Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

The focus of this study has been to plumb social workers experiences regarding their work methods in two institutions regarding unaccompanied children through a perspective of gender. In this study, four semi-structured qualitative interviews has been contrived in purpose to gather in material which has been presented in five themes to ease an analyse focusing of gender. In all the interviews both authors participated with one in charge and one taking notes. One of the main focuses has been the relations between both social workers and their clients and between colleges in the institutions. The other main focus has been the activities, both planed and spontaneous, which the clients been offered, for example football, bowling and boxing. The idea of analyse this main focuses is based in our knowledge through recent science studies that shows that gender is created both in relations, language and everyday actions.

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.

No description available.

Parenting plans are a new concept for professionals in South Africa working in the field of divorce. Emphasis has been placed on the development of parenting plans by including the concept of parenting plans in section 33 of the Children’s Act 38 of 2005. Professionals are now confronted with formulating adequate parenting plans which are focussed on the best interest standard of the child(ren) and that will assist the family with its functioning post–divorce. The aim of this research was to develop substantive guidelines that can assist the professional in drafting parenting plans. To achieve this aim, the following objectives guided the study: * To provide the legal context of parenting plans in South Africa. A legal context of parenting plans in South Africa is discussed by way of a literature study through an intensive analysis and critical discussion on a number of legal instruments pertaining to children, such as the Children’s Act 38 of 2005; the Constitution of the Republic of South Africa, 1996; the United Nations Convention on the Rights of the Child (UNCRC) and the African Charter on the Rights and Welfare of the Child (ACRWC). * To ascertain the views of mental health professionals (social workers and psychologists) and legal professionals (attorneys and family advocates) with regard to the divorcing family and parenting plans. The views of mental health professionals and legal professionals with regard to the divorcing family in respect of parenting plans were established. Professionals completed an electronic questionnaire that consisted of open questions to establish their views. Further telephonic interviews with the professionals were facilitated to gain further insight into the professional view points on the issue of divorcing families and parenting plans. * To ascertain the needs of the divorcing family (parents and child(ren)) and to present these needs in the structuring of a parenting plan. The needs of the divorcing family (parents and child(ren)) were established through an explorative approach by means of a document study and electronic questionnaires that were completed by the family members. Through this exploration the researcher obtained an understanding of the needs of the divorcing family related to parenting plans. * To provide professionals with comprehensive and clear guidelines on the basic structure and general content of a parenting plan. Comprehensive and clear guidelines on the basic structure and general content of a parenting plan were constructed for professionals working in this area, based on findings of articles 1, 2 and 3 of this research. The researcher also relied on her background and training as a qualified social worker to construct these guidelines. It was concluded that professionals are not necessarily well equipped to structure high quality parenting plans. Guidelines ought to assist the professionals to structure plans that are well organised, practical, child–centred, developmentally appropriate, and representative of the divorcing family needs. The researcher believes that as parenting plans are obligatory for parents that divorce, there is a need for South African research on this subject matter. This study is a contribution in the research field as to assist the professionals with the task of compiling parenting plans for the divorcing family. In addition this study adds value to the forensic field by setting guidelines on the structuring of parenting plans that empower the professional to work in this challenging field. / Thesis (Ph.D. (Social Work))--North-West University, Potchefstroom Campus, 2011.

Cardiac function is regulated by the molecular components of the sarco/endoplasmic reticulum (ER/SR). Disruptions in homeostatic balance of these proteins and calcium regulation results in activation of ER stress response. Sarcolemmal membrane-associated proteins (SLMAPs) are found in cell membrane, SR/ER, and mitochondria. Overexpression of SLMAP in the myocardium has shown to impair excitation-contraction (E-C) coupling in the transgenic (Tg) mice. ER stress response was examined in Tg mice overexpressing SLMAP in the myocardium. In Tg hearts, changes observed in the expression of proteins involved in ER stress were dependent on the age and sex. SLMAP overexpression results in maladaptive ER stress response, as the mice age. Neonatal cardiomyocytes isolated from the Tg hearts showed decreased viability, upregulation of ER stress response proteins, which were sensitized to thapsigargin-induced stress, and desensitized to palmitate-induced oxidative stress. These findings suggest that normal SLMAP levels are important for proper cardiac function, and cell viability.

Parenting plans are a new concept for professionals in South Africa working in the field of divorce. Emphasis has been placed on the development of parenting plans by including the concept of parenting plans in section 33 of the Children’s Act 38 of 2005. Professionals are now confronted with formulating adequate parenting plans which are focussed on the best interest standard of the child(ren) and that will assist the family with its functioning post–divorce. The aim of this research was to develop substantive guidelines that can assist the professional in drafting parenting plans. To achieve this aim, the following objectives guided the study: * To provide the legal context of parenting plans in South Africa. A legal context of parenting plans in South Africa is discussed by way of a literature study through an intensive analysis and critical discussion on a number of legal instruments pertaining to children, such as the Children’s Act 38 of 2005; the Constitution of the Republic of South Africa, 1996; the United Nations Convention on the Rights of the Child (UNCRC) and the African Charter on the Rights and Welfare of the Child (ACRWC). * To ascertain the views of mental health professionals (social workers and psychologists) and legal professionals (attorneys and family advocates) with regard to the divorcing family and parenting plans. The views of mental health professionals and legal professionals with regard to the divorcing family in respect of parenting plans were established. Professionals completed an electronic questionnaire that consisted of open questions to establish their views. Further telephonic interviews with the professionals were facilitated to gain further insight into the professional view points on the issue of divorcing families and parenting plans. * To ascertain the needs of the divorcing family (parents and child(ren)) and to present these needs in the structuring of a parenting plan. The needs of the divorcing family (parents and child(ren)) were established through an explorative approach by means of a document study and electronic questionnaires that were completed by the family members. Through this exploration the researcher obtained an understanding of the needs of the divorcing family related to parenting plans. * To provide professionals with comprehensive and clear guidelines on the basic structure and general content of a parenting plan. Comprehensive and clear guidelines on the basic structure and general content of a parenting plan were constructed for professionals working in this area, based on findings of articles 1, 2 and 3 of this research. The researcher also relied on her background and training as a qualified social worker to construct these guidelines. It was concluded that professionals are not necessarily well equipped to structure high quality parenting plans. Guidelines ought to assist the professionals to structure plans that are well organised, practical, child–centred, developmentally appropriate, and representative of the divorcing family needs. The researcher believes that as parenting plans are obligatory for parents that divorce, there is a need for South African research on this subject matter. This study is a contribution in the research field as to assist the professionals with the task of compiling parenting plans for the divorcing family. In addition this study adds value to the forensic field by setting guidelines on the structuring of parenting plans that empower the professional to work in this challenging field. / Thesis (Ph.D. (Social Work))--North-West University, Potchefstroom Campus, 2011.

Com o objetivo de testar a qualidade das restaurações realizadas após a remoção de cárie de raiz com laser de alta potência, realizamos esse trabalho. Lesões de cárie radicular foram induzidas em fragmentos dentais humanos (n=154) através de contaminação com o S. mutans. Obtidas as lesões, essas foram removidas com instrumento cortante rotatório (ICR) em baixa rotação (grupo controle) ou com o laser de Er,Cr:YSGG, (?=2,78 µm, taxa de repetição= 20 Hz, duração de pulso na ordem de 140 a 150 µs, energia por pulso de 0 a 300 mJ) em 13 potências diferentes entre 1 e 4 W, em intervalos de 0,25 W entre cada grupo (n=11 por grupo). Para a remoção de cárie, foi utilizada a peça de mão 2415 e a fibra de safira modelo ?G? (Ø= 600 µm) posicionada a 1 mm das amostras (modo não contato). Após a remoção das lesões de cárie e mensuração do tempo necessário para este passo operatório, uma amostra de cada grupo foi preparada para microscopia eletrônica de varredura (MEV). Em seguida, as demais foram condicionadas com Clearfil SE Bond, restauradas com resina composta de baixa viscosidade e submetidas à termociclagem (1.000 ciclos de 5°C e 55°C) e então imersas em solução de azul de metileno a 2 % para teste de microinfiltração. Depois desta etapa, as amostras foram seccionadas e analisadas em microscopia luz para a obtenção do índice de microinfiltração, assim como de outras características, como a área da interface dente/restauração, a profundidade das cavidades, e a presença de cárie residual sob as restaurações. Os dados foram comparados estatisticamente (p?0,05). Os resultados indicaram que o ICR removeu as lesões de cárie mais rapidamente que o laser (p<0,05). As eletromicrografias de varredura mostraram que o grupo controle apresentou as margens cavitárias sem irregularidades e a superfície dentinária coberta pela camada de esfregaço, obliterando os túbulos dentinários. Todas as cavidades preparadas com o laser apresentaram margens irregulares. Quando parâmetros inferiores a 2,0 W foram utilizados, observou-se a presença de material amorfo sobre a superfície dentinária, com pouca exposição dos túbulos dentinários. Já nos parâmetros acima de 2,0 W, as cavidades apresentaram superfícies com aspecto de escamas, ausência da camada de esfregaço, túbulos dentinários abertos e crateras, distribuídas de forma irregular. Quando analisadas em microscopia de luz, as amostras irradiadas com potências superiores a 3,0 W apresentaram cavidades mais profundas em relação às demais. Graças às irregularidades promovidas pelo laser, as amostras irradiadas com potências maiores que 1,5 W apresentaram maior área de interface dente/restauração do que aquelas do grupo controle ou irradiadas com os parâmetros mais baixos. O índice de infiltração foi semelhante entre todos os grupos irradiados, e significantemente maiores que o do grupo controle. Houve correlação fortemente positiva entre a quantidade de cárie residual presente nas amostras e os índices de microinfiltração. Nas condições deste estudo pôde-se concluir que a remoção de cárie radicular com laser de Er,Cr:YSGG seguida de restauração com resina composta mostrou pobre selamento marginal em função da presença de cárie residual nas paredes das cavidades. / The present study aimed to compare the sealing ability of root caries restoration after caries removal with bur or Er,Cr:YSGG laser. Seventy two extracted human carious-free molars and premolars were used. After the in vitro root caries induction using S. mutans, the carious lesions were removed either by the conventional technique using burs (control), or by the Er,Cr:YSGG laser ((?=2,78 µm, repetition rate= 20 Hz, pulse duration around 150 µs, energy per pulse of 0 to 300 mJ, non-contact mode), using 13 different parameters, between 1 and 4,0 W. For the irradiation procedures, a 600 µm tip was used. During caries removal, preparation time was recorded for all groups. After that, one sample of each group was observed in scanning electron microscopy (MEV), and the others were conditioned with Clearfil SE Bond and restored with a flowable composite. Then, the samples were termocycled (1000 Cycles) and immersed into a 2 % methylene blue solution for microlekage analysis. These samples were sectioned and analyzed under light microscopy for microleakage index calculation. Other characteristics, such as the dentin/composite interface area, the cavity depth and the presence of residual caries under the restorations, were also evaluated. The data were statistically analyzed by either ANOVA complemented by the Tukey?s test or Kruskall-Wallis complemented by the Dunn?s test (p = 0.05). The caries lesions were significantly faster removed by the conventional method (p<0.05). Amongst the lased groups, the caries removal time was inversely proportional to the parameters used. SEM revealed that the surfaces treated by air turbine were smooth, but with debris. When parameters under 2.0 W were used, an amorphous material covering the dentin surface was observed. Using parameters above of 2.0 W, SEM revealed surfaces with smooth undulations, with absence of smear layer, and some irregular-distributed craters. No signs of carbonization were observed. The light microscopy analysis showed that the samples irradiated with power outputs over 3.0 W were deeper than those irradiated with lower parameters and those prepared with bur. The samples irradiated with power outputs higher than 1.5 W presented dentin/composite interface area larger than those of the control group. Finally, the infiltration index was similar in all lased groups, and significantly higher than those of control group (p<0.05). The microleakage index and the presence of residual caries, only detected by light microscopy, at the lateral walls of the cavities obtained by Er, Cr:YSGG laser was significantly higher compared to control cavities. In the conditions of this study, we can conclude that the carious removal by Er, Cr:YSGG laser, independently of the parameters used, creates dentin surfaces characteristics that promotes a poor marginal sealing.

Objetivos: avaliar o efeito de 3 parâmetros de condicionamento da dentina utilizando o laser de Er,Cr;YSGG (2,78m) na resistência de união (RU) de cimento de ionômero de vidro convencional (CIV). Materiais e métodos: Foram utilizados neste estudo 68 terceiros molares humanos, divididos aleatoriamente em 4 grupos (n=17/ grupo): G1 (grupo controle): nenhum tipo de condicionamento; G2: condicionamento com laser de Er,Cr;YSGG 0,5W, 25mJ, 20Hz ou G3: 1,0W, 50mJ, 20Hz e G4: 1,5W, 75mJ, 20Hz. Dois dentes em cada um dos grupos foram observados em microscopia eletrônica de varredura (MEV) para identificar as características morfológicas dos diferentes condicionamentos de superfície realizados. Os outros 15 dentes/grupo, foram preparados para ensaio de RU, obtendo uma superfície plana de dentina onde foram construídos 3 (n=45/grupo) corpos de prova em forma de cilindro (1mm de diâmetro X 0,5mm de altura) utilizando o ionômero de vidro Ketac Molar Easymix- 3M ESPE. Após o armazenamento em água destilada por 24h a 37oC os espécimes foram ensaiados pelo teste de microcisalhamento. Os valores finais de resistência adesiva foram calculados e expressos em Mega Pascal (MPa). Os dados foram coletados e analisados por ANOVA (p<0,001). Resultados: Os espécimes do G2 apresentaram os maiores resultados de RU (10,50MPa ±0,84), seguido pelo G1 (4.77MPa ±0.59), G3 (3.32MPa ±0.39) e G4 (2,94MPa ±0,50) que não apresentaram diferença estatisticamente significante quanto comparados entre si. A análise em MEV do G1 mostrou camada de smear ao longo de toda a superfície de dentina, enquanto nos outros grupos observou-se túbulos dentinários abertos, característicos na irradiação com laser de Er,Cr:YSGG. Os pulsos do laser no G2 eram facilmente distinguidos, o que não acontecia nos grupos G3 e G4, sugerindo que parâmetros de energia maiores podem causar efeitos mais extensos à estrutura da dentina. Na análise do padrão de fratura, todos os grupos apresentaram maior porcentagem de fratura adesiva G1:73,3%, G2:48,9% G3:66,7% e G4:64,4%. O tipo de fratura mista foi proporcional ao aumento da RU (G1:24,4%, G2:46,7%, G3:20,0% e G4:17,8%). Conclusão: O condicionamento da dentina com o laser de Er,Cr:YSGG na potência de 0,5W aumentou significativamente a RU do CIV à dentina, enquanto os demais parâmetros testados reduziram a RU. / Objective: To evaluate the effect of 3 dentin conditioning parameters of Er,Cr:YSGG laser (2,78 m) on the bond strength (BS) of conventional glass ionomer cement (CGIC). Methods: A total of 68 third molars were divided into four groups and was subjected to one of the following pretreatments: G1- the control group, no pretreatment was realized; G2, G3 and G4 were pretreated with Er,Cr:YSGG laser irradiation at 0.5 W, 25 mJ, 9 J/cm2, and 20 Hz (G2); 1.0 W, 50 mJ, 18 J/cm2, and 20 Hz (G3); and at 1.5 W, 75 mJ, 27 J/cm2, and 20 Hz (G4). A scanning electron microscopy (SEM) evaluation (n=2) of pretreated dentine and a microshear bond strength test (n=15) were performed after 24h of water immersion. To build the specimens (each of which was 1 mm in diameter) for the microshear test, CGIC (Ketac Molar Easy Mix) was used. The data were analysed by one-way ANOVA (p<0.001). Results: The G2 specimens presented the highest BS results (10.50 MPa ± 0.84), and G1 had the next highest average BS values (4.77 MPa ± 0.59). G3 (3.32 MPa ± 0.39) and G4 (2.94 MPa ± 0.50) demonstrated the lowest BS values, although the BS difference between these groups was not statistically significant (p>0.001). The SEM analysis of G1 revealed that the smear layer covered the entire dentine surface, whereas in groups G2, G3, and G4, irregular dentine was detected that possessed the open dentinal tubules and protruded peritubular dentine that are the characteristic results of laser irradiation. Laser pulses could easily be distinguished in G2 but not in G3 and G4, suggesting that higher energy parameters can broadly disrupt dentine structure and thereby affect the bonding of dentine to GIC. Conclusion: Dentine pretreatment with an Er,Cr:YSGG laser at a power of 0.5 W increased the BS of conventional GIC.

Cardiac function is regulated by the molecular components of the sarco/endoplasmic reticulum (ER/SR). Disruptions in homeostatic balance of these proteins and calcium regulation results in activation of ER stress response. Sarcolemmal membrane-associated proteins (SLMAPs) are found in cell membrane, SR/ER, and mitochondria. Overexpression of SLMAP in the myocardium has shown to impair excitation-contraction (E-C) coupling in the transgenic (Tg) mice. ER stress response was examined in Tg mice overexpressing SLMAP in the myocardium. In Tg hearts, changes observed in the expression of proteins involved in ER stress were dependent on the age and sex. SLMAP overexpression results in maladaptive ER stress response, as the mice age. Neonatal cardiomyocytes isolated from the Tg hearts showed decreased viability, upregulation of ER stress response proteins, which were sensitized to thapsigargin-induced stress, and desensitized to palmitate-induced oxidative stress. These findings suggest that normal SLMAP levels are important for proper cardiac function, and cell viability.