| 251. |
- Yu, Zhengquan, 1965-
(författare)
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Proton trapping in the cellular acidic vacuolar compartment : lysosomal mechanisms in apoptosis/necrosis and iron chelation
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Under ischemic conditions, a number of cytotoxic metabolic products are formed. Reactive oxygen species are known to be important mediators of progressive ischemic cell injury, and the synergistic damage to cells caused by the combination of such oxygen species and redox-active iron is well appreciated. The acidic interior of lysosome leads to the trapping of substances with high pK4 values. A large variety of molecules, being weak bases, may thus concentrate within this acidic vacuolar compartment, potentially leading to both beneficial and detrimental effects. A major part of the intracellular pool of redoxactive iron is likely to be located in the lysosomal compartment, and iron chelators that are lysosomotropic due to high pK4 values may prove to be important pharmacological tools to protect the brain from oxidative stress. Among a variety of substances formed in the ischemic penumbra zone is the polyamine metabolite, 3-aminopropanal (3-AP), a substance of extreme neurotoxicity. 3-AP is a weak base and may theoretically exert its toxic action through induction of cell death after intralysosomal accumulation.On the 1774 mouse histiocytic lymphoma cell line, we used the common lysosomotropic agent NH3 to increase lysosomal pH, the lysosomotropic iron chelator, 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (LAP) and the lysosomotropic iron binder, WR-1065, a metabolite of amifostine, as tools to determine that proton trapping within the lysosomal acidic vacuolar compartment plays an important role in oxidative stress-induced apoptosis. We also used another lysosomotropic agent, 3-AP, on the J774 cell line and on the SH-SY5Y human neuroblastoma cell line. The results indicate that proton trapping of this toxin within the lysosome might explain its toxicity to cells.Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in the lysosomal content of redox-active iron following reduced acidity of the lysosome, and electron spin-resonance studies showed that no hydroxyl radicals [OH•] were formed from hydrogen peroxide under these conditions. This suggests that lysosomes contain most of the free, redox-active iron. In further support of this idea, the lysosomotropic agents LAP and WR-1065 were found to be 5000 and 2500 times more effective, respectively, in protecting cells from oxidative stress, compared with the well-known iron chelator desferrioxamine [DFO]. Evidence was obtained that LAP and WR-1065 exerted their effect on intralysosomal redox-active iron, and that the effect was linked to the acidity of the lysosome. Being weak bases (LAP, pKa = 8.0; WR-1065, pKa = 9.2), these compounds accumulate intralysosomally by proton trapping. The neurotoxic effect of 3-AP (pKa = 9.3) could be linked to a dose-dependent induction of cell death, most likely based on intralysosomal proton trapping of this molecule followed by lysosomal rupture. The lysosomal rupture seems to induce a chain of intracellular events (including generation of oxidative stress), leading to mitochondrial damage directly or indirectly caused by the release of lysosomal proteases.We conclude that the low pH of the lysosome may both serve to attract basic toxins, such as 3-AP, and promote the accumulation of protective agents, such as LAP and WR-1065. Prevention of lysosomal damage from both oxidants and neurotoxins by lysosomotropic agents has great potential therapeutic utility.
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| 252. |
- Zdolsek, Joachim, 1960-
(författare)
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Water physiology in burn victims
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Patients who sustain a burn injury of more than 20 - 30 % TBSA will, if untreated, develop burn shock within a couple of hours. Flcid is lost fi·om the vascular compartment due to suction from the interstitium of the wound and due to a generalised increase in vascular permeability. To prevent bum shock intravenous fluids are given during the first two days after a bum in order to ensure adequate organ perfusion. Although organ perfusion will be improved when blood volume is restored, the fluid provided will add to the continuing leak into the tissues. With fluid treatment the patient survives the acute stage, but the resulting oedema interferes with the healing of the burn wound and partially damaged skin may be further compromised.To describe, follow and understand the development of the general oedema in burn injured patients we used four different techniques. Ethanol dilution was used for measurement of total body water (TBW), iohexol dilution in order to estimate extracellular volume (ECV), bioimpedance analysis (BIA) to register TBW as well as electrical membrane properties and the impression method (IM) for the measurement of visco-elastic properties in non-burned tissues. In order to further examine the non-invasive techniques (BIA and IM) these were used in patients subjected to haemodialysis.The excess fluid was found to be accumulated in the extracellular space. Interesting alterations in the visco-clastic properties of the skin and cellular electrical membrane properties could also be detected. These alterations were not related to the actual tluid volume in the tissues. Instead, they were related to changes in the fluid equilibrium of the tissues. Still, one week postbum an excess of tissue fluid, altered cellular electrical membrane properties and changed visco-elastic properties of the skin remained.Albumin supplementation did not influence the amount or distribution of the excess tissue fluid, measured with dilution techniques.
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| 253. |
- Zhao, Ming, 1966-
(författare)
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The Lysosomal-Mitochondrial Axis Theory of Apoptosis
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In many cases, apoptosis may be initiated by a minor lysosomal destabilization, which some time later is followed by a secondary, more pronounced, lysosomal rupture. After exposure to low concentrations of sphingosine, a lysosomotropic detergent, Jurkat and J774 cells underwenr apoptotic cell death, while cells exposed to higher concentrations of this agent showed necrosis. Sphingosine-induced apoptosis was partly prevented by the inhibitors of lysosomal aspartic or cysteine proteases, pepstatin A or E64d. Under these conditions, caspase-3 like activity was reduced 40-55%, suggesting that lysosomal enzymes could be upstream activators of caspase-3.In J774 cells over-expressing Bcl-2, the early oxidant-induced lysosomal destabilization takes place, but the delayed secondary lysosomal rupture and ensuing apoptosis are both suppressed. Phosphorylation of Bcl-2 seems to be required for this anti-apoptotic effect because the protection is amplified by pre-treatment with phorbol 12-myristate 13-acetate, which promotes protein kinase C (PKC)-dependent phosphorylation of Bcl-2. In contrast, cells over-expressing the Bcl-2 mutant S70A (which cannot be phosphorylated and is inactive) are not protected. Transfection with Bcl-2(S70E), a constitutively active Bcl-2 mutant, which does not require phosphorylation, is protective independent of PKC activation. In contrast, C2- ceramide, a putative protein phosphatase 2A (PP2A)-activator, abolishes the protective effects of wild-type Bcl-2 over-expression but does not diminish protection afforded by Bcl-2(S70E).It may be that Bcl-2 directly blocks the effects of initially released lysosomal enzymes and/or prevents down-stream activation of cytosolic pro-apoptotic enzymes by released lysosomal hydrolases. Short-term (1 h) exposure of cells to a low steadystate concentration of H202 causes no immediate cell death, but apoptosis occurs several hours later when cells have been returned to standard culture conditions. This delayed cell death seems to arise from activation of phospholipases, in particular phospholipase A2 (PLA2), which may dcstabilize lysosomal as well as mitochondrial membranes. Indeed, the specific inhibition of PLA2 by 4-bromophenacyl bromide (BPB ), diminishes both delayed lysosomal rupture and apoptosis. Furthermore, PLA2 activation by mellitin, or direct micro-injection of PLA2, causes lysosomal rupture and apoptosis. Finally, Bcl-2 over-expression prevents oxidant-induced activation of PLA2, and delays lysosomal destabilization as well as apoptosis.Exogenous oxidative stress may induce apoptosis, but enhanced endogenous production of oxidants is also often found during apoptosis caused by other agonists, raising the question of whether this latter actually contributes to apoptosis or is simply a by-product. Our data show that leak to the cytosol of lysosomal enzymes results in mitochondria-mediated oxidative stress, release of cytochrome c, and further lysosomal rupture. In mixed lysosome-mitochondria preparations, the lysosomotropic detergent, 0-methyl-serine dodecylamide hydrochloride (MSDH), selectively lyses lysosomes, while PLA2 attacks lysosomes as well as mitochondria. Released lysosomal enzymes, and aclivated PLA2, cause mitochondria to produce enhanced amounts of hydrogen peroxide and to release cytochrome c. Purified lysosomal cathepsins B and D have the same effects on mitochondrial oxidant production but do not destabilize lysosomal membranes in these mixed preparations of mitochondria and lysosomes. In intact cells, MSDH induces lysosomal rupture, oxidative stress and apoptosis.These data allow us to propose the following lysosomal-mitochondrial axis theory of apoptosis:1. Limited lysosomal rupture induces activation of PLA2 (probably often mediated by the lysosomal enzyme, cathepsin B).2. Released lysosomal enzymes, and activated PLA2, cause enhanced mitochondrial production of reactive oxygen as wel1 as release of cytochrome c.3. This cascade of events is accompanied by further lysosomal rupture (by the combined effects of oxidative stress and PLA2), initiating full-blown apoptosis.4. Through presently unknown mechanisms, phosphorylated Bcl-2 preserves the integrity of both mitochondria and lysosomes, preventing further release of lysosomal enzymes and of mitochondrial pro-apoptotic proteins.
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| 254. |
- Öhman, Daniel, 1973-
(författare)
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Bioanalytical development for application in therapeutic drug monitoring : focus on drugs used in psychiatry
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Since the 1950s and onwards, many new psychoactive drugs have been successfully introduced in clinical practice. A common feature of these drugs is a profound metabolism before clearance from the body. Genetic and phenotypic factors among patients can be anticipated to interact with the metabolism and therefore a major variance in the drug concentration between patients upon the same dose may be found. In later years there has been an increasing awareness, that the individual drug metabolism and drug body disposition do not only refer to the parent compound and major metabolites but also to the separate enantiomers in the case of chiral drugs.Bioanalytical methods for determination of drug concentrations have constantly been developed to assess the large pharmacokinetic variability of these drugs. Moreover, in the psychopharmacological practice these methods are often extended into a "Therapeutic Drug Monitoring (TDM) Service". A TDM service is used to determine the patient-specific drug analytical outcome in order to assess the precision in dose prescription as well as to recommend adequate dose adjustments for a particular patient.Aim: The aim was to develop bioanalytical methods, utilising high performance liquid chromatography, for serum determinations in patients under chronic dosing schedules for some recently introduced drugs used in psychiatry.Firstly, for the antidepressant drug reboxetine by an achiral methodology (paper I) consecutively extended to an enantioselective methodology for the individual enantiomers of the drug (paper II and III). Thereafter, after the evaluation of on-line extraction as an automated alternative to manually performed solid phase extraction for sample preparation (paper IV), apply on-line extraction in combination with ion-trap mass spectrometry detection for serum determination of the novel antipsychotic drug ziprasidone (paper V).Results and Conclusion: The methodology described in paper I has been applied on more than 500 patient samples from the naturalistic clinical practice as well as from controlled clinical trials. The methodology has proven robust and reliable and, although manually performed, easy to handle. The kinetic outcome display a great variability in concentration outcome even within the same prescribed dose, i.e. through level samples in steady-state were found to be 660±400 nM on 8 mg/day.Paper II, which describes the development of three approaches for direct chiral separation of reboxetine and O-deethylreboxetine enantiomers in the reversed phase mode, is in itself new and important. It is however, after applying MS2 detection, in paper III that the methodology is extended to include patients, i.e. patients from two separate clinical trials. Trial I comprised 23 patients on monotherapy with reboxetine and trial II comprised 47 patient from a naturalistic clinical setting. The pharrnacokinetic outcome displayed a S,S- over R,R-reboxetine ratio of about 0.5 with a rather pronounced inter-individual ratio (i.e. 0.2 to 0.9). The enantiomeric ratio did not correlate to the overall reboxetine concentration and the enantiomeric ratio was about 30% higher in females than in males. Repeated samples were analysed for trial I patients displaying an inter-individual coefficient of variation (CV) of about 17% meanwhile the intra-individual CV was about 4%. Calculating the noradrenaline reuptake inhibition (NARI) -activity out of the individual enantiomeric ratios showed that females may have a higher NARI-activity than males at a given reboxetine concentration.Paper IV proves that online extraction offers a robust, reliable and, for the analyst, time saving alternative (above 80%) to manually performed off-line solid phase extraction for sample preparation (citalopram and its demethylated metabolites were used as model substances).Paper V describes the development of a TDM process for ziprasidone and its S-methylated metabolite. The sample handling as well as the analytical process was subjected to a solid and satisfactory validation. It was proved that the lack of selectivity during on-line extraction can be compensated for by a selective form of detection, i.e. mass spectrometric detection.
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| 255. |
- Östlund, Gunnel, 1956-
(författare)
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Promoting return to work : lay experiences after sickness absence with musculoskeletal diagnoses
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Musculoskeletal disorders constitute the greatest cause of sickness absence from work. Despite research and efforts at rehabilitation, sickness absence due to these disorders has not decreased, but has instead increased, particularly in women. Clients’ perceptions of care and rehabilitation, i.e. knowledge generated from a lay perspective, is a neglected area of research. This thesis deals with lay experiences of rehabilitation following sickness absence due to back, neck or shoulder problems, termed musculoskeletal disorders (MSD).Aim: The overall aim was to examine hindering and promoting processes in rehabilitation after sickness absence due to MSD from a lay perspective. Specific aims were to study how lay persons experience rehabilitation agents and rehabilitation activities (paper I), how they describe themselves and their experience in relation to work (paper II), the significance of the private arena regarding return to work (paper III), and how clients who have experienced sickness absence due to MSD perceive contact with rehabilitation agents (paper IV).Method: The study population in the four papers is part of a cohort of persons living in the same municipality and who in 1985 were aged 25-34 years and were sick-listed due to back, neck or shoulder diagnoses for 28 days or more, n=213. During 1995, 148 persons in the cohort responded to a questionnaire, and in 1997-1998, 20 of these persons were interviewed concerning their experiences with rehabilitation. In papers I, II and III the qualitative method of Grounded Theory was used with a focus on creating an empirically-based theory concerning the area under study. Data collection was strategic and analysis of the tape-recorded interviews was done on a continual basis. How previously sick-listed persons experienced contact with professional rehabilitation agents in t he health care sector and social insurance office was investigated in paper IV. Factor analysis and multiple regression analysis were used to analyse the data in this study.Results: The interview study shed light on lay persons’ experiences with medical, social and work-related measures in rehabilitation, their perceptions of rehabilitation actors and family members in relation to rehabilitation, and their self-presentations. The descriptions of lay persons concerned three arenas, the health care arena, the occupational arena, and the private arena. Dilemmas and difficulties in these arenas were described, such as handling the duty to work, experiencing domestic strain, and the experience of lacking socioemotional support from significant persons during the rehabilitation process. In paper I some ideal types of rehabilitation agents emerged from the interviewees’ descriptions concerning the health care arena, and we called these the routine bureaucrat, the empathic administrator, the distant technician, and the professional mentor. The latter agent was requested and was described as a person who could provide socioemotional support, who had professional competence, and who could function as a unifying link during the rehabilitation process. The results from paper II showed that in their self-presentations, the interviewees expressed having a duty to work and that there were differences in how they handled this sense of duty. The selfpresentations contained descriptions of work as a part of personal identity and could be summarised in the following ideal types: the work manic, the workhorse, the workaholic and the relaxed worker. The latter used a strategy that can be considered to promote rehabilitation in that the individual himself/herself had control over his/her work and worked in accordance with his/her own needs rather than those of others. Paper III focused on the private arena. Different patterns were found in the experiences of men and women. Women related that their responsibility for the home and domestic work seldom left any time for themselves, including any time for rehabilitation. Men more often reported having time for themselves that could be used for leisure activities and rehabilitation. Some of the women said that they lacked socioemotional support from their partner and that they had a great deal of responsibility for housework, which seemed to be a hindrance in returning to work after sickness absence. Furthermore, these women, like most of the men, had little education, which could make finding other work alternatives more difficult. Based on the interviews, a hypothesis was developed regarding domestic strain that is related to the distribution of domestic work, the distribution of responsibility for the home, and the quality of the marital relationship. Paper IV dealt with clients’ perceptions of contact with rehabilitation agents in health care and the social insurance office. Three latent dimensions were found in the respondents’ ratings of these contacts: supportive treatment, distant treatment, and empowering treatment. Sex, disability pension status, mental health and diagnostic group were significantly related to how these dimensionswere rated. Women perceived the treatment from both types of rehabilitation agents as more supportive than men. Contact with the social insurance offices were rated higher by persons with disability pensions than by those who had returned to work. Men rated their contact with rehabilitation agents at social insurance offices high on the dimension of distant treatment. Respondents with mental health problems rated the contact as distant for both types of rehabilitation agents, but contact with health care was also scored low on the supportive dimension. Finally, respondents with neck/shoulder diagnoses rated contact with rehabilitation agents in health care as more empowering than was done by persons with back diagnoses.Conclusions: From a lay perspective rehabilitation following sickness absence due to MSD occured in three arenas, the health care arena, the occupational arena and the private arena, where the quality of relationships both with rehabilitation agents, persons at work and in one’s private life was described as important regarding the rehabilitation process. This thesis also showed that both sex and health were important factors regarding how lay persons’ perceived contacts with rehabilitation agents during the rehabilitation process following sickness absence due to MSD.
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