The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.

The seasonal reproduction of the three-spined stickleback is stimulated by long day photoperiod. As in other vertebrates, the reproductive system of stickleback is regulated by the brain-pituitary-gonadal (BPG) axis which is largely controlled by feedback effects. Both negative and positive feedback effects on the BPG axis have been found in fish. So far, the roles feedback effects on the BPG axis play in the photoperiodic regulation of seasonal reproduction are still unclear. This thesis focused on the photoperiodic regulation and gonadal feedback effects on the gene expressions of gonadotropin (GtH) and gonadotropin-releasing hormones (GnRH) in the brain and pituitary, and how gonadal feedback regulated the steroid homeostasis in stickleback.Both GnRH2 and GnRH3 mRNA was found in the hypothalamus. Higher expression levels of both GnRH2 and 3 in breeding than in post-breeding males suggested that they are both involved in seasonal reproduction. There was no evidence for a role of GnRH3, which may be the dominating form, in the photoperiodic control of reproduction. However, the polarity of the feedback effect on gnrh3 gene expression may turn from positive to be negative when the males went into post-breeding state. Tapeworm, Schistocephalus solidus, infection inhibited the reproduction of sticklebacks. However, the infection caused higher expression levels of both GnRHs and GtHs genes, which may be due to feedback effect on the BPG axis.Under short day, both lh-β and fsh-β were suppressed by low androgen levels. This negative feedback may inhibit maturation completely, unless a rise of androgens triggers positive feedback under long day. The change in feedback polarity may result in all or nothing maturation. Furthermore, the androgen inhibitory effect on lh-β and fsh-β under short day could be abolished by aromatase inhibitor, which means the estrogen may cause negative feedback in males under short day.There was no compensation effect on plasma androgen level in fully mature hemi-castrated fish. However, both testosterone and 11-ketoandrostenedione treatments increased plasma levels much less in sham-operated fish than in castrated ones, indicating that homeostatic mechanisms are nevertheless present. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows<strong>:  </strong>Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.</p>

Group A Streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces and is capable of producing a variety of diseases. This dissertation investigates the function of a metalloregulator named MtsR in GAS physiology and disease process. An mtsR mutant was constructed and analyzed. Consistent with MtsR role in iron uptake regulation, the mtsR mutant accumulates more iron (80 ± 22.5%) than the wild type strain. Inactivation of mtsR results in constitutive transcription of the sia (Streptococcal Iron Acquisition) operon, which is negatively regulated by iron in the parent strain. We identified the promoter that controls the expression of the sia operon (Pshr) and used it as a model to study MtsR interaction with DNA. Electrophoretic mobility gel shift assays (EMSAs) demonstrated that MtsR binds to the shr upstream region specifically and in an iron and manganese dependent manner. DNase I footprint analysis revealed that MtsR protects a 69 bp segment in Pshr that includes 2 inverted repeats, overlapping the core promoter elements. A global transcriptional analysis determined that MtsR modulates the expression of 64 genes, of which 44 were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including immune evasion, colonization, dissemination, metal homeostasis, nucleic acid and amino acid metabolism, and protein stability. MtsR functions as a dual regulator as it binds to the promoters of the repressed genes ska, aroE, and nrdF.2, as well as the upstream region of the positively regulated genes mga, emm49, and pyrF. A 16 bp MtsR-binding consensus region was identified in all of the promoters that are directly regulated by MtsR. In conclusion, we have demonstrated that MtsR is a global regulator in GAS that controls the expression of vital virulence factors and genes involved in metal transport, virulence and metabolic pathways.

<p>Mechanisms of copper homeostasis are of great interest partly due to their connection to debilitating genetic and neurological disorders. The family of high-affinity copper transporters (Ctr) is responsible for extracellular copper acquisition and internalization in yeast, plants, and mammals, including human. The extracellular domain of the human high-affinity copper transporter (hCtr1) contains essential Cu-binding methionine-rich MXXM and MXM (Mets) motifs that are important for copper acquisition and transport. The hCtr1 extracellular domain also contains potential copper binding histidine (His) clusters, including a high-affinity Cu(II) ATCUN site. As of yet, extracellular His clusters have no established significance for hCtr1 function. We have made model peptides based on the extracellular copper acquisition domain of hCtr1 that is rich in His residues and Mets motifs. The peptides' Cu(I) and Cu(II) binding properties have been characterized by UV-Vis and mass spectrometry. Our findings have been extended to a mouse cell model and we show that His residues are important for hCtr1 function likely because of their contribution to strong copper-binding sites in the hCtr1 extracellular domain responsible for copper acquisition. </p> <p>Copper's pro-oxidant property is also medicinally promising if it can be harnessed to induce oxidative stress as a cancer chemotherapy strategy. Our lab has designed a photocleavable caged copper complex that can selectively release redox-active copper in response to light. The thermodynamic copper binding properties of these potential chemotherapeutics have been characterized</p> / Dissertation

This is the first research by using suppression subtractive hybridization (SSH) to analysis the gene expression in marine macroalga Ulva fasciata Delile against excess copper toxicity, and it gives us a comprehensive understanding of the tolerant mechanism while macroalgae face to the excess copper. Suppression subtractive hybridization was used to identify genes differentially expressed following exposure to 50 £gM CuSO4 for 6- 12h in a marine macroalga Ulva fasciata Delile. In this work, 69 genes were identified, of which 55 were up-regulated and 14 were down-regulated. According to the database of Gene Ontology (GO), these genes were classified into 10 categories as follows: 1. Transcription; 2. Translation, ribosomal structure and biogenesis; 3. Posttranslational modification, protein turnover, chaperones; 4. Photosynthesis; 5. Cell redox homeostasis; 6. Stress; 7. Metabolism; 8. Energy production and conversion; 9. Transport; 10. Function unknown. According to the results, we suggest that the responses of U. fasciata against excess copper toxicity are mainly through increase of the energy production for providing sufficient energy to many metabolic pathways, and control of the Fe homeostasis and redox form of thiol groups for maintaining the cellular redox homeostasis, moreover, expression of photosynthetic genes for letting the photosynthesis work. In addition, to scavenge the ROS is by expression of stress-related genes, meanwhile, the proteins, DNA and lipids damaged by ROS (reactive oxygen species) and copper are repaired by expression of the other categorical genes. Over and above, the genes expressing in the metabolism category might maintain the amino acids homeostasis and increase the purine content, and subsequently increase the tolerant capacity of U. fasciata against excess copper toxicity. In addition, the concentrations of antioxidants and the activities and gene expression of antioxidant enzymes were determined in Ulva fasciata Delile by a 4-day exposure to 0, 5, 10, 20 and 50 £gM CuSO4. These results demonstrate that the maintenance of antioxidant homeostasis and the induction of activities of antioxidant enzymes via enhanced gene expression are used by U. fasciata to cope with the Cu-induced oxidative stress, but the defense capacity cannot sufficiently alleviate oxidative damage occurring under the condition of higher Cu concentrations. Moreover, according to the results from the expression of genes involved in the control of redox homeostasis and antioxidant defense was studied in macroalga Ulva fasciata Delile in response to CuSO4 (5 and 50 £gM) and ROS (H2O2 and O2£»-), we suggest that ROS involved in up-regulation of antioxidant defense-related genes and the expression of genes of antioxidant defense enzymes and UfMsrA (methionine sulfoxide reductase A) are associated with long-term adaptation of U. fasciata to Cu excess and transcription of redox- related genes and UfGr (glutathione reductase) is up-regulated for short-term acclimation. Promoters play a key role in regulating gene expression. Based on the analysis of cis-acting elements on UfMsr promoters, we suggested that the signal transduction pathway of copper stress in U. fasciata is related to that of other stresses and of defense-related plant hormones, however, Ca2+ and calmodulin might participate in it. To sum up, U. fasciata could resist oxidative damage caused by excessive copper through the regulation on the molecular level.

<p>Vi gör dagligen bedömningar av risker i situationer av alla de slag. Trafiken är en av de situationer där riskbedömningar är särskilt viktiga och hur korrekt bedömningen blir beror på hur vi upplever riskerna, vilket i hög grad varierar från person till person. Hypotesen i detta examensarbete är: Kvinnliga bilister upplever större subjektiv risk i trafiken än manliga bilister. Undersökningen av hypotesens giltighet utfördes med hjälp av intervjuer av 20 män och kvinnor. Frågorna syftade till att ta reda på deltagarnas subjektiva upplevelser av trafikrisker. Resultaten pekade på att det finns en tendens till skillnad i hur de båda könen upplever risker i trafiken. Kunskap om hur kvinnor och män upplever risker i trafiken är användbara för att göra olika trafiksituationer säkrare och därmed minska antalet olyckor. Ännu ett steg i denna strävan är att undersöka hur stressfaktorer påverkar riskuppfattningen i en trafiksituation.</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010. / Härtill 3 uppsatser.

Development of a Tool for Pressure Ulcer Risk Assessment and Preventive Interventions in Ancillary Services Patients Monica S. Messer Abstract The incidence of nosocomial pressure ulcers has increased 70 percent in U.S. hospitals over the past 15 years despite implementation of preventive guidelines and the wide-spread use of validated risk assessment tools. Most preventive efforts have been focused primarily on patients who are bed-ridden or immobile for extended periods. What has not been well studied or identified is the risk for pressure injury to patients undergoing diagnostic procedures in hospital ancillary units where extrinsic risk factors such as high interface pressures on procedure tables and friction and shear from positioning and transport can greatly magnify the effect of patient-specific intrinsic risk factors which might not otherwise put these patients at high risk on an inpatient unit. The purpose of this study was to develop a risk assessment tool designed explicitly to quantify the combination of these intrinsic and extrinsic risk factors in individual patients undergoing ancillary services procedures, and to identify targeted preventive interventions based on the individual level of risk. Empirically and theoretically-derived risk factors for the tool were tested in a nation-wide hospital database of over 6 million patient discharge records using bivariate and multivariate analysis to identify significant predictors of pressure ulcer outcomes. The statistically significant factors emerging were then used to develop the risk assessment scale. These predictors included; advanced age, diabetes, human immunodeficiency virus infection, sepsis, and fever. The scale was tested for internal validity using the split-sample cross-validation method, and for accuracy using the area under the Receiver Operating Characteristics curve. The optimum score cut point was identified to provide a predictive accuracy of 71 percent. Interventions for the tool were identified from national clinical practice guidelines and aligned in sets based on patient levels of risk identified by the scoring portion of the tool. The entire tool was evaluated for content validity by a panel of five international nurse experts in pressure ulcer prevention and tool development. The content validity index calculated from their ratings was .91 indicating excellent agreement on content validity.

The small intestine is the main organ for food digestion and nutrient absorption. It is constantly exposed to antigen and immunomodulatory agents from diet and commensal microbiota. Thus, the intestine is the largest compartment of the immune system in the body. Peanuts and many other allergen resources contain triglycerides, which may affect the antigen absorption through the intestine, but their effects on sensitization and anaphylaxis are unknown. We found that medium chain triglycerides (MCT) promoted antigen absorption into Peyer’s Patches, rather than into the blood directly. Both gavage and feeding of MCT plus peanut protein induced spontaneous allergic sensitization. MCT-sensitized mice experienced the IgG-dependent anaphylaxis from systemic challenges and the IgE-dependent anaphylaxis from oral challenges. Furthermore, MCT alone had direct pharmaceutical effect on enterocytes, like stimulating Jejunal-epithelial Th2 cytokine responses compared with what was seen in the long chain triglycerides (LCT) treated group. Moreover, the oral challenges conducted with peanut protein in MCT significantly exacerbated anaphylaxis compared with the LCT challenges. The intestine also plays an important role in whole body cholesterol homeostasis due to its exclusive function in cholesterol absorption. The researchers found that the intestine function in cholesterol secretion and elimination, but it has not been proven directly until recently. This pathway that facilitate the cholesterol secretions through intestine was named the Transintestinal Cholesterol Efflux (TICE) and has not been well studied yet. To find the possible transporter candidates involved in TICE, we compared both biliary and intestinal cholesterol excretion rates in wild-type (WT) and G5G8 deficient (KO) mice of both sexes. All mice were maintained on a plant-sterol free diet beginning at weaning to prevent the development of secondary phenotypes associated with Sitosterolemia. We found that WT mice had higher biliary cholesterol excretion rates compared to their G5G8 KO littermates as previously reported. However, this difference is significantly greater in females compared to males. Interestingly, intestinal cholesterol excretions increased in female KO mice compared to their WT littermates, a difference not observed in males. This data suggests a sexually dimorphic adaptive mechanism to maintain cholesterol elimination in the absence of G5G8. Whereas male mice maintain a greater level of biliary output in the absence of G5G8, female mice upregulate an alternate intestinal elimination route. To determine the origin of intestinally secreted cholesterol, we compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (CETP TG) mice at the age of 12 weeks. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma and alters the lipoprotein distribution of plasma cholesterol. We found that WT and CETP TG mice did not differ in either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, CETP TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to a Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to bile and intestinal lumen. Unlike bulk cholesterol secretions, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP TG mice. This data suggests that CETP alter the routes of total and HDL cholesterol elimination from the body in mice.

No description available.