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Sökning: WFRF:(Tuck Simon Professor)

  • Resultat 1-7 av 7
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1.
  • Zhao, Lina, 1990- (författare)
  • Oxygen sensing in Caenorhabditis elegans
  • 2023
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Sufficient supply of oxygen (O2) to tissue is essential for survival of aerobicanimals. In mammals, there are constant homeostatic regulation mechanisms that act on different time scales to maintain optimal O2 delivery to tissues. The ability to detect and respond to acute oxygen shortages is indispensable to aerobic life. However, the molecular mechanisms and circuits underlying this capacity are poorly understood.We characterize the locomotory response of feeding Caenorhabditis elegans (C. elegans) to 1% O2. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. Increasing cGMP signaling inhibits escape from 1% O2, and that cGMP activates the protein kinase G, EGL-4, which in turn enhances neuroendocrine secretion to inhibit acute response to 1% O2. A primary source of cGMP is the guanylyl cyclase, GCY-28. In addition, increasing mitochondrial reactive oxygen species (ROS), abrogate acute hypoxia response. Up-regulation of mitochondrial ROS increases cGMP levels, which contribute to the reduced hypoxia response. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute response to hypoxia by C. elegans.In addition, we found that FMRFamide-related peptides FLP-1 plays a role in hypoxia evoked locomotory response. Our data showed that FLP-1 secretion from AVK interneurons acts on AVA and other neurons through DMSR-4, DMSR7, and DMSR-8 GPCR receptors to maintain baseline speed and to promote locomotory response to hypoxia.We also found that hypoxia could induce food leaving behavior in C. elegans. Animals quickly escaped from the bacterial lawn when exposed to 1% O2. The known O2 response mechanisms cannot explain this phenotype, instead, neuropeptidergic signalling seems to be required for this behaviour.It's known that pro-inflammatory cytokine ILC-17.1, the homologue of mammalian IL-17s, act as a neuromodulator involved in hyperoxia sensing in C. elegans. We found that it was not involved in acute hypoxia response. Instead, ILC-17.1 could modulate lifespan and damage defense mechanisms against stress in C. elegans by triggering an inhibitory network to constrain the activity of the nuclear hormone receptor, NHR-49.In summary, our research can provide molecular and neurological understanding of how O2 is sensed by animals. Additionally, it further emphasis C. elegans as a good model to understand oxygen sensing
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2.
  • Pu, Longjun, 1990- (författare)
  • A molecular exploration of sensory responses in c. elegans
  • 2023
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Sensation provides a pivotal ability, allowing animals to survive in complex environments. The cues sensed by animals are represented by external stimuli and internal signals. However, the mechanisms mediating sensations in molecular and cellular level are still not well-studied. In this thesis, by using free-living nematodes C. elegans with relatively simple nerve system, we are trying to get better understandings of molecular mechanisms by which animals sense and interpret external cues and internal signals.G protein-coupled receptors (GPCRs), as one of the major families of transmembrane proteins, participate in a variety of physiological responses to both external stimuli and internal cues. Previous studies have shown that GPCR signals are broadly involved in many processes in C. elegans, such as olfactory sensing, nociceptive responses, social behavior, pathogen responses, and mating. However, the complexity and diversity of GPCRs pose significant challenges to systematic dissection of their function as well as identification of receptor-ligand pairs which play crucial roles for animals´ sensory behaviors. Interestingly, the genome of C. elegans encodes one of the largest GPCR repertoires among any sequenced organisms, indicating a dramatical expansion and high degree of gene redundancy. To comprehensively dissect GPCR signaling in C. elegans and gain more insights into their roles in sensations, we developed an approach by employing CRISPR/Cas9-based gene editing to mutate closely related GPCRs and neuropeptide genes (internal signals) in a single strain on a genome-wide scale, resulting in disrupting nearly all the GPCR and neuropeptide genes (more than 1800 genes in total) and eliminating high degree of gene redundancy as well. Then using these two genetic libraries, we successfully identified neuropeptide (FLP -1) and cognate receptors (DMSR-4, DMSR-7 and DMSR-8) required for hypoxia-evoked locomotory responses, obtained a set of novel regulators of the pathogen-induced immune response including FMI-1 and DOP-6, and especially identified receptors (SRX-64) in AWA neurons for the volatile odorant pyrazine and redundant receptors (SRX-1, SRX-2 and SRX-3) in AWCOFF neuron for 2,3-pentanedione.In nature, animals often experience and sense constantly changing gas environments. And human bodies also generate internal gas as gasotransmitters for signal transduction, such as CO, NO and H2S. For the mechanism governing sensory and adaptive responses to different gaseous cues, extensive studies are still needed. Here, taking advantage of the robust locomotory responses to H2S in C. elegans, we delineated the molecular mechanisms of H2S sensation and adaptation. We found that C. elegans exhibited transiently increased locomotory and turning activity as a strategy to escape the noxious H2S. The behavioral responses to H2S were modulated by a complex network of signaling pathways, ranging from cyclic GMP signaling in ciliated sensory neurons, calcineurin, nuclear hormone receptors, to the major starvation regulators such as insulin and TGF-β signaling. Prolonged exposure to H2S robustly evoked H2S detoxification and reprogrammed gene expression, where genes involved in iron homeostasis, including ftn-1 and smf-3, were robustly modified, implying that labile iron levels are affected by H2S. In addition, the roles of labile iron for modulating H2S response were further investigated by using genetic studies and chemical applications. Interestingly, the response to H2S was substantially affected by the ambient O2 levels and their prior experience in low O2 environments, suggesting an intricate interplay between O2 and H2S sensing. The crosstalk is often seen between different experiences and sensations. In addition to the interplay between O2 and H2S sensing, we found hypoxia challenge could induce food leaving behavior in C. elegans. The alteration of food behavior by hypoxia experience was independent of the known mechanisms involved in O2 response, including pathways in acute hypoxia and HIF-1 signaling for chronic hypoxia response. The robust failure of induced food avoidance in egl-3 and egl-21 mutants suggested that neuropeptidergic signaling was required for this response. And future work is needed for comprehensively understanding the roles of neuropeptide signaling in the crosstalk between hypoxia experience and food leaving behavior.In summary, our studies shed light on the molecular and cellular mechanisms of how animals sense and interpret the signals, allowing them to survive in a complex environment niche. More specifically, 1) we demonstrated the dissection of genetic landscape of GPCR signaling through phenotypic profiling in C. elegans. And as a powerful genetic resource, our libraries can greatly expedite the analyses of GPCR signaling in multiple additional contexts. 2) we provided molecular insights into how C. elegans detects and adapts its response to H2S and modulates behaviors through ambient environment and experience. 
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3.
  • Rahmani, Shapour, 1975- (författare)
  • Studies on lipid transport and extracellular vesicle production in Caenorhabditis elegans ciliated neurons
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The cilium is a protrusion of cell membrane. Both the protein and lipid contents of cilia are different from those of other parts of the cell membrane. While the transport of proteins into and out of cilia has been intensively studied, much less is known about how the lipid content of ciliary membranes is regulated. TAT-6 is a P4-family ATPase that is expressed in C. elegans ciliated neurons whose endings are exposed to the environment. To study the function of TAT-6 and that other translocases in lipid transport in C. elegans ciliated neurons, I developed a technique to allow labelling of cilia with lipids. For the first time I used fusogenic liposomes to study the roles of all the TAT proteins in this organism in maintaining the lipid asymmetry in this organelle. Assessment the cilia with these liposomes showed that TAT-5 and TAT-1 translocase activities promote the transport of phosphatidylethanolamine (PE) and phosphatidylserine (PS) respectively and TAT-6 has an overlapping function in transporting both phospholipds. In C. elegans males, certain ciliated neurons release extracellular vesicles (EVs). The cilium is a site of EV biogenesis and shedding. I found that ciliated neurons in tat-6 mutant males produced significantly fewer EVs than those in wild type. tat-1, tat-5 and pad-1 mutants, however, produced far more EVs than those in wild type. PPK-3, CUP-5 and LMP-1 are proteins necessary for endolysosomal trafficking and lysosomes biogenesis, a process in which TAT-1 has previously been shown to function in C. elegans intestinal cells. I found that, like tat-1 mutants, ppk-3, lmp-1 and cup-5 mutant males release significantly greater numbers of EVs from cilia compared with wild-type. I found that increasing and decreasing the cGMP signaling cause defects in the response and turning behavior in male C. elegans respectively. Exposing wild-type males to high levels of 8-Bromoguanosine 3′,5′-cyclic monophosphate strongly reduced response behavior. Males mutant for odr-3, which encodes a G protein were defective in response. Overall my investigations indicate that the regulation of lipid asymmetry and phospholipid transport is required for proper cilia function in C. elegans, that intercellular trafficking and lipid composition have important roles in EVs biogenesis, and that different TAT proteins can affect the size and number of EVs produced. I also showed that in male animals, cGMP is one of the mediators in mating transduction signal and that a high level of cGMP inhibits mating response behavior in male C. elegans. 
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4.
  • Zhao, Yani, 1983- (författare)
  • Systemic RNAi Relies on the Endomembrane System in Caenorhabditis elegans
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The membrane system of a eukaryotic cell is a large and complex system handling the transport, exchange and degradation of many kinds of material. Recent research shows that double-stranded RNA (dsRNA) mediated gene silencing (RNA interference) is a membrane related process. After long dsRNA is processed to small interfering RNA (siRNA) by Dicer, the guide strand and passenger strand are separated in the RNA induced silencing complex (RISC) by Argonaute. The process of loading siRNA into RISC has been suggested to occur at the rough Endoplasmic Reticulum (rER).The components of RISC also associate with late endosomes/multivesicular bodies (MVBs). Furthermore, disturbing the balance between late endosomes/MVBs and lysosomes has been shown to affect the efficiency of silencing.We use the nematode Caenorhabditis elegans as our model organism to study two questions: how does membrane transport affect RNAi and spreading of RNAi from the recipient cells to other tissues (systemic RNAi); and how does RNA transport contribute to the multigenerational silencing induced by dsRNA (RNAi inheritance)? Using SID-5, a protein required for efficient systemic RNAi, as bait in a yeast two-hybrid (Y2H) screen, we got 32 SID-5 interacting candidate proteins. Two of these are the SNARE protein SEC-22 and the putative RNA binding protein C12D8.1. In two additional Y2H screens, we found that SID-5 interacts with multiple syntaxin SNAREs, including SYX-6, whereas SEC-22 only interacts with SYX-6. SNAREs usually function in vesicle fusion processes. We found the two SNARE proteins SEC-22 and SYX-6 to be negative regulators of RNAi and to localize to late endosomes/MVBs. In addition, loss of sid-5 leads to an endosome maturation defect. Finally, we found that the putative RNA binding protein C12D8.1 negatively regulates RNAi inheritance and that C12D8.1 mutant animals show impaired RNAi upon targeting a new gene. Taken together, the results presented in this thesis provide us with more evidence for the connection of the membrane transport system and RNAi. The identification of a putative negative regulator of RNAi inheritance further enriches this research field.
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5.
  • Friberg, Josefin, 1974- (författare)
  • The control of growth and metabolism in Caenorhabditis elegans
  • 2006
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • 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.
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6.
  • Goergen, Philip, 1986- (författare)
  • The Molecular Mechanism of Aggression and Feeding Behaviour in Drosophila melanogaster
  • 2014
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Obesity is a complex disorder which has become a growing health concern. Twin studies have demonstrated a strong genetic component to the development of obesity and genome wide association studies have identified several genetic loci associated with it. However, most of these loci are still poorly understood in a functional context. Interestingly, many of the hormones and neurobiological messengers responsible for regulating feeding behaviour and metabolism are also linked to controlling aggression, but it is still not understood how they interact to maintain metabolic homeostasis. In this thesis, the model organism Drosophila melanogaster was employed to dissect the molecular mechanisms of the genetic cascades regulating aggressive behaviour and metabolic homeostasis.In paper I and II, the role of transcription factor AP-2 (TfAP-2) and Tiwaz Twz, Drosophila homologues of two human obesity-linked genes were investigated in aggression and feeding behaviour. Paper I demonstrated that TfAP-2 and Twz genetically interact in octopaminergic neurons to modulate male aggression by controlling the expression of genes necessary for octopamine (fly analogue of noradrenaline) production and secretion. Moreover, it was revealed that octopamine in turn regulates aggression through the Drosophila cholecystokinin (CCK) satiation hormone homologue Drosulfakinin (Dsk). Paper II revealed that TfAP-2 and Twz also initiate feeding through regulation of octopamine poduction and secretion. Octopamine then induces Dsk expression leading to inhibition of feeding.Paper III established that the activity of the small GTPase Ras-related C3 botulinum toxin substrate 2 (Rac2) is required in Drosophila for the proper regulation of metabolic homeostasis, as well as overt behaviours. Rac2 mutants were starvation susceptible, had less lipids and exhibited disrupted feeding behaviour. Moreover, they displayed aberrant aggression and courtship behaviour towards conspecifics.Paper IV studied Protein kinase D (PKD), the homologue of a third obesity-linked gene PRKD1, and another kinase Stretchin-Mlck (Strn-Mlck). Reducing PKD transcript levels in the insulin producing cells led to flies with increased starvation susceptibility, decreased levels of lipids and diminished insulin signalling compared to controls. Reduced Strn-Mlck expression resulted in a starvation phenotype and slight reduction in insulin signalling and lipid content. These findings imply a function for PKD and Strn-Mlck in insulin release.
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7.
  • Sheng, Ming, 1981- (författare)
  • Regulation of energy balance in Caenorhabditis elegans 
  • 2015
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Obesity is a medical condition in which excess body fat has been accumulated. It is most commonly caused by imbalance between energy intake and energy expenditure (lack of physical activity and lower metabolic rate, etc.). The control of energy metabolism involves multiple tissues and signalling pathways and there is a great need for further understanding of these different interactions.In this study, I use Caenorhabditis elegans to study these complex pathways at the level of a whole organism. The downstream target of mTOR, p70 S6 kinase (S6K), has been implicated in the phosphorylation of multiple substrates and the regulation of growth and metabolism. In this study the worm homolog of S6K, rsks-1, found to be important for fat metabolism. Previous work in our lab found that RSKS-1::GFP is expressed at high levels in a set of sensory neurons and upregulated in ASJ, ASE and BAG sensory neurons in starved worms or mutants with low insulin activity. In this study, I found that the upregulation of rsks-1 expression was affected by serotonin, but not by the other neurotransmitters. Combined with the result that rsks-1 is required for the expression of TGFβ and insulin in ASI, rsks-1 may control dietary sensing by affecting insulin and TGFβ signalling within nervous system. Quantification of fat accumulation by TLC/GC revealed that in comparison to wild type worms, rsks-1 mutants have more than two-fold higher levels of triglycerides. This was confirmed by FT-IR microspectroscopy analysis. rsks-1 mutants also contain disproportionately high levels of C16:1n9 and C18:1n9 lipids compared with wild type worms. Genetic analysis has shown that rsks-1 acts either downstream of, or in parallel to the insulin and TGFβ pathways to affect fat levels. My studies showed that rsks-1 affects fat metabolism by influencing mRNA levels of genes encoding proteins in the β-oxidation pathway. Combined with defects in dietary sensing, fatty acid absorption, fertility and mitochondria function, the loss of rsks-1 activity induced much more energy storage than wild type by making a profound metabolic shift. These results are consistent with the metabolomics data analysis. Tissue specific RNAi showed that rsks-1 was required in many different tissues to regulate fat metabolism. Taken together, it can be concluded that RSKS-1 activity is needed for co-ordination of metabolic states in C. elegans. In order to understand more about the physiology behind fat accumulation, I analysed a mutant, aex-5, that has significantly lowered lipid levels. I found that this defect is associated with a significant reduction in the rate at which dietary fatty acids are taken up from the intestinal lumen. The aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase, is required for a discrete step in an ultraradian rhythmic phenomenon called the defecation motor program (DMP). Combined with other results, we conclude that aex-5 and other defecation genes may affect fat uptake by promoting the correct distribution of acidity within the intestinal lumen.This dissertation also described how to use Fourier transform infrared (FT-IR) microspectroscopy to detect lipids, proteins and carbohydrates directly in single worm. In conclusion, in this thesis I have uncovered several components that play roles in dietary sensing, fatty acid synthesis, adiposity regulation and fatty acid absorption in C. elegans.   
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