Dynamic interplay between the intermediate filament protein nestin and Cyclin-dependent kinase 5

Cellen har ett s.k. cytoskelett som bl.a. ger stadga åt cellen och deltar i dess form- och rörelsefunktioner. Intermediärfilamenten är en viktig del av cytoskelettet och de har länge varit kända för sina väsentliga roller i att upprätthålla den cellulära organisationen och vävnadernas integritet. På senare år har man insett att intermediärfilamenten har en större funktionell mångsidighet än man tidigare tänkts sig, i och med att en rad olika studier har visat betydelsen av intermediärfilamenten vid olika signaleringprocesser. Dessa proteinnätverk samverkar nämligen med kinaser och andra viktiga signalfaktorer och deltar därmed i cellens signaleringmaskineri. Intermediärfilamentproteinet nestin används ofta som en markör för stamceller men dess fysiologiska funktioner är i stort sett okända. Interaktion mellan nestin och ett signalkomplex bestående av cyklin-beroende kinas 5 (eng. Cyclin-dependent kinase, Cdk5) och dess aktivatorprotein p35 upptäcktes i vårt laboratorium före denna avhandling påbörjades. Därför var syftet med min avhandling att undersöka den funktionella betydelsen av nestin i regleringen av Cdk5/p35 komplexet. Cdk5 är ett multifunktionellt kinas som reglerar både utvecklingen och stressreaktioner i nerver och muskler. Vi visade att nestin skyddar neuronala stamceller under oxidativ stress genom dess förmåga att hämma Cdk5s skadliga aktivitet. Genom att förankra Cdk5/p35 komplexet, reglerar nestin den subcellulära lokaliseringen av Cdk5/p35 och minskar klyvningen av p35 till den mer stabila aktivatorn p25. Vi demonstrerade också aktiveringsmekanismen för Cdk5 under differentiering av muskelceller. Proteinkinas C zeta (PKCzeta) avslöjades ha en förmåga att accelera klyvningen av p35 till p25, och därmed öka aktiviteten hos Cdk5. Nestin kunde genom sin förmåga att reglera Cdk5 signalkomplexet styra muskelcellernas differentiering. Denna doktorsavhandling har på ett avgörande vis ökat förståelsen av de reglerande mekanismer som styr Cdk5 aktivering. Avhandling presenterar nestin och PKCzeta som kritiska faktorer i denna reglering. Vidare innehåller avhandlingen ny information om de cellulära funktionerna hos nestin som vi har visat vara en viktig reglerare av cellernas överlevnad och differentiering.

Methods for construction and analysis of computational models in systems biology : applications to the modelling of the heat shock response and the self-assembly of intermediate filaments

Systems biology is a new, emerging and rapidly developing, multidisciplinary research field that aims to study biochemical and biological systems from a holistic perspective, with the goal of providing a comprehensive, system- level understanding of cellular behaviour. In this way, it addresses one of the greatest challenges faced by contemporary biology, which is to compre- hend the function of complex biological systems. Systems biology combines various methods that originate from scientific disciplines such as molecu- lar biology, chemistry, engineering sciences, mathematics, computer science and systems theory. Systems biology, unlike “traditional” biology, focuses on high-level concepts such as: network, component, robustness, efficiency, control, regulation, hierarchical design, synchronization, concurrency, and many others. The very terminology of systems biology is “foreign” to “tra- ditional” biology, marks its drastic shift in the research paradigm and it indicates close linkage of systems biology to computer science. One of the basic tools utilized in systems biology is the mathematical modelling of life processes tightly linked to experimental practice. The stud- ies contained in this thesis revolve around a number of challenges commonly encountered in the computational modelling in systems biology. The re- search comprises of the development and application of a broad range of methods originating in the fields of computer science and mathematics for construction and analysis of computational models in systems biology. In particular, the performed research is setup in the context of two biolog- ical phenomena chosen as modelling case studies: 1) the eukaryotic heat shock response and 2) the in vitro self-assembly of intermediate filaments, one of the main constituents of the cytoskeleton. The range of presented approaches spans from heuristic, through numerical and statistical to ana- lytical methods applied in the effort to formally describe and analyse the two biological processes. We notice however, that although applied to cer- tain case studies, the presented methods are not limited to them and can be utilized in the analysis of other biological mechanisms as well as com- plex systems in general. The full range of developed and applied modelling techniques as well as model analysis methodologies constitutes a rich mod- elling framework. Moreover, the presentation of the developed methods, their application to the two case studies and the discussions concerning their potentials and limitations point to the difficulties and challenges one encounters in computational modelling of biological systems. The problems of model identifiability, model comparison, model refinement, model inte- gration and extension, choice of the proper modelling framework and level of abstraction, or the choice of the proper scope of the model run through this thesis.

"Dov'è la sinistra?" Hemispatial neglect in stroke

As long as the incidence of stroke continues to grow, patients with large right hemisphere lesions suffering from hemispatial neglect will require neuropsychological evaluation and rehabilitation. The inability to process information especially that coming from the left side accompanied by the magnetic orientation to the ipsilesional side represents a real challenge for rehabilitation. This dissertation is concerned with crucial aspects in the clinical neuropsychological practice of hemispatial neglect. In studying the convergence of the visual and behavioural test batteries in the assessment of neglect, nine of the seventeen patients, who completed both the conventional subtests of the Behavioural Inattention Test and the Catherine Bergego Scale assessments, showed a similar severity of neglect and thus good convergence in both tests. However, patients with neglect and hemianopia had poorer scores in the line bisection test and they displayed stronger neglect in behaviour than patients with pure neglect. The second study examined, whether arm activation, modified from the Constraint Induced Movement Therapy, could be applied as neglect rehabilitation alone without any visual training. Twelve acute- or subacute patients were randomized into two rehabilitation groups: arm activation training or traditional voluntary visual scanning training. Neglect was ameliorated significantly or almost significantly in both training groups due to rehabilitation with the effect being maintained for at least six months. In studying the reflections of hemispatial neglect on visual memory, the associations of severity of neglect and visual memory performances were explored. The performances of acute and subacute patients with hemispatial neglect were compared with the performances of matched healthy control subjects. As hypothesized, encoding from the left side and immediate recall of visual material were significantly compromised in patients with neglect. Another mechanism of neglect affecting visual memory processes is observed in delayed visual reproduction. Delayed recall demands that the individual must make a match helped by a cue or it requires a search for relevant material from long-term memory storage. In the case of representational neglect, the search may succeed but the left side of the recollected memory still fails to open. Visual and auditory evoked potentials were measured in 21 patients with hemispatial neglect. Stimuli coming from the left or right were processed differently in both sensory modalities in acute and subacute patients as compared with the chronic patients. The differences equalized during the course of recovery. Recovery from hemispatial neglect was strongly associated with early rehabilitation and with the severity of neglect. Extinction was common in patients with neglect and it did not ameliorate with the recovery of neglect. The presence of pusher symptom hampered amelioration of visual neglect in acute and subacute stroke patients, whereas depression did not have any significant effect in the early phases after the stroke. However, depression had an unfavourable effect on recovery in the chronic phase. In conclusion, the combination of neglect and hemianopia may explain part of the residual behavioural neglect that is no longer evident in visual testing. Further research is needed in order to determine which specific rehabilitation procedures would be most beneficial in patients suffering the combination of neglect and hemianopia. Arm activation should be included in the rehabilitation programs of neglect; this is a useful technique for patients who need bedside treatment in the acute phase. With respect to the deficit in visual memory in association with neglect, the possible mechanisms of lateralized deficit in delayed recall need to be further examined and clarified. Intensive treatment induced recovery in both severe and moderate visual neglect long after the first two to first three months after the stroke.

Risk management measures for REACH registered intermediate substances in pulp mill

Tämän työn tavoitteena on antaa kuvaus riskinhallintamenetelmistä viidelle välituotekemikaalille, joita käytetään Stora Enson Imatran tehtailla. Välituotekemikaalit ovat mustalipeä, viherlipeä, valkolipeä, natriumbisulfiitti ja natriumsulfiitti. Nämä kemikaalit ovat jo rekisteröityjä ECHA:an ja rekisteröintiin liittyen ECHA:an on toimitettava myös kuvaus riskinhallintamenetelmistä. Työn alussa kuvaillaan työn kannalta olennaiset säädökset ja viranomaiset, jotka valvovat kemikaalien käyttöä ja valmistusta Euroopan Unionin alueella. Tämän jälkeen kerrotaan yleisesti välituotekemikaalien rekisteröintikriteereistä. Työn loppuosa käsittää kuvauksen riskinhallintamenetelmistä jokaiselle kemikaalille. Riskinhallintamenetelmät sisältävät eristyksen teknisin keinoin, menettelytapa- ja valvontatekniikat, johtamistavat ja henkilökunnan koulutuksen ja välituotekemikaalien kuljetuksen. Myös jokaisen kemikaalin ominaisuudet on kuvattu ja lyhyt prosessikuvaus kemikaalien valmistuksesta ja käytöstä on esitetty helpottamaan ymmärtämistä.

Models to study intermediate filament dynamics and functions

Intermediate filaments are part of the cytoskeleton and nucleoskeleton; they provide cells with structure and have important roles in cell signalling. The IFs are a large protein family with more than 70 members; each tightly regulated and expressed in a cell type-specific manner. Although the IFs have been known and studied for decades, our knowledge about their specific functions is still limited, despite the fact that mutations in IF genes cause numerous severe human diseases. In this work, three IF proteins are examined more closely; the nuclear lamin A/C and the cytoplasmic nestin and vimentin. In particular the regulation of lamin A/C dynamics, the role of nestin in muscle and body homeostasis as well as the functions and evolutionary aspects of vimentin are investigated. Together this data highlights some less well understood functions of these IFs. We used mass-spectrometry to identify inter-phase specific phosphorylation sites on lamin A. With the use of genetically engineered lamin A protein in combination with high resolution microscopy and biochemical methods we discovered novel roles for this phosphorylation in regulation of lamin dynamics. More specifically, our data suggests that the phosphorylation of certain amino acids in lamin A determines the localization and dynamics of the protein. In addition, we present results demonstrating that lamin A regulates Cdk5-activity. In the second study we use mice lacking nestin to gain more knowledge of this seldom studied protein. Our results show that nestin is essential for muscle regeneration; mice lacking nestin recover more slowly from muscle injury and show signs of spontaneous muscle regeneration, indicating that their muscles are more sensitive to stresses and injury. The absence of nestin also leads to decreased over-all muscle mass and slower body growth. Furthermore, nestin has a role in controlling testicle homeostasis as nestin-/- male mice show a greater variation in testicle size. The common fruit fly Drosophila melanogaster lacks cytoplasmic IFs as most insects do. By creating a fly that expresses human vimentin we establish a new research platform for vimentin studies, as well as provide a new tool for the studies of IF evolution.

The role of keratin intermediate filaments in the colon epithelial cells

Keratins (K) are cytoskeletal proteins mainly expressed in the epithelium and constitute the largest subgroup of intermediate filaments (IFs). Simple epithelial keratins (SEKs) K7-K8 and K18-K20 are the major IF elements in the colon. SEK mutations are known to cause around 30 human diseases, mainly affecting liver and skin. However, so far no strong associations between K8 mutations and the development of human colitis have been found. The keratin contribution to colonic health comes from the K8 knock-out (K8-/-) mouse model, which develops an early chronic inflammation and hyperproliferation in the colon. The aim of this thesis was to investigate how keratins contribute to intestinal health and disease mainly by the experimental analysis using the K8-/- mouse colon and cell culture models. The work described here is divided into three studies. The first study revealed involvement of keratins in Notch1 signaling, which is the master regulator of cell fate in the colon. Immunoprecipitation and immunostaining, both in vitro and in vivo showed that K8 binds and co-localizes with Notch1. Interestingly, overexpression of keratins enhanced Notch1 levels and stabilized Notch intracellular domain (NICD), leading to higher activity of Notch signaling. The dramatic decrease in Notch activity in the K8-/- colon resulted in a differentiation shift towards goblet and enteroendocrine cells. The second study focused on the involvement of keratins in colitis-associated cancer (CAC). Although, the K8-/- inflamed colon did not develop colorectal cancer (CRC) spontaneously, it was dramatically more susceptible to induced CRC in two CRC models: azoxymethane (AOM) and multiple intestinal neoplasia (ApcMin/+). To understand how the loss of K8 contributes to CAC, the epithelial inflammasome signaling pathway was analyzed. The released component of active inflammasome, cleaved caspase-1 and its downstream protein, interleukin (IL)-18, were significantly increased in K8-/- and K8-/-ApcMin/+ colons. The inflammasome pathway has recently been suggested to control the levels of IL-22 binding protein (IL-22BP), which is a negative regulator of IL-22 activity. Interestingly, the activated inflammasome correlated with an upregulation of IL-22 and a complete loss of IL-22BP in the K8-null colons. The activation of IL-22 was confirmed by increased levels of downstream signaling, which is phosphorylated signal transducer and activator of transcription 3 (P-STAT3), a transcription factor promoting proliferation and tissue regeneration in the colon. The objective of the third study, was to examine the role of keratins in colon energy metabolism. A proteomic analysis identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) as the major ownregulated protein in the K8-/- colonocytes. HMGCS2 is the rate-limiting enzyme in ketogenesis, where energy from bacterially produced short chain fatty acids (SCFAs), mainly butyrate, is converted into ketone bodies in colonic epithelium. Lower levels and activity of HMGCS2 in the K8-/- colon resulted in a blunted ketogenesis. The studies upstream from HMGCS2, identified decreased levels of the SCFA-transporter monocarboxylate transporter 1 (MCT1), which led to increased SCFA content in the stool suggesting impaired butyrate transport through the colonic epithelium. Taken together, the results of the herein thesis indicate that keratins are essential regulators of colon homeostasis, in particular epithelial differentiation, tumorigenesis and energy metabolism.