Under unfavorable environmental conditions Caenorhabditis elegans larvae enter a dauer stage which is a specialized non-feeding larval stage. In the dauer stage, worms display astonishingly low metabolism, which allows them to adapt themselves to environmental stress and to dwell without food for several months. Dauer larvae can enter into the reproductive larval stage, when environmental conditions become favorable. In this study, the metabolic transition of dauers into the reproductive larval stage is analyzed in detail: a. During the exit of dauers, several metabolic traits were examined. Primarily, dauer larva initiates the metabolic transition by activating feeding, which is followed by upregulated oxygen consumption and mitochondrial remodeling, as well as enhanced protein synthesis. b. To better understand the metabolic transition, inhibitors of the dauer exit were introduced. Lithium ions were shown to inhibit the transition of dauers to reproductive larvae and prevent the upregulation of metabolic activities required for this process. c. In liquid culture, the transition from the dauer to the reproductive larva is also inhibited, presumably because of the hypoxic character of the liquid culture. Thus, hypoxia has a negative effect on the metabolic transition. d. In the course of our investigation we discovered that the dauer larva is not a closed system but indeed, it can dwell on the externally available ethanol as a carbon source by incorporating it into the energy metabolism. This allows dauers to survive for longer periods in the absence of bacteria, the preferred food of worms. These findings clarify the nature of dauers, how they utilize distinct pathways during the metabolic transition and how they take advantage of the externally available carbon source. These results may in the future enable us to elucidate the complex pathways of metabolism, as well as the ways in which it can be regulated.

The control of growth is a poorly understood aspect of animal development. This thesis focuses on body size regulation in Caenorhabditis elegans, and in particular, how worms grow to a certain size. In C. elegans, a key regulator of size is the TGFβ homologue DBL-1. Mutations that deplete the worm of DBL-1 result in a small body size, whereas overexpression of the gene renders long animals. The small mutants have the same number of cells as wild type suggesting that some or all cells are smaller. DBL-1 activates a TGFβ receptor leading to the nuclear localization of three Smad proteins which then initiate a transcriptional program for size control whose targets are mainly unknown. In order to learn more about how body size in C. elegans is regulated, we set up EMS mutagenesis screens to identify new loci that caused a long phenotype. A subset of the genes we have identified might function in the TGFβ signaling pathway regulating growth while others likely function in parallel pathways. One gene that we found in this screen, lon-3, encodes a cuticle collagen that genetically lies downstream of the DBL-1 TGFβ signaling pathway. Interestingly, loss of function mutations in lon-3 result in a Lon phenotype, whereas increasing the amount of LON-3 protein cause the worms to be dumpy, i.e. shorter, but slightly fatter than wild type. LON-3 is expressed in the hypodermis, the tissue from which the cuticle is synthesized and in which TGFβ signaling, regulating body size, has its focus. This study and previous work have shown that DBL-1 may affect body volume via effects on hypodermal nuclear ploidy, however this is unaffected in lon-3 mutants. Consistent with this finding, the volume of lon-3 mutant worms is not different from wild type. Taken together, our results suggest that another mechanism, by which TGFβ signaling can regulate body length, is by altering the shape of the cuticle via its effect on lon-3 and possibly other cuticle collagens. Studies in worms, flies and mice show that body size and nutrient allocation are closely connected. p70 S6-kinase (S6K) is a known regulator of cell and body size that also plays a role in metabolism. In mice and flies S6K mutants are much smaller than wild type. Our work on the worm homolog, rsks-1, shows that in worms as well, this gene is important for growth regulation and cell size. However, this effect seems to be at least in part independent of DBL-1 TGFβ signaling. Furthermore, rsks-1mutants have a 50 % increase in the amount of stored fat. Fatty acid metabolism has been shown to play an important role in environmental adaptation, especially in regards to temperature changes. Consistent with this idea, rsks-1 mutants appear to have difficulties in adjusting to such changes, reflected in a much-decreased fecundity at 15 and 25 °C compared to their cultivation temperature (20 °C). Within the nervous system the gene is specifically expressed in a subset of the chemosensory neurons that, when nutrients are abundant, secrete signals that promote growth. Intriguingly, this expression seems to be negatively regulated by insulin- like signaling, in contrast to the positive regulation of S6K by insulin in Drosophila and mice. Taken together we show that rsks-1 is an important regulator of growth and fat metabolism in Caenorhabditis elegans.

Suisse, Univ., Diss.--Fribourg, 1994.

Univ., Diss.--Münster, 2003.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Under unfavorable environmental conditions Caenorhabditis elegans larvae enter a dauer stage which is a specialized non-feeding larval stage. In the dauer stage, worms display astonishingly low metabolism, which allows them to adapt themselves to environmental stress and to dwell without food for several months. Dauer larvae can enter into the reproductive larval stage, when environmental conditions become favorable. In this study, the metabolic transition of dauers into the reproductive larval stage is analyzed in detail: a. During the exit of dauers, several metabolic traits were examined. Primarily, dauer larva initiates the metabolic transition by activating feeding, which is followed by upregulated oxygen consumption and mitochondrial remodeling, as well as enhanced protein synthesis. b. To better understand the metabolic transition, inhibitors of the dauer exit were introduced. Lithium ions were shown to inhibit the transition of dauers to reproductive larvae and prevent the upregulation of metabolic activities required for this process. c. In liquid culture, the transition from the dauer to the reproductive larva is also inhibited, presumably because of the hypoxic character of the liquid culture. Thus, hypoxia has a negative effect on the metabolic transition. d. In the course of our investigation we discovered that the dauer larva is not a closed system but indeed, it can dwell on the externally available ethanol as a carbon source by incorporating it into the energy metabolism. This allows dauers to survive for longer periods in the absence of bacteria, the preferred food of worms. These findings clarify the nature of dauers, how they utilize distinct pathways during the metabolic transition and how they take advantage of the externally available carbon source. These results may in the future enable us to elucidate the complex pathways of metabolism, as well as the ways in which it can be regulated.

Previous studies that examined human judgments of frequency and duration found an asymmetrical relationship: While frequency judgments were quite accurate and independent of stimulus duration, duration judgments were highly dependent upon stimulus frequency. A potential explanation for these findings is that the asymmetry is moderated by the amount of attention directed to the stimuli. In the current experiment, participants\' attention was manipulated in two ways: (a) intrinsically, by varying the type and arousal potential of the stimuli (names, low-arousal and high-arousal pictures), and (b) extrinsically, by varying the physical effort participants expended during the stimulus presentation (by lifting a dumbbell vs. relaxing the arm). Participants processed stimuli with varying presentation frequencies and durations and were subsequently asked to estimate the frequency and duration of each stimulus. Sensitivity to duration increased for pictures in general, especially when processed under physical effort. A large effect of stimulus frequency on duration judgments was obtained for all experimental conditions, but a similar large effect of presentation duration on frequency judgments emerged only in the conditions that could be expected to draw high amounts of attention to the stimuli: when pictures were judged under high physical effort. Almost no difference in the mutual impact of frequency and duration was obtained for low-arousal or high-arousal pictures. The mechanisms underlying the simultaneous processing of frequency and duration are discussed with respect to existing models derived from animal research. Options for the extension of such models to human processing of frequency and duration are suggested.

Herdecke, Privatuniv., Diss--Witten, 2006.

Herdecke, Universiẗat, Diss., 2006--Witten.