TMS and X = -C≡C-TMS, -C≡C-C6H4-4-NMe2, -C≡C-C≡C-C6H4-4-NPh2, Me, Cl).47,49 Unexpectedly, these compounds show intense fluorescence rather than phosphorescence (ɸf = 0.33-0.69, t = 1.2 3.0 ns). The substituent R has a significant influence on the photophysical properties, as absorption and emission are both bathochromically shifted compared to R = H, especially for R = π-acceptor.
To clarify the mechanism of the formation of the rhodacyclopentadienes, and to investigate further their unique photophysical properties, a series of novel, luminescent rhodacyclopentadienes with dithiocarbamate as a bidentate ligand at the rhodium centre has been synthesised and characterised (R = NO2, CO2Me, Me, NMe2, SMe, Ar = C6F4-4-OMe). The rhodacyclopentadienes have been formed via reductive coupling of diaryl undecatetraynes with [Rh(k2-S,S`-S2CNEt2)(PMe3)2]. The structures of a series of such compounds were solved by single crystal X-ray diffraction and are discussed in this work. The compounds were fully characterised via NMR, UV/Vis and photoluminescence spectroscopy as well as by elemental analysis, high-resolution mass spectrometry (HRMS) and X-ray diffraction.
When heating the reactions, another isomer is formed to a certain extent. The so-called dibenzorhodacyclopentadienes already appeared during earlier studies of Marder et al., when acetylacetonate (acac) was employed as the bidentate ligand at the Rh-centre. They are probably formed via a [4+2] cycloaddition reaction and C-H activation, followed by a β-H shift.
Use of the perfluorinated phenyl moiety Ar = C6F4-4-OMe provided a total new insight into the mechanism of formation of the rhodacyclopentadiene isomers and other reactions. Besides the formation of the expected rhodacyclopentadiene, a bimetallic compound was generated, isolated and characterised via X-ray crystallography and NMR spectroscopy, elemental analysis and high resolution mass spectrometry.
For further comparison, analogous reactions with [Rh(k2 S,S` S2CNEt2)(PPh3)2] and a variety of diaryl undecatetraynes (R = NO2 CO2Me, Me, NMe2, SMe, Ar = C6F4-4-OMe) were carried out. They also yield the expected rhodacyclopentadienes, but quickly react with a second or even third equivalent of the tetraynes to form, catalytically, alkyne cyclotrimerisation products, namely substituted benzene derivatives (dimers and trimers), which are highly luminescent. The rhodacyclopentadienes (R = NO2, CO2Me, Me, SMe, Ar = C6F4-4-OMe) are stable and were isolated. The structures of a series of these compounds were obtained via single crystal X-ray crystallography and the compounds were fully characterised via NMR, UV/Vis and photoluminescence spectroscopy as well as by elemental analysis and HRMS.
Another attempt to clarify the mechanism of formation of the rhodacyclopentadienes involved reacting a variety of diaryl 1,3-butadiynes (R = CO2Me, Me, NMe2, naphthyl) with [Rh(k2 S,S` S2CNEt2)(PMe3)2]. The reactions stop at an intermediate step, yielding a 1:1 trans π-complex, confirmed by single crystal X-ray diffraction and NMR spectroscopy. Only after several weeks, or under forcing conditions (µw / 80 °C, 75 h), the formation of another major product occurs, having bound a second diaryl 1,3-butadiyne. Based on earlier results of Murata, the product is identified as an unusual [3+2] cycloaddition product, ϭ-bound to the rhodium centre.
The molecular architecture of the meiotic chromosome axis as revealed by super-resolution microscopy
(2018)
During meiosis proteins of the chromosome axis are important for monitoring chromatin structure and condensation, for pairing and segregation of chromosomes, as well as for accurate recombination. They include HORMA-domain proteins, proteins of the DNA repair system, synaptonemal complex (SC) proteins, condensins and cohesins. To understand more about their function in shaping the meiotic chromosome it is crucial to establish a defined model of their molecular architecture. Up to now their molecular organization was analysed using conventional methods, like confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). Unfortunately, these techniques are limited either by their resolution power or their localization accuracy. In conclusion, a lot of data on the molecular organization of chromosome axis proteins stays elusive. For this thesis the molecular structure of the murine synaptonemal complex (SC) and the localization of its proteins as well as of three cohesins was analysed with isotropic resolution, providing new insights into their architecture and topography on a nanoscale level. This was done using immunofluorescence labelling in combination with super-resolution microscopy, line profiles and average position determination. The results show that the murine SC has a width of 221.6 nm ± 6.1 nm including a central region (CR) of 148.2 nm ± 2.6 nm. In the CR a multi-layered organization of the central element (CE) proteins was verified by measuring their strand diameters and strand distances and additionally by imaging potential anchoring sites of SYCP1 (synaptonemal complex protein 1) to the lateral elements (LEs). We were able to show that the two LEs proteins SYCP2 and SYCP3 do co-localize alongside their axis and that there is no significant preferential localization towards the inner LE axis of SYCP2.
The presented results also predict an orderly organization of murine cohesin complexes (CCs) alongside the chromosome axis in germ cells and support the hypothesis that cohesins in the CR of the SC function independent of CCs.
In the end new information on the molecular organization of two main components of the murine chromosome axis were retrieved with nanometer precision and previously unknown details of their molecular architecture and topography were unravelled.
In recent decades the international migration has increased worldwide. The influx of people from different cultures and ethnic groups poses new challenges to the labor market and the welfare state of the host countries and causes changes in the social fabric. In general, immigration benefits the economy of the host country. However, these gains from immigration are unevenly distributed among the native population. Natives who are in direct competition with the new workers expect wage losses and a higher probability of getting unemployed, whereas remaining natives foresee either no feedback effects or even wage gains. On the other hand, the tax and transfer system benefits disproportionally from an influx of highly skilled immigrants. Examinations of 20 European countries in 2010 show that a higher proportion of low-skilled immigrants in the immediate neighborhood of the natives increases the difference in the demand for redistribution between high-skilled and low-skilled natives. Thus, high-skilled natives are more opposed to an expansion of the governmental redistribution. On the one hand, a higher proportion of low-skilled immigrants generates a higher fiscal burden on the welfare state. On the other hand, high-skilled natives' wages increase due to an influx of low-skilled immigrants, since relative supply of high-skilled labor increases.
In addition to the economic impact of immigration, the inflow of new citizens is accompanied by natives' fear of changes in the social environment as well as in symbolic values, such as cultural identity or natives' set of values. The latter might generate negative attitudes towards immigrants and increase the demand for a more restrictive immigration policy. On the other hand, more interethnic contact due to a higher ethnic diversity could reduce natives' information gaps, prejudices and stereotypes. This, in turn, could enhance more tolerance and solidarity towards immigrants among natives. Examinations of 18 European countries in 2014 show that more interethnic contact during everyday life reduces both the natives' social distance from immigrants and their fear of social upheaval by the presence of immigrants. However, natives' social distance from immigrants has no effect on their preference for redistribution, but their perceived threat to the national culture and social life by the presence of immigrants has a significantly negative impact on their demand for redistribution. Thus, natives’ concern about the preservation of symbolic norms and values affects the solidarity channel of their redistribution preference.
An individual's upward mobility over time or in relation to his or her parents determines his or her attitude towards the welfare state as well as the transfer of his or her opinions to his or her own children. With regard to intergenerational income mobility, Germany shows a value in the international midfield; higher than the United States (lower mobility) and lower than the Scandinavian countries (higher mobility). For example, if a father's lifetime income increases by 10 percent, his son's lifetime income increases by 4.9 percent in the United States and by 3.1 percent in Germany. Additionally, in Germany, fathers' lifetime income tends to show a higher impact on their sons' income if their incomes are higher. In the United States, fathers' lifetime incomes have a stronger influence on their sons' income at the lower and the upper end of the income distribution compared to the middle.
Taking a closer look at the intragenerational wage mobility and wage inequality in Germany, the development at the current edge is rather sobering. Since 2000 there is a steady decline in wage mobility. Furthermore, wage mobility in the services sector has been significantly lower than in the manufacturing sector since the beginning of the 2000s. This result is mainly driven by the decrease of wage mobility in the health care and social services sector. Moreover, a worker's unemployment spells and occupation have become more important in the meantime. Since 2006 the increase in the German wage inequality has markedly slowed down and wage growth between 2006 and 2013 has been even polarized, i.e. wages at the lower and at the upper end of the wage distribution have increased more than wages in the middle. However, this development can be partly attributed to the computerization and automation of the production processes. Although, there was substitution of manual routine tasks between 2001 and 2013, cognitive routine tasks are still more pronounced in the middle and at the upper end of the wage distribution. Furthermore, the latter experienced an increase in wage mobility since 2000. On the other hand, manual non-routine tasks are localized disproportionally in the middle and at the lower end of the wage distribution. Thus, the wage gains of these occupations at the lower end were compensated for by the wage losses in the middle.
Solitary bees in seasonal environments have to align their life-cycles with favorable environmental conditions and resources. Therefore, a proper timing of their seasonal activity is highly fitness relevant. Most species in temperate environments use temperature as a trigger for the timing of their seasonal activity. Hence, global warming can disrupt mutualistic interactions between solitary bees and plants if increasing temperatures differently change the timing of interaction partners. The objective of this dissertation was to investigate the mechanisms of timing in spring-emerging solitary bees as well as the resulting fitness consequences if temporal mismatches with their host plants should occur. In my experiments, I focused on spring-emerging solitary bees of the genus Osmia and thereby mainly on O. cornuta and O. bicornis (in one study which is presented in Chapter IV, I additionally investigated a third species: O. brevicornis).
Chapter II presents a study in which I investigated different triggers solitary bees are using to time their emergence in spring. In a climate chamber experiment I investigated the relationship between overwintering temperature, body size, body weight and emergence date. In addition, I developed a simple mechanistic model that allowed me to unite my different observations in a consistent framework. In combination with the empirical data, the model strongly suggests that solitary bees follow a strategic approach and emerge at a date that is most profitable for their individual fitness expectations. I have shown that this date is on the one hand temperature dependent as warmer overwintering temperatures increase the weight loss of bees during hibernation, which then advances their optimal emergence date to an earlier time point (due to an earlier benefit from the emergence event). On the other hand I have also shown that the optimal emergence date depends on the individual body size (or body weight) as bees adjust their emergence date accordingly. My data show that it is not enough to solely investigate temperature effects on the timing of bee emergence, but that we should also consider individual body conditions of solitary bees to understand the timing of bee emergence.
In Chapter III, I present a study in which I investigated how exactly temperature determines the emergence date of solitary bees. Therefore, I tested several variants degree-day models to relate temperature time series to emergence data. The basic functioning of such degree-day models is that bees are said to finally emerge when a critical amount of degree-days is accumulated. I showed that bees accumulate degree-days only above a critical temperature value (~4°C in O. cornuta and ~7°C in O. bicornis) and only after the exceedance of a critical calendar date (~10th of March in O. cornuta and ~28th of March in O. bicornis). Such a critical calendar date, before which degree-days are not accumulated irrespective of the actual temperature, is in general less commonly used and, so far, it has only been included twice in a phenology model predicting bee emergence. Furthermore, I used this model to retrospectively predict the emergence dates of bees by applying the model to long-term temperature data which have been recorded by the regional climate station in Würzburg. By doing so, the model estimated that over the last 63 years, bees emerged approximately 4 days earlier.
In Chapter IV, I present a study in which I investigated how temporal mismatches in bee-plant interactions affect the fitness of solitary bees. Therefore, I performed an experiment with large flight cages serving as mesocosms. Inside these mesocosms, I manipulated the supply of blossoms to synchronize or desynchronize bee-plant interactions. In sum, I showed that even short temporal mismatches of three and six days in bee-plant interactions (with solitary bee emergence before flower occurrence) can cause severe fitness losses in solitary bees. Nonetheless, I detected different strategies by solitary bees to counteract impacts on their fitness after temporal mismatches. However, since these strategies may result in secondary fitness costs by a changed sex ratio or increased parasitism, I concluded that compensation strategies do not fully mitigate fitness losses of bees after short temporal mismatches with their food plants. In the event of further climate warming, fitness losses after temporal mismatches may not only exacerbate bee declines but may also reduce pollination services for later-flowering species and affect populations of animal-pollinated plants.
In conclusion, I showed that spring-emerging solitary bees are susceptible to climate change as in response to warmer temperatures bees advance their phenology and show a decreased fitness state. As spring-emerging solitary bees not only consider overwintering temperature but also their individual body condition for adjusting emergence dates, this may explain differing responses to climate warming within and among bee populations which may also have consequences for bee-plant interactions and the persistence of bee populations under further climate warming. If in response to climate warming plants do not shift their phenologies according to the bees, bees may experience temporal mismatches with their host plants. As bees failed to show a single compensation strategy that was entirely successful in mitigating fitness consequences after temporal mismatches with their food plants, the resulting fitness consequences for spring-emerging solitary bees would be severe. Furthermore, I showed that spring-emerging solitary bees use a critical calendar date before which they generally do not commence the summation of degree-days irrespective of the actual temperature. I therefore suggest that further studies should also include the parameter of a critical calendar date into degree-day model predictions to increase the accuracy of model predictions for emergence dates in solitary bees. Although our retrospective prediction about the advance in bee emergence corresponds to the results of several studies on phenological trends of different plant species, we suggest that more research has to be done to assess the impacts of climate warming on the synchronization in bee-plant interactions more accurately.
The rotation of the earth around its axis causes recurring and predictable changes in the environment. To anticipate those changes and adapt their physiology and behavior accordingly, most organisms possess an endogenous clock. The presence of such a clock has been demonstrated for several ant species including Camponotus ants, but its involvement in the scheduling of daily activities within and outside the ant nest is fairly unknown. Timing of individual behaviors and synchronization among individuals is needed to generate a coordinated collective response and to maintain colony function. The aim of this thesis was to investigate the presence of a circadian clock in different worker castes, and to determine the daily timing of their behavioral tasks within the colonies of two nectar-collecting Camponotus species.
In chapter I, I describe the general temporal organization of work throughout the worker life in the species Camponotus rufipes. Continuous tracking of behavioral activity of individually- marked workers for up to 11 weeks in subcolonies revealed an age-dependent division of labor between interior and exterior workers. After eclosion, the fairly immobile young ants were frequently nurtured by older nurses, yet they started nursing the brood themselves within the first 48 hours of their life. Only 60% of workers switched to foraging at an age range of one to two weeks, likely because of the reduced needs within the small scale of the subcolonies. Not only the transition rates varied between subcolonies, but also the time courses of the task sequences between workers did, emphasizing the timed allocation of workers to different tasks in response to colony needs.
Most of the observed foragers were present outside the nest only during the night, indicating a distinct timing of this behavioral activity on a daily level as well. As food availability, humidity and temperature levels were kept constant throughout the day, the preference for nocturnal activity seems to be endogenous and characteristic for C. rufipes. The subsequent monitoring of locomotor activity of workers taken from the subcolonies revealed the presence of a functional endogenous clock already in one-day old ants. As some nurses displayed activity rhythms in phase with the foraging rhythm, a synchronization of these in-nest workers by social interactions with exterior workers can be hypothesized.
Do both castes use their endogenous clock to schedule their daily activities within the colony? In chapter II, I analyzed behavioral activity of C. rufipes foragers and nurses within the social context continuously for 24 hours. As time-restricted access to food sources may be one factor affecting daily activities of ants under natural conditions, I confronted subcolonies with either daily pulses of food availability or ad libitum feeding. Under nighttime and ad libitum feeding, behavioral activity of foragers outside the nest was predominantly nocturnal, confirming the results from the simple counting of exterior workers done in chapter I. Foragers switched to diurnality during daytime feeding, demonstrating the flexible and adaptive timing of a daily behavior. Because they synchronized their activity with the short times of food availability, these workers showed high levels of inactivity. Nurses, in contrast, were active all around the clock independent of the feeding regime, spending their active time largely with feeding and licking the brood. After the feeding pulses, however, a short bout of activity was observed in nurses. During this time period, both castes increasingly interacted via trophallaxis within the nest. With this form of social zeitgeber, exterior workers were able to entrain in-nest workers, a phenomenon observed already in chapter I. Under the subsequent monitoring of locomotor activity under LD conditions the rhythmic workers of both castes were uniformly nocturnal independent of the feeding regime. This endogenous activity pattern displayed by both worker castes in isolation was modified in the social context in adaption to task demands.
Chapter III focuses on the potential factors causing the observed plasticity of daily rhythms in the social context in the ant C. rufipes. As presence of brood and conspecifics are likely indicators of the social context, I tested the effect of these factors on the endogenous rhythms of otherwise isolated individuals. Even in foragers, the contact to brood triggered an arrhythmic activity pattern resembling the arrhythmic behavioral activity pattern seen in nurses within the social context. As indicated in chapter I and II, social interaction could be one crucial factor for the synchronization of in nest activities. When separate groups were entrained to phase-shifted light-dark-cycles and monitored afterwards under constant conditions in pairwise contact through a mesh partitioning, both individuals shifted parts of their activity towards the activity period of the conspecific. Both social cues modulated the endogenous rhythms of workers and contribute to the context dependent plasticity in ant colonies.
Although most nursing activities are executed arrhythmically throughout the day (chapter II), previous studies reported rhythmic translocation events of the brood in Camponotus nurses. As this behavior favors brood development, the timing of the translocations within the dark nest seems to be crucial. In chapter IV, I tracked translocation activity of all nurses within subcolonies of C. mus. Under the confirmed synchronized conditions of a LD-cycle, the daily pattern of brood relocation was based on the rhythmic, alternating activity of subpopulations with preferred translocation direction either to the warm or to the cold part of the temperature gradient at certain times of the day. Although the social interaction after pulse feeding had noticeable effects on the in-nest activity in C. rufipes (chapter I and II), it was not sufficient to synchronize the brood translocation rhythm of C. mus under constant darkness (e.g. when other zeitgebers were absent). The free-running translocation activity in some nurses demonstrated nevertheless the involvement of an endogenous clock in this behavior, which could be entrained under natural conditions by other potential non-photic zeitgebers like temperature and humidity cycles.
Daily cycling of temperature and humidity could not only be relevant for in-nest activities, but also for the foraging activity outside the nest. Chapter V focuses on the monitoring of field foraging rhythms in the sympatric species C. mus and C. rufipes in relation to abiotic factors. Although both species had comparable critical thermal limits in the laboratory, foragers in C. mus were strictly diurnal and therefore foraged under higher temperatures than the predominant nocturnal foragers in C. rufipes. Marking experiments in C. rufipes colonies with higher levels of diurnal activity revealed the presence of temporally specialized forager subpopulations. These results suggest the presence of temporal niches not only between the two Camponotus species, but as well between workers within colonies of the same species.
In conclusion, the temporal organization in colonies of Camponotus ants involves not only the scheduling of tasks performed throughout the worker life, but also the precise timing of daily activities. The necessary endogenous clock is already functioning in all workers after eclosion. Whereas the light-dark cycle and food availability seem to be the prominent zeitgebers for foragers, nurses may rely more on non-photic zeitgeber like social interaction, temperature and humidity cycles.
BD is a severe and highly prevalent psychiatric illness characterized by oscillating mood episodes, where patients express either depressed mood, anhedonia, decreased activation along with concentration difficulties and sleep disturbances, or elevated mood with hyperactivity and loss of inhibitions. Between mood episodes, patients return to a relatively normal state of functioning without mood symptoms. Previous research on underlying neuronal mechanisms has led to a model of neuronal dysfunction in BD which states that BD arises from disruption in early development within brain networks that modulate emotional behavior. These abnormalities in the structure and function of key emotional control networks then lead to decreased connectivity among ventral prefrontal networks and limbic brain regions. This in turn creates a loss of emotional homeostasis, putting bipolar patients at risk for developing extreme mood states and switching among mood states. Two core components for BD have been identified, a hyperactive emotion processing system and a hypoactive cognitive functions system. It is controversial whether these deficits are still detectable in euthymia, so it is unclear if hyper- and hypoactivations represent state or trait-like characteristics. The aim of this study was to research both core components of BD with a paradigm eliciting differential activations in both cognitive and emotion processing networks. For this, an emotional word working memory paradigm was constructed to test for differences between manic, depressive, and remitted patients as well as a healthy control group. Differences were assessed in behavior, brain activation (as a correlate for the hypoactive cognitive functions system), measured with near-infrared spectroscopy (fNIRS), and electrophysiological changes in the late positive potential (as a correlate for the hyperactive emotion processing system), an event-related potential (ERP) measured with electroencephalography. 47 patients in the acutely ill phase and 45 healthy controls were measured. Of the 47 patients, 18 returned to the clinic for a second testing while in remission for at least 3 months. Acutely ill patients were classified into 4 groups according to their disorder status: a mildly depressed group, a depressed group, a manic group, and a mixed group along DSM-IV criteria. Analyses were calculated for 3 load conditions (1-back, 2-back and 3-back) and 3 valence conditions (negative, neutral, positive) for behavioral measures reaction time and omission errors, for brain activation and event related potential changes.
Results indicate that ill patients differed from controls in their behavioral performance, but the difference in performance was modulated by the mood state they were in. Depressed patients showed the most severe differences in all behavioral measures, while manic and mixed patients differed from controls only upon different valence conditions. Brain activation changes were most pronounced in mildly depressed and manic patients, depressed patients and mixed patients did not differ as much from controls. ERP changes showed a significant difference only between mixed patients and controls, where mixed patients had an overall much higher ERP amplitude. When remitted patients were compared to controls, no differences in behavior, brain activation or ERP amplitude could be found. However, the same was true for differences in patients between acutely ill and remitted state. When looking at the overall data, the following conclusion can be drawn: assuming that the brain activation seen in the prefrontal cortex is part of the dorsal cognitive system, then this is the predominantly disturbed system in depressed patients who show only small changes in the ERP. In contrast, the predominantly disturbed system in manic and mixed patients is the ventral emotion processing system, which can be seen in a hyper-activation of ERP related neural correlates in mixed and hypo-activated neural correlates of the LPP in manic patients. When patients are remitted, the cognitive system regains temporary stability, and can be compared to that of healthy controls, while the emotion processing system remains dysfunctional and underlies still detectable performance deficits.
Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite’s surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG’Y’ and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG’Y’ fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG’Y’ as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that
target the parasites VSG coat.
During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat.
Several transition metal ions, like Fe2+, Co2+, Ni2+, and Zn2+ complex to the ditopic ligand 1,4-bis(2,2’:6’,2’’-terpyridin-4’-yl)benzene. Due to the high association constant, metal ion induced self-assembly of Fe2+, Co2+, and Ni2+ leads to extended, rigid-rod like metallo-supramolecular coordination polyelectrolytes (MEPEs) even in aqueous solution. Here, the kinetics of coordination and the kinetics of growth of MEPEs are presented. The species in solutions are analyzed by stopped-flow fluorescence spectroscopy, light scattering, viscometry and cryogenic transmission electron microscopy. At near-stoichiometric amounts of the reactants, high molar masses are obtained, which follow the order Ni-MEPE ~ Co-MEPE < Fe-MEPE. Furthermore, a way is presented to adjust the average molar mass, chain-length and viscosity of MEPEs using the monotopic chain stopper 4’-(phenyl)-2,2’:6’,2’’-terpyridine.
Staphylococcus aureus is a versatile human pathogen that normally develops acute or chronic infections. The broad range of diseases caused by this bacterium facilitates the escape from the host's immune response as well as from target-specific antimicrobial therapies. Nevertheless, the underlying cellular and molecular mechanisms that enable S. aureus to cause these disparate types of infections are largely unknown. In this work, we depicted a novel genetic program involved in the development of cell-fate decision, which promotes the differentiation of the staphylococcal cells into two genetically identical but differently heritable cell lines capable of defining the course of an infection, by simultaneously progressing to (i) a biofilm-associated chronic infection or (ii) a disperse acute bacteremia. Here, S. aureus growing in architecturally complex multicellular communities harbored different cell types that followed an exclusive developmental plan, resulting in a clonal heterogeneous population. We found that these cell types are physiologically specialized and that, this specialization impacts the collective behavior within the multicellular aggregates. Whereas one cell line that we named BRcells, promotes biofilm formation that engenders chronic infections, the second cell line, which we termed DRcells is planktonic and synthetizes virulence factors, such as toxins that can drive acute bacteremia. We identified that the positive feedback loop present in Agr quorum sensing system of S. aureus acts a bimodal switch able to antagonistically control the divergence of these two physiologically distinct, heritable cell lines. Also, we found that this bimodal switch was triggered in response to environmental signals particularly extracellular Mg2+, affecting the size of the subpopulations in specific colonization environments. Specifically, Mg2+-enriched environments enhanced the binding of this cation to the staphylococcal teichoic acids, increasing the rigidity of the cell wall and triggering a genetic program involving the alternative sigma factor σB that downregulated the Agr bimodal switch, favoring the enrichment of the BRcells type. Therefore, colonization environments with different Mg2+ content favored different outcomes in the bimodal system, defining distinct ratio in the BRcells/DRcells subpopulations and the S. aureus outcome in our in vitro model of development of multicellular aggregates and, the infection outcome in an in vivo mice infection model. In this prime human pathogen cell-fate decision-making generates a conserved pattern of heritable, physiological heterogeneity that actively contributes to determine the course of an infection through the emergence and spatio-temporal dynamics of distinct and specialized cell types. In conclusion, this work demonstrates that cell differentiation in pathogenic bacteria is a fundamental phenomenon and its understanding, is central to understand nosocomial infections and to designing new anti-infective strategies