Compared to our current knowledge of neuronal excitation, little is known about the development and maturation of inhibitory circuits. Recent studies show that inhibitory circuits develop and mature in a similar way like excitatory circuit. One such similarity is the development through excitation, irrespective of its inhibitory nature. Here in this current study, I used the inhibitory projection between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) as a model system to unravel some aspects of the development of inhibitory synapses. In LSO neurons of the rat auditory brainstem, glycine receptor-mediated responses change from depolarizing to hyperpolarizing during the first two postnatal weeks (Kandler and Friauf 1995, J. Neurosci. 15:6890-6904). The depolarizing effect of glycine is due to a high intracellular chloride concentration ([Cl-]i), which induces a reversal potential of glycine (EGly) more positive than the resting membrane potential (Vrest). In older LSO neurons, the hyperpolarizing effect is due to a low [Cl-]i (Ehrlich et al., 1999, J. Physiol. 520:121-137). Aim of the present study was to elucidate the molecular mechanism behind Clhomeostasis in LSO neurons which determines polarity of glycine response. To do so, the role and developmental expression of Cl-cotransporters, such as NKCC1 and KCC2 were investigated. Molecular biological and gramicidin perforated patchclamp experiments revealed, the role of KCC2 as an outward Cl-cotransporter in mature LSO neurons (Balakrishnan et al., 2003, J Neurosci. 23:4134-4145). But, NKCC1 does not appear to be involved in accumulating chloride in immature LSO neurons. Further experiments, indicated the role of GABA and glycine transporters (GAT1 and GLYT2) in accumulating Cl- in immature LSO neurons. Finally, the experiments with hypothyroid animals suggest the possible role of thyroid hormone in the maturation of inhibitory synapse. Altogether, this thesis addressed the molecular mechanism underlying the Cl- regulation in LSO neurons and deciphered it to some extent.
Proteins of the intermembrane space of mitochondria are generally encoded by nuclear genes that are synthesized in the cytosol. A group of small intermembrane space proteins lack classical mitochondrial targeting sequences, but these proteins are imported in an oxidation-driven reaction that relies on the activity of two components, Mia40 and Erv1. Both proteins constitute the mitochondrial disulfide relay system. Mia40 functions as an import receptor that interacts with incoming polypeptides via transient, intermolecular disulfide bonds. Erv1 is an FAD-binding sulfhydryl oxidase that activates Mia40 by re-oxidation, but the process how Erv1 itself is re-oxidized has been poorly understood. Here, I show that Erv1 interacts with cytochrome c which provides a functional link between the mitochondrial disulfide relay system and the respiratory chain. This mechanism not only increases the efficiency of mitochondrial inport by the re-oxidation of Erv1 and Mia40 but also prevents the formation of deleterious hydrogen peroxide within the intermembrane space. Thus, the miochondrial disulfide relay system is, analogous to that of the bacterial periplasm, connected to the electron transport chain of the inner membrane, which possibly allows an oxygen-dependend regulation of mitochondrial import rates. In addition, I modeled the structure of Erv1 on the basis of the Saccharomyces cerevisiae Erv2 crystal structure in order to gain insight into the molecular mechanism of Erv1. According to the high degree of sequence homologies, various characteristics found for Erv2 are also valid for Erv1. Finally, I propose a regulatory function of the disulfide relay system on the respiratory chain. The disulfide relay system senses the molecular oxygen levels in mitochondria and, thus, is able to adapt respiratory chain activity in order to prevent wastage of NADH and production of ROS.
Cyanobacteria are the only prokaryotes with the ability to conduct oxygenic photosynthesis,
therefore having major influence on the evolution of life on earth. Their diverse morphology
was traditionally the basis for taxonomy and classification. For example, the genus
Chroococcidiopsis has been classified within the order Pleurocapsales, based on a unique
reproduction modus by baeocytes. Recent phylogenetic results suggested a closer
relationship of this genus to the order Nostocales. However, these studies were based
mostly on the highly conserved 16S rRNA and a small selection of Chroococcidiopsis
strains. One aim of this present thesis was to investigate the evolutionary relationships of
the genus Chroococcidiopsis, the Pleurocapsales and remaining cyanobacteria using
16S rRNA, rpoC1 and gyrB gene. Including the single gene, as the multigene analyses of
97 strains clearly showed a separation of the genus Chroococcidiopsis from the
Pleurocapsales. Furthermore, a sister relationship between the genus Chroococcidiopsis
and the order Nostocales was confirmed. Consequently, the monogeneric family
Chroococcidiopsidaceae Geitler ex. Büdel, Donner & Kauff familia nova is justified. The
phylogenetic analyses also revealed the polyphyly of the remaining Pleurocapsales, due to
the fact that the strain Pleurocapsa PCC 7327 was always separated from other strains.
This is supported by differences in their metabolism, ecology and physiology.
A second aim of this study was to investigate the thylakoid arrangement of
Chroococcidiopsis and a selection of cyanobacterial strains. The investigation of 13 strains
with Low Temperature Scanning Electron Microscopy revealed two unknown thylakoidal
arrangements within Chroococcidiopsis (parietal and stacked). This result revised the
knowledge of the thylakoid arrangement in this genus. Previously, only a coiled
arrangement was known for three strains. Based on the data of 66 strains, the feature
thylakoid arrangement was tested as a potential feature for morphological identification of
cyanobacteria. The results showed a strong relationship between the group assignment of
cyanobacteria and their thylakoid arrangements. Hence, it is in general possible to
conclude from this certain phenotypic character the affiliation to a particular family, order
The third aim of this study was to investigate biogeographical patterns of the worldwide
distributed genus Chroococcidiopsis. The phylogenetic analysis suggested that the genus do not have biogeographical patterns, which is in contrast with a recent study on hypolithic
living Chroococcidiopsis strains and the majority of phylogeographic analysis of
microorganisms. Further analysis showed no separation of different life-strategies within
the genus. These results could be related to the genetic markers utilized, which may not
contain biogeographical information. Hence the present study can neither exclude nor
prove the possibility of biogeographic and life-strategy patterns in the genus
Future research should be focused on finding appropriate genetic markers investigate of
evolutionary relationships and biogeographical patterns within Chroococcidiopsis.
Biological Soil Crusts (BSCs), composed of lichens, mosses, green algae, microfungi and cyanobacteria are an ecological important part of the perennial landcover of many arid and semiarid regions (Belnap et al. 2001a), (Büdel 2002). In many arid and hyperarid areas BSCs form the only perennial "vegetation cover" largely due to their extensive resistance to drought (Lange et al. 1975). For the Central Namib Desert (Namibia), BSCs consisting of extraordinary vast lichen communities were recently mapped and classified into six morphological classes for a coastal area of 350 km x 60 km. Embedded into the project "BIOTA" (www.biota-africa.org) financed by the German Federal Ministry of Education and Research the study was undertaken in the framework of the PhD thesis by Christoph Schultz. Some of these lichen communities grouped together in so called "lichen fields" have already been studied concerning their ecology and diversity in the past (Lange et al. 1994), (Loris & Schieferstein 1992), (Loris et al. 2004), (Ullmann & Büdel 2001a), (Wessels 1989). Multispectral LANDSAT 7 ETM+ and LANDSAT 5 TM satellite imagery was utilized for an unitemporal supervised classification as well as for the establishment of a monitoring based on a combined retrospective supervised classification and change detection approach (Bock 2003), (Weiers et al. 2003). Results comprise the analysis of the mapped distribution of lichen communities for the Central Namib Desert as of 2003 as well as reconstructed distributions for the years 2000, 1999, 1992 and 1991 derived from retrospective supervised classification. This allows a first monitoring of the disturbance, destruction and recovery of the lichen communities in these arid environments including the analysis of the major abiotic processes involved. Further analysis of these abiotic processes is key for understanding the influence of Namib lichen communities on overall aeolian and water induced erosion rates, nutrient cycles, water balance and pedogenic processes (Belnap & Gillette 1998), (Belnap et al. 2001b), (Belnap 2001c), (Evans & Lange 2001), (McKenna Neumann & Maxwell 1999). In order to aid the understanding of these processes SRTM digital elevation model data as well as climate data sets were used as reference. Good correlation between geomorphological form elements as well as hydrological drainage system and the disturbance patterns derived from individual post classification change comparisons between the timeframes could be observed. Conjoined with the climate data sets sporadic foehn-like windstorms as well as extraordinary precipitation events were identified to largely affect the distribution patterns of lichen communities. Therefore the analysis and monitoring of the diversity, distribution and spatiotemporal change of Central Namib BSCs with the means of Remote Sensing and GIS applications proof to be important tools to create further understanding of desertification and degradation processes in these arid regions.
The hypoxia inducible factor-1 (HIF-1), a heterodimer composed of HIF-1alpha and HIF-1beta, is activated in response to low oxygen tension and serves as the master regulator for cells to adapt to hypoxia. HIF-1 is usually considered to be regulated via degradation of its a-subunit. Recent findings, however, point to the existence of alternative mechanisms of HIF-1 regulation which appear to be important for down-regulating HIF-1 under prolonged and severe oxygen depletion. The aims of my Ph.D. thesis, therefore, were to further elucidate mechanisms involved in such down-regulation of HIF-1. The first part of the thesis addresses the impact of the severity and duration of oxygen depletion on HIF-1alpha protein accumulation and HIF-1 transcriptional activity. A special focus was put on the influence of the transcription factor p53 on HIF-1. I found that p53 only accumulates under prolonged anoxia (but not hypoxia), thus limiting its influence on HIF-1 to severe hypoxic conditions. At low expression levels, p53 inhibits HIF-1 transactivity. I attributed this effect to a competition between p53 and HIF-1alpha for binding to the transcriptional co-factor p300, since p300 overexpression reverses this inhibition. This assumption is corroborated by competitive binding of IVTT-generated p53 and HIF-1alpha to the CH1-domain of p300 in vitro. High p53 expression, on the other hand, affects HIF-1alpha protein negatively, i.e., p53 provokes pVHL-independent degradation of HIF-1alpha. Therefore, I conclude that low p53 expression attenuates HIF-1 transactivation by competing for p300, while high p53 expression negatively affects HIF-1alpha protein, thereby eliminating HIF-1 transactivity. Thus, once p53 becomes activated under prolonged anoxia, it contributes to terminating HIF-1 responses. In the second part of my study, I intended to further characterize the effects induced by prolonged periods of low oxygen, i.e., hypoxia, as compared to anoxia, with respect to alterations in HIF-1alpha mRNA. Prolonged anoxia, but not hypoxia, showed pronounced effects on HIF-1alpha mRNA. Long-term anoxia induced destabilization of HIF-1alpha mRNA, which manifests itself in a dramatic reduction of the half-life. The mechanistic background points to natural anti-sense HIF-1alpha mRNA, which is induced in a HIF-1-dependent manner, and additional factors, which most likely influence HIF-1alpha mRNA indirectly via anti-sense HIF-1alpha mRNA mediated trans-effects. In summary, the data provide new information concerning the impact of p53 on HIF-1, which might be of importance for the decision between pro- and anti-apoptotic mechanisms depending upon the severity and duration of hypoxia. Furthermore, the results of this project give further insights into a novel mechanism of HIF-1 regulation, namely mRNA down-regulation under prolonged anoxic incubations. These mechanisms appear to be activated only in response to prolonged anoxia, but not to hypoxia. These considerations regarding HIF-1 regulation should be taken into account when prolonged incubations to hypoxic or anoxic conditions are analyzed at the level of HIF-1 stability regulation.
In my doctoral thesis, I present new information about the developmental expression pattern of the potassium chloride cotransporter KCC2 in the rat auditory brain stem and the morphometrical effects caused by KCC2 gene silencing in mice. The thesis is divided into 3 Chapters. Chapter 1 is a general introduction which gives a brief outline of the primary ascending auditory pathway in mammals. Also, it provides information about the presence of a large number of inhibitory inputs in the auditory system and how these inputs develop; the involvement of inhibition in the acoustic processing is mentioned. In addition, the role of the KCC2 cotransporter in the shift of GABA/glycine transmission, and thus, in maintaining the normal level of inhibition in the mature brain, is described. The focus of Chapter 2 was to investigate the KCC2 immunofluorescent signal from postnatal day (P) 0 to P60 in four major nuclei of the rats superior olivary complex (SOC), namely the medial nucleus of the trapezoid body (MNTB), the medial superior olive (MSO), the lateral superior olive (LSO), and the superior paraolivary nucleus (SPN). The lack of a correlation between the continuous presence of KCC2 mRNA/protein in the postnatal rat brain stem on one side, and the shift in GABA/glycinergic polarity (i.e. KCC2 functionality) on the other side, prompted me to search for a specific cellular expression pattern of the KCC2 protein that might correlate with the switch in GABA/glycine signalling. To do so, the KCC2 immunoreactivity was analysed using high-resolution confocal microscopy in three cellular regions of interest: the soma surface, the soma interior, and the neuropil. In the soma surface, I observed an increase of the KCC2 immunofluorescent signal intensity, yet with a moderate magnitude (1.1 to 1.6-fold). Therefore, I conclude that the change in the soma surface signal is only of minor importance and does not explain the change in KCC2 functionality. The KCC2 signal intensity in the soma interior decreased in all nuclei (1.4 to 2-fold) with the exception of the MNTB where no statistically significant change was found. The decrease in the soma interior was probably related to the increase in the soma surface immunoreactivity and the proposed (weak) intracellular trafficking process of the KCC2 protein. The main developmental reorganization (in qualitative as well as in quantitative aspects) of the KCC2 immunofluorescence in the SOC nuclei was observed in the neuropil. The signal changed its pattern from a diffusely stained neuropil early in development (P0-P4) to a crisp and membrane-confined signal later on (P8-P60), with single dendrites becoming apparent. The exception was found in the MNTB, where the neuropil became almost unlabeled. Quantification revealed a statistically significant decrease (2.2 to 3.8-fold) in the neuropil immunoreactivity in all four nuclei, although the remaining KCC2-stained dendrites became thicker and the signal became stronger. I suppose that, at least in part, the neuropil reorganization can be explained by an age-related reduction of dendritic branches via a pruning mechanism and with the absence of an abnormal Cl- load via extrasynaptic GABAA receptors. This is consistent with the proposed additional role of KCC2, namely to maintain the cellular ionic homeostasis and to prevent dendritic swelling (Gulyás et al., 2001). In conclusion, neither the increase in the KCC2 soma surface signal intensity, nor the reorganization in the neuropil can be strictly related to the developmental switch in the GABA/glycine polarity and the onset of KCC2 function, although some correlation (the appearance of a specific membrane-confined dendritic pattern) between structure and function was found. Further implication of different molecular methods, regarding the proposed posttranslational modification of KCC2, will shed light upon the question of what leads to the functional activation of the cotransporter. In Chapter 3, the advantage of loss-of-function KCC2 mice made it possible, via manipulating the duration of the depolarizing phase of GABA/glycine transmission, to analyse the effect of disturbed Cl- regulation and, thus, the effect of disrupted GABA/glycine neurotransmission (lack of inhibition). I asked the following question: how important is the Cl- homeostasis to maintain general aspects (brain weight) and specific aspects (nucleus volume, neuron number, and soma cross-sectional area) of brain development? Brain stem slices from KCC2 knock-out animals (-/-), with a trace amount of transporter (~5%), as well as from wild type animals (+/+) at P3 and P12 were stained for Nissl substance and the analyses were performed with the help of basic morphometrical and stereological methods. In KCC2 (-/-) animals, body growth impairment was observed, in part related to the seizure activity preventing normal feeding (Woo et al., 2002). However, their brains, in terms of brain weight, were less affected. Therefore, I conclude that Cl- homeostasis is not essential per se to maintain the brain weight. Four auditory nuclei (MNTB, MSO, LSO, and ventral cochlear nucleus (VCN)), were compared with respect to the KCC2 null mutation. The SOC nuclei were not influenced by the lack of KCC2 at P3 considering the morphometric parameters. A difference in the number of neurons occurred in the VCN at P3. I suggest to perform additional immunohistochemical studies of glial presence related to its involvement in the structural and functional support of the neurons and their survival. At P12, the volume of the auditory nuclei in KCC2 (-/-) animals was smaller than in (+/+) animals. However, this is likely to be an epiphenomenon since the brain weight increase was also impaired with the same magnitude. Therefore, I suppose that the Cl- homeostasis is not crucial for the nucleus volume increase in the VCN, the MNTB and the MSO during development. An exception was found for the LSO. Regarding the other morphometric parameters at P12, the four nuclei behaved in a different way: (1) in the VCN, after P3, no parameter underwent a disproportional change due to impaired Cl- homeostasis; (2) the MNTB and the LSO showed less pronounced neuropil in mutants in comparison to age-matched controls and two reasons were proposed: first, the depolarizing GABA/glycine transmission in mutants may contribute to excessive Ca2+ load, excitotoxicity and dendrite damage; second, a decrease of some trophic factors may prevent dendrite development in addition to impaired normal body growth; (3) the MSO neurons in P12 (-/-) animals had smaller soma cross-sectional area than in P12 (+/+) animals. I conclude that the normal Cl- homeostasis is required in the MSO at older ages (P12) to achieve and maintain a proper soma size; (4) the lack of KCC2 did not prevent the process of neuronal differentiation in the VCN and the MNTB during development in both mutant and control animals. In conclusion, the various auditory nuclei have to be discussed independently regarding the influence of Cl- homeostasis on some morphometric parameters. Presumably, this is related to the different time of the shift in the GABA/glycine polarity i.e., the onset of KCC2 function (Srinivasan et al., 2004a). Taken together, my thesis accumulated data about the immunohistological expression pattern of KCC2 in various auditory brain stem nuclei and the influence of impaired Cl- homeostasis on some morphometric features in these nuclei. This information will be helpful for further investigations involved to discover the mechanisms and the events that govern the inhibition and the inhibitory pathway in the central auditory system.
In this study, 27 marine bacteria were screened for production of bioactive metabolites. Two strains from the surface of the soft coral Sinularia polydactyla, collected from the Red Sea, and three strains from different habitats in the North Sea were selected as a promising candidates for isolation of antimicrobial substances. A total of 50 compounds were isolated from the selected bacterial strains. From these metabolites 25 substances were known from natural sources, 10 substances were known as synthetic chemical and herein are reported as new natural products, and 13 metabolites are new. Two substances are still under elucidation. All new compounds were chemically and biologically characterized. Pseudoalteromonas sp. T268 produced simple phenol and oxindole derivatives. Production of homogentisic acid and WZ 268S-6 from this bacteria was affected by the salinity stress. WZ 268S-6 shows antimicrobial and cytotoxic activities. Its target is still unclear. Isolation of isatin from this strain points out for the possibility of using this substance as a chemotaxonomical marker for Alteromonas-like bacteria. A large number of nitro-substituted aromatic compounds were isolated from both Salegentibacter sp. T436 and Vibrio sp. WMBA1-4. They may be derived from metabolism of phenylalanine or tyrosine. From Salegentibacter sp. T436, 24 compounds were isolated, of which four compounds are new and six compounds were known as synthetic chemicals. WZ 436S-16 (dinitro-β-styrene) is the most potent antimicrobial and cytotoxic compound. It inhibits the oxygen uptake by N. coryli and causes apoptosis in the human promyelocytic leukaemia (HL-60 cells). From Vibrio sp. WMBA1-4, 13 new alkaloids were isolated, of which four were known as synthetic products and herein are reported as new substances from natural sources. The majority of these compounds show antimicrobial and cytotoxic activities. The cytotoxic activity of WMB4S-11 against the mouse lymphocytic leukaemia (L1210 cells) is due to the inhibition in the protein biosynthesis, while the remaining cytotoxic alkaloids have no effect on the synthesis of macromolecules in this cell line. The antibacterial activity of WMB4S-2, -11, -12, -13 and the antifungal activity of WMB4S-9 are not due to the inhibition in the macromolecules biosynthesis or in the oxygen uptake by the microorganisms. The biological activity of these nitro-aromatic compounds from Salegentibacter sp. T436 and Vibrio sp. WMBA1-4 is influenced by the presence of a nitro group and its position in respect to the hydroxyl group, number of the nitro groups, and the type of substitutions on the side chain. In diaryl-maleimide derivatives, types and position of substitution on the aryl rings, on the maleimide moity, and the hydrophobicity of the aryl ring itself lead to variations in the extent of the bioactivity of these derivatives. This is the first time that vibrindole (WMB4S-14) and turbomycin B or its noncationic form (WMB4S-15), isolated from Vibrio sp., are reported as cytotoxic compounds. WMB4S-15 inhibits the biosynthesis of macromolecules in L1210 cells. The structural similarity between some of the metabolites in this study and previously reported compounds from sponges, ascidians, and bryozoan indicates that the microbial origin of these compounds must be considered.
The study provides insights into the dynamic processes of vascular epiphyte vegetation in two host tree species of lowland forest in Panama. Further, a novel approach is presented to examine the possible role of host tree identity in the structuring of vascular epiphyte communities: For three locally common host tree species (Socratea exorrhiza, Marila laxiflora, Perebea xanthochyma) we created null models of the expected epiphyte assemblages assuming that epiphyte colonization reflected random distribution of epiphytes in the forest. In all three tree species, abundances of the majority of epiphyte species (69 – 81 %) were indistinguishable from random, while the remaining species were about equally over- or underrepresented compared to their occurrence in the entire forest plot. Permutations based on the number of colonized trees (reflecting observed spatial patchiness) yielded similar results. Finally, a Canonical Correspondence Analysis also confirmed host-specific differences in epiphyte assemblages. In spite of pronounced preferences of some epiphytes for particular host trees, no epiphyte species was restricted to a single host. We conclude that the epiphytes on a given tree species are not simply a random sample of the local species pool, but there are no indications of host specificity either. To determine the qualitative and quantitative long-term changes in the vascular epiphyte assemblage of the host tree Socratea exorrhiza, in the lowland forest of the San Lorenzo Crane Plot, we followed the fate of the vascular epiphyte assemblage on 99 individuals of this palm species, in three censuses over the course of five years. The composition of the epiphyte assemblage changed little during the course of the study. While the similarity of epiphyte vegetation decreased on single palm individuals through time, the similarity analyzed over all palms increased. Even well-established epiphyte individuals experienced high mortality with only 46 % of the originally mapped individuals surviving the following five years. We found a positive correlation between host tree size and epiphyte richness and detected higher colonization rates of epiphytes per surface area on larger trees. Epiphyte assemblages on single Socratea exorrhiza trees were highly dynamic while the overall composition of the epiphyte vegetation on the host tree species in the study plot was rather stable. We suggest that higher recruitment rates due to localized seed dispersal by already established epiphytes on larger palms promote the colonization of epiphytes on larger palms. Given the known growth rates and mortality rates of the host tree species, the maximum time available for colonization and reproduction of epiphytes on a given Socratea exorrhiza tree is estimated to be about 60 years. Changes in the epiphyte vegetation of c. 1000 individuals of the host tree species Annona glabra at Barro Colorado Island over the course of eight year were documented by means of repeated censuses. Considerable increase in the abundance of the dominating epiphyte species and ongoing colonization of the host tree species suggests that the epiphyte vegetation has not reached a steady state in the maximal 80 years since the establishment of the host tree. Epiphyte species composition as a whole was rather stable. We disentangled the relationship between epiphyte colonization and tree size/available time for colonization with the finding that tree size explained only a low proportion of colonization while other factors like connectivity to dispersal source and time explain may explain a larger part. Epiphyte populations are patchily distributed and examined species exhibit properties of a metapopulation with asynchronous local population growth, high local population turnover, a positive relationship between regional occurrence and patch population size, and negatively correlated relationship between extinction and patch occupancy. The documented metapopulation processes highlight the importance of not colonized suitable habitat for the conservation of epiphytes.
The heart is reported to show a net consumption of lactate. This may contribute up to 15% to the total body lactate disposal. In this work, the consumption of lactate was shown for the first
time on the single cell level with the new FRET-based lactate sensor Laconic.
Research published until today, almost exclusively reports the monocarboxylate transporter 1
(MCT1) as the transporter responsible for myocardial lactate uptake. As this membrane
transporter transports lactate together with H+ in a stoichiometry of 1:1, lactate transport is
coupled to pH regulation. Consequently, interactions of MCT1 and acid/base regulating proteins
(carbonic anhydrases (CAs and sodium bicarbonate co-transporters (NBCs)) are described in
the oocyte expression system, skeletal muscle and cancer cells.
In this work it is shown that activity of extracellular CA increases lactate uptake into mouse
cardiomyocytes by 27% and lactate induced JA/B by 42.8% to 46.2%. This effect is most likely
mediated via NBC/CA interaction because inhibition of extracellular CA reduces HCO3--
dependent acid extruding JA/B by 53.3% to 78.4%. This may link lactate uptake to cellular
respiration. When lactate was applied in medium gassed with 100% N2, lactate induced
acidification was 12.6% faster than in medium gassed with 100% O2. Thus, CO2 produced on
the pathway transferring redox energy from substrates like glucose and lactate to ADP and
phosphate via oxidative phosphorylation, may support further lactate uptake. The findings of
this work suggest an auto regulation of lactate uptake via CO2 release in ventricular mouse
Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell. Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell.