Cellular Mechanisms of Sleep Homeostasis : Studies on Brain Energy Metabolism and Aging

Sleep is governed by a homeostatic process in which the duration and quality of previous wake regulate the subsequent sleep. Active wakefulness is characterized with high frequency cortical oscillations and depends on stimulating influence of the arousal systems, such as the cholinergic basal forebrain (BF), while cessation of the activity in the arousal systems is required for slow wave sleep (SWS) to occur. The site-specific accumulation of adenosine (a by-product of ATP breakdown) in the BF during prolonged waking /sleep deprivation (SD) is known to induce sleep, thus coupling energy demand to sleep promotion. The adenosine release in the BF is accompanied with increases in extracellular lactate and nitric oxide (NO) levels. This thesis was aimed at further understanding the cellular processes by which the BF is involved in sleep-wake regulation and how these processes are affected by aging. The BF function was studied simultaneously at three levels of organization: 1) locally at a cellular level by measuring energy metabolites 2) globally at a cortical level (the out-put area of the BF) by measuring EEG oscillations and 3) at a behavioral level by studying changes in vigilance states. Study I showed that wake-promoting BF activation, particularly with glutamate receptor agonist N-methyl-D-aspatate (NMDA), increased extracellular adenosine and lactate levels and led to a homeostatic increase in the subsequent sleep. Blocking NMDA activation during SD reduced the high frequency (HF) EEG theta (7-9 Hz) power and attenuated the subsequent sleep. In aging, activation of the BF during SD or experimentally with NMDA (studies III, IV), did not induce lactate or adenosine release and the increases in the HF EEG theta power during SD and SWS during the subsequent sleep were attenuated as compared to the young. These findings implicate that increased or continuous BF activity is important for active wake maintenance during SD as well as for the generation of homeostatic sleep pressure, and that in aging these mechanisms are impaired. Study II found that induction of the inducible NO synthase (iNOS) during SD is accompanied with activation of the AMP-activated protein kinase (AMPK) in the BF. Because decreased cellular energy charge is the most common cause for AMPK activation, this finding implicates that the BF is selectively sensitive to the metabolic demands of SD as increases were not found in the cortex. In aging (study III), iNOS expression and extracellular levels of NO and adenosine were not significantly increased during SD in the BF. Furthermore, infusion of NO donor into the BF did not lead to sleep promotion as it did in the young. These findings indicated that the NO (and adenosine) mediated sleep induction is impaired in aging and that it could at least partly be due to the reduced sensitivity of the BF to sleep-inducing factors. Taken together, these findings show that reduced sleep promotion by the BF contributes to the attenuated homeostatic sleep response in aging.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

On cluster approximations of cellular automata

In this thesis I examine one commonly used class of methods for the analytic approximation of cellular automata, the so-called local cluster approximations. This class subsumes the well known mean-field and pair approximations, as well as higher order generalizations of these. While a straightforward method known as Bayesian extension exists for constructing cluster approximations of arbitrary order on one-dimensional lattices (and certain other cases), for higher-dimensional systems the construction of approximations beyond the pair level becomes more complicated due to the presence of loops. In this thesis I describe the one-dimensional construction as well as a number of approximations suggested for higher-dimensional lattices, comparing them against a number of consistency criteria that such approximations could be expected to satisfy. I also outline a general variational principle for constructing consistent cluster approximations of arbitrary order with minimal bias, and show that the one-dimensional construction indeed satisfies this principle. Finally, I apply this variational principle to derive a novel consistent expression for symmetric three cell cluster frequencies as estimated from pair frequencies, and use this expression to construct a quantitatively improved pair approximation of the well-known lattice contact process on a hexagonal lattice.

Use of Document Management Systems - a Case Study of the Finnish Construction Industry

Triggered by the very quick proliferation of Internet connectivity, electronic document management (EDM) systems are now rapidly being adopted for managing the documentation that is produced and exchanged in construction projects. Nevertheless there are still substantial barriers to the efficient use of such systems, mainly of a psychological nature and related to insufficient training. This paper presents the results of empirical studies carried out during 2002 concerning the current usage of EDM systems in the Finnish construction industry. The studies employed three different methods in order to provide a multifaceted view of the problem area, both on the industry and individual project level. In order to provide an accurate measurement of overall usage volume in the industry as a whole telephone interviews with key personnel from 100 randomly chosen construction projects were conducted. The interviews showed that while around 1/3 of big projects already have adopted the use of EDM, very few small projects have adopted this technology. The barriers to introduction were investigated through interviews with representatives for half a dozen of providers of systems and ASP-services. These interviews shed a lot of light on the dynamics of the market for this type of services and illustrated the diversity of business strategies adopted by vendors. In the final study log files from a project which had used an EDM system were analysed in order to determine usage patterns. The results illustrated that use is yet incomplete in coverage and that only a part of the individuals involved in the project used the system efficiently, either as information producers or consumers. The study also provided feedback on the usefulness of the log files.