Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (Dym). The collapse of Dym along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Mobiilipelien kehitys ja julkaisu monialustakehitystyökaluja hyödyntäen

Tässä kandidaatintyössä esitellään mobiilimarkkoiden pirstaloitumisongelma, ja se millaisia haasteita kehittäjät voivat kokea kehittäessään sovelluksia, jotka tukevat useampia mobiilialustoja. Lisäksi työssä esitellään erilaisia mobiilisovelluksissa käytettäviä monetisaatiomalleja. Työssä tarkastellaan myös monialustakehitystyökalujen ja HTML5-tekniikan soveltuvuutta mobiilialustojen kehityshaasteiden ratkaisemiseen. Työn lopuksi toteutetaan ja julkaistaan käytännön pelisovellus monialustatyökalulla eri alustoja tukien. Tämän demosovelluksen avulla perehdytään julkaisuprosesseihin käytännössä, tuoden esille eri alustojen asettamia vaatimuksia kehittäjälle.

Histomorphometric analysis of the response of rat skeletal muscle to swimming, immobilization and rehabilitation

The objective of the present study was to determine to what extent, if any, swimming training applied before immobilization in a cast interferes with the rehabilitation process in rat muscles. Female Wistar rats, mean weight 260.52 ± 16.26 g, were divided into 4 groups of 6 rats each: control, 6 weeks under baseline conditions; trained, swimming training for 6 weeks; trained-immobilized, swimming training for 6 weeks and then immobilized for 1 week; trained-immobilized-rehabilitated, swimming training for 6 weeks, immobilized for 1 week and then remobilized with swimming for 2 weeks. The animals were then sacrificed and the soleus and tibialis anterior muscles were dissected, frozen in liquid nitrogen and processed histochemically (H&E and mATPase). Data were analyzed statistically by the mixed effects linear model (P < 0.05). Cytoarchitectural changes such as degenerative characteristics in the immobilized group and regenerative characteristics such as centralized nucleus, fiber size variation and cell fragmentation in the groups submitted to swimming were more significant in the soleus muscle. The diameters of the lesser soleus type 1 and type 2A fibers were significantly reduced in the trained-immobilized group compared to the trained group (P < 0.001). In the tibialis anterior, there was an increase in the number of type 2B fibers and a reduction in type 2A fibers when trained-immobilized rats were compared to trained rats (P < 0.001). In trained-immobilized-rehabilitated rats, there was a reduction in type 2B fibers and an increase in type 2A fibers compared to trained-immobilized rats (P < 0.009). We concluded that swimming training did not minimize the deleterious effects of immobilization on the muscles studied and that remobilization did not favor tissue re-adaptation.

Delta sleep instability in children with chronic arthritis

The objective of the present study was to evaluate the expression of a cyclic alternating pattern (CAP) in slow wave sleep (SWS) in children with the well-defined chronic syndrome juvenile idiopathic arthritis (JIA). Twelve patients (9-17 years of age), 7 girls, with JIA were compared to matched controls by age, pubertal stage and gender. After one night of habituation in the sleep laboratory, sleep measurements were obtained by standard polysomnography with conventional sleep scoring and additional CAP analyses. The sleep parameters of the JIA and control groups were similar for sleep efficiency (91.1 ± 6.7 vs 95.8 ± 4.0), sleep stage in minutes: stage 1 (16.8 ± 8.5 vs 17.8 ± 4.0), stage 2 (251.9 ± 41 vs 262.8 ± 38.1), stage 3 (17.0 ± 6.0 vs 15.1 ± 5.7), stage 4 (61.0 ± 21.7 vs 77.1 ± 20.4), and rapid eye movement sleep (82.0 ± 27.6 vs 99.0 ± 23.9), respectively. JIA patients presented nocturnal disrupted sleep, with an increase in short awakenings, but CAP analyses showed that sleep disruption was present even during SWS, showing an increase in the overall CAP rate (P < 0.01). Overall CAP rate during non-rapid eye movement sleep was significantly higher in pediatric patients who were in chronic pain. This is the first study of CAP in pediatric patients with chronic arthritis showing that CAP analyses can be a powerful tool for the investigation of disturbance of SWS in children, based on sleep EEG visual analysis.

GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia

White matter injury characterized by damage to myelin is an important process in hypoxic-ischemic brain damage (HIBD). Because the oligodendrocyte-specific isoform of neurofascin, neurofascin 155 (NF155), and its association with lipid rafts are essential for the establishment and stabilization of the paranodal junction, which is required for tight interaction between myelin and axons, we analyzed the effect of monosialotetrahexosyl ganglioside (GM1) on NF155 expression and its association with lipid rafts after HIBD in Sprague-Dawley rats, weighing 12-15 g, on day 7 post-partum (P7; N = 20 per group). HIBD was induced on P7 and the rats were divided into two groups: one group received an intraperitoneal injection of 50 mg/kg GM1 three times and the other group an injection of saline. There was also a group of 20 sham-operated rats. After sacrifice, the brains of the rats were removed on P30 and studied by immunochemistry, SDS-PAGE, Western blot analysis, and electron microscopy. Staining showed that the saline group had definite rarefaction and fragmentation of brain myelin sheaths, whereas the GM1 group had no obvious structural changes. The GM1 group had 1.9-2.9-fold more GM1 in lipid rafts than the saline group (fraction 3-6; all P < 0.05) and 0.5-2.4-fold higher expression of NF155 in lipid rafts (fraction 3-5; all P < 0.05). Injection of GM1 increased the content of GM1 in lipid rafts as well as NF155 expression and its lipid raft association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of NF155 (or other important proteins) with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage, suggesting a novel mechanism for its neuroprotective properties.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and ofXpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

A phthalide derivative isolated from endophytic fungiPestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniaeisolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation ofp73, JunB, FKHR, andBim. The results indicate that MP may be a potential treatment for cervical cancer.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13±0.05 V and 52.31±1.95 µs (control group, n=13) to 2.84±0.06 V and 59.71±2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63±7.49 to 79.14±5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Presence of central nervous system tissues as bovine spongiform encephalopathy specified risk material in Turkish raw meat ball (cig kofte)

Abstract Bovine Spongiform Encephalopathy (BSE) is a virulent disease which may infect by affecting the central nervous system (CNS) tissues in cattle and causes degeneration in nerves. Central nervous system tissues such as brain and spinal cord which are classified as specified risk materials (SRMs) are regarded to be main source of infection. The contamination of the meat with the specific risk materials (SRMs) can occur in phases of slaughter, fragmentation of carcass and processing. This study was conducted in order to investigate the existence of CNS tissues in raw meat ball (cig kofte) which is commonly consumed in the Southeastern Region of Turkey, particularly in Şanlıurfa. For this purpose, 145 samples of raw meat ball were tested. The enzyme-linked immunosorbent assay (ELISA) kits (Ridascreen risk material 10/5, R-biofarm GmbH) which determine glial fibrillary acidic protein (GFAP) as determinant were used. As a result of the analyses, positivity was detected in 21 of totally 145 samples of raw meat ball (14.48%). 6 (4.14%) of the samples gave low level of positivity (≥ 0.1 standard absorbance), 10 (6.90%) gave medium level of positivity (>0.2 standard absorbance) and 5 (3.45%) gave high level of positivity (≥0.5 standard absorbance). As a consequence, meats are contaminated in any phase of both slaughter and meat production even if accidentally. Regarding this matter, necessary measures should be taken and hygiene rules should be applied.

Business Ecosystem Change in Financial Services – Case Study

The thesis explores how the business ecosystem of financial services has changed and what its drivers of change are. Existing literature in the field of financial industry is concerned with financial innovations and their features, determinants and factors, but also with how to organize innovation activities such as open innovation principles. Thus, there is a clear need for understanding changes in financial service ecosystem. First, the comprehensive theory framework is conducted in order to serve the reader’s necessary understanding of basic theoretical concepts that are related to ecosystem changes. Second, the research is carried out by using qualitative research methods; the data is collected by interviewing 11 experts from the field of financial services in Finland. According to the results of this thesis, the most significant changes in the financial service ecosystem are the new market players. They have increased competition, created new courses of action, set new requirements for financial services, and first and foremost, they have shifted customers into the heart of the whole ecosystem. These new market players have a willingness to cooperate with external partners, which means a shift towards the world of open innovation. In addition, the economic environment has changed which has resulted in tighter regulation for incumbents making them even unyielding. Technology change, together with digitalization, has lead new financial innovations and new digital service channels, which have challenged the traditional business models in the financial industry. They have improved transparency, openness and efficiency, but also lead to the fragmentation of financial services. Thus, customers search for financial services from different sources and different service providers, and finally combine them into a coherent whole, which meets their own needs. The change of customers’ behavior and social environment has enabled and boosted these changes in the financial ecosystem. All in all, the change of the financial ecosystem is not a result of one or a few change forces, but instead it is a combination of many different factors.

Cleavages in contemporary Finland : a study on party-voter ties

Cleavages have been central in understanding the relationship between political parties and voters but the credibility of cleavage approach has been increasingly debated. This is because of decreasing party loyalty, fewer ideological differences between the parties and general social structural change amongst other factors. By definition, cleavages arise when social structural groups recognize their clashing interests, which are reflected in common values and attitudes, and vote for parties that are dedicated to defend the interests of the groups concerned. This study assesses relevance of cleavage approach in the Finnish context. The research problem in this study is “what kind of a cleavage structure exists in Finland at the beginning of the 21st century? Finland represents a case that has traditionally been characterized by a strong and diverse cleavage structure, notable ideological fragmentation in the electorate and an ideologically diverse party system. Nevertheless, the picture of the party-voter ties in Finland still remains incomplete with regard to a thorough analysis of cleavages. In addition, despite the vast amount of literature on cleavages in political science, studies that thoroughly analyze national cleavage structures by assessing the relationship between social structural position, values and attitudes and party choice have been rare. The research questions are approached by deploying statistical analyses, and using Finnish National Election Studies from 2003, 2007 and 2011as data. In this study, seven different social structural cleavage bases are analyzed: native language, type of residential area, occupational class, education, denomination, gender and age cohorts. Four different value/attitudinal dimensions were identified in this study: economic right and authority, regional and socioeconomic equality, sociocultural and European Union dimensions. This study shows that despite the weak overall effect of social structural positions on values and attitudes, a few rather strong connections between them were identified. The overall impact of social structural position and values and attitudes on party choice varies significantly between parties. Cleavages still exist in Finland and the cleavage structure partly reflects the old basis in the Finnish party system. The cleavage that is based on the type of residential area and reflected in regional and socioeconomic equality dimensions concerns primarily the voters of the Centre Party and the Coalition Party. The linguistic cleavage concerns mostly the voters of the Swedish People’s Party. The classic class cleavage reflected in the regional and socioeconomic equality dimension concerns in turn first and foremost the blue-collar voters of the Left Alliance and the Social Democratic Party, the agricultural entrepreneur voters of the Centre Party and higher professional and manager voters of the Coalition Party. The conflict with the most potential as a cleavage is the one based on social status (occupational class and education) and it is reflected in sociocultural and EU dimensions. It sets the voters of the True Finns against the voters of the Green League and the Coalition Party. The study underlines the challenges the old parties have met after the volatile election in 2011, which shook the cleavage structure. It also describes the complexity involved in the Finnish conflict structure and the multidimensionality in the electoral competition between the parties.

Customer Knowledge Transfer in MNCs

The purpose of this Master’s thesis was to study customer knowledge transfer processes in multinational corporations (MNCs). The main objective was to examine how customer knowledge is transferred in MNCs and what kind of factors enhance or inhibit the knowledge transfer process, and to create a framework on the basis of the existing literature and the empirical findings. In this thesis the factors were organized according to whether they are properties of the unit involved in knowledge management, properties of relationships between the units or properties of the knowledge itself. There are various properties that influence knowledge transfer but in this thesis the focus was on examining the relevant findings from the customer knowledge viewpoint. Empirical results show that internal fragmentation in the MNC seems to be inherent in this type of organization, and may cause many problems in customer knowledge transfer and utilization. These knowledge transfer inhibitors rise from the organization’s properties: it’s absorptive capacity, motivation, organizational culture, and the two dimensions of knowledge. However, in spite of the inherent forces causing internal fragmentation and inhibiting knowledge transfer, moderate customer knowledge and expertise codification, cooperative working practices among the experts, and socialization mechanisms posed by the headquarters seem to help maintain customer knowledge transfer, and value creation in the long-term relationship. This value creation can be seen to be based on accessing and integrating a wide variety of knowledge resources in order to create a coherent product and service offering.