In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gabg, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the b-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gbg and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function.

Consumers now more than ever are concerned by a teeming pool of social and cultural powers-Social Media Influencers (SMI)-that influence their purchasing decisions. Fashion as one of the most extensive industries in the world has spurred the rise of fashion SMIs on Instagram. As such , identifying that previous literature has studied female and male consumers jointly, this thesis thus, by employing a psycho-social methodological approach and a Consumer Culture theory perspective, takes a rather explorative modus operandi in trying to understand the perception of Swedish female consumer toward fashion SMIs while identifying the factor related to fashion SMIs that contour Swedish female consumers perceptions and facilitate purchasing decisions. Some of the findings are 1) fashion SMIs that are always presenting their perfect self are considered mischievous, 2) fashion SMIs consumers to discover their true selves, 3) fashion SMIs expertise is highly valued when seen outside a collaboration, 4) fashion SMIs trustworthiness is enhanced when they also present the flaws of their lives,5)consumers are rational buyers and regardless of the great influence of fashion SMIs, they finalize their purchasing decisions based on their needs, product attributes, and opinions received from friends and family.

Syfte: Studiens syfte var att kontrollera repeterbarhet och jämförbarhet hos tre kliniska instrument som används för att mäta hornhinnans tjocklek i friska ögon. Instrumenten som användes har flera olika användningsområden men fokus i denna studie ligger specifikt på pakymetri: mätning av hornhinnetjocklek. Metod: Det var 64 personer som deltog i studien. Alla var över 18 år och befann sig på Linnéuniversitetet i Kalmar vid tidpunkterna för mättillfällena. De tre optiska pakymetri instrumenten som användes var Topcon TRK-2P, Bon Sirius samt Essilor Wavefront Analyzer Medica 700+. Tre mätningar utfördes vid varje instrument på deltagarnas båda ögon. All data sammanställdes i Google Kalkyl och analyserades vidare i Graphpad Prism (version 9). Resultat: Repeterbarheten testades med ICC vilket var över 0,98 på samtliga instrument, CR var mellan 5,28-8,80 μm och CV var mellan 6,29-6,74%. Jämförbarheten visade att det finns en signifikant skillnad mellan de tre instrumenten (One-way RM- ANOVA,  p<0,0001). Slutsats: Samtliga instrument som användes inom studien visade på en utmärkt repeterbarhet vid mätning av friska ögon, däremot förekom signifikanta skillnader dem emellan. Detta innebär att man bör hålla sig till ett och samma instrument vid uppföljning av patienter. / Purpose: The purpose of this study was to examine the repeatability and comparability of three clinical instruments used to measure the corneal thickness in healthy eyes. The different instruments all have various fields of usage but the focus in this study is pachymetry: measurement of the corneal thickness. Method: In total, 64 people participated in this study. Everyone that participated were above the age of 18 and were located at Linneaus university in Kalmar, Sweden at the time the measurements took place. The three optical pachymetry instruments used were Topcon TRK-2P, Bon Sirius and Essilor Wavefront Analyzer Medica 700+. Three measurements were taken on each instrument on the participants’ both eyes. All collected data was collated in Google Sheets and analyzed in Graphpad Prism (version 9).  Results: The repeatability was tested with ICC that was above 0,98 on all the instruments. CR was between 5,28-8,80 μm and CV was between 6,29-6,74%. The comparability showed that there was a significant difference between the three instruments (One-way RM- ANOVA,  p<0,0001). Conclusion: All three instruments that were used during this study showed an excellent repeatability when measuring healthy eyes. However, there were significant differences between the instruments themselves. This means that you should stick to the same instrument while following up patients.

No description available.

Phosducin-like protein (PhLP) has been shown to interact with the cytosolic chaperonin containing TCP-1 (CCT), and the βγ subunit dimer of heterotrimeric G proteins (Gβγ). Here we provide details obtained from cryo-electron microscopic and biochemical studies on the structure of the complex between the cytosolic chaperonin CCT and PhLP. Binding of PhLP to CCT occurs through only one of the two chaperonin rings, making multiple contacts with CCT through both its N- and C-terminal domains. In addition, we show that PhLP acts as a co-chaperonin along with CCT in mediating the assembly of the G protein βγ subunit and that assembly is dependant upon the phosphorylation of PhLP by the protein kinase CK2. Variants of PhLP lacking the CK2 phosphorylation sites, or variants with an inability to bind Gβγ block the assembly process and inhibit G protein signaling. PhLP forms a complex with CCT and nascent Gβ prior to the release of Gβγ from the ternary complex and subsequent association with the Gγ subunit to form the Gβγ dimer. In order to understand the mechanism of Gβγ dimer assembly and the role of PhLP phosphorylation in the assembly process, we provide here a method for the purification of the PhLP·CCT·Gβ ternary complex of sufficient purity for structural studies.

A fundamental question in biology is how proteins, which are synthesized by the ribosome as a linear sequence of amino acids, fold into their native functional state. Many proteins require the assistance of molecular chaperones to maneuver through the folding process to protect them from aggregation and to help them reach their native state in the very concentrated protein environment of the cell. This study focuses on the roles of Phosducin-like Protein 1 (PhLP1) and Programmed Cell Death Protein 5 (PDCD5) as molecular co-chaperones of the Cytosolic Chaperonin Complex (CCT).Signaling in retinal photoreceptors is mediated by canonical G protein pathways. Previous in vitro studies have demonstrated that Gβ subunits rely on CCT and its co-chaperone PhLP1 to fold and assemble into Gβγ and RGS-Gβ5 heterodimers. The importance of PhLP1 in the assembly process was first demonstrated in vivo in a retinal rod photoreceptor-specific deletion of PhLP1. To test whether this mechanism applied to other cell types, we prepared a second mouse line that specifically disrupts the PhLP1 gene in cone photoreceptor cells and measured the effects on G-protein expression and cone visual signal transduction. In PhLP1 depleted cones, Gt2 and RGS9-Gβ5 levels were dramatically reduced, resulting a 60-fold decrease in cone sensitivity and a 50-fold increase in cone photoresponse recovery time. These results demonstrate a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, underlining the significance of PhLP1/CCT-mediated folding in G protein signaling.PDCD5 has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for PhLP1 binding partners revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5-CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 binds the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

All cells depend on properly folded proteins for survival and function. Misfolding of proteins results in loss of critical functions and may trigger the misfolding of other nearby proteins leading to toxic aggregation. While many proteins can fold on their own, others with complicated domain structures require assistance from protein folding machines called chaperones. The most complex and highly specialized of all chaperones is the eukaryotic chaperonin complex CCT which is necessary for the folding of a wide variety of essential proteins. These include the cytoskeletal proteins actin and tubulin as well as the Gβ subunit of the G protein heterotrimer. However, CCT activity can also drive diseases such as cancer and viral infections and could represent a high value therapeutic target if the mechanisms by which it folds its different client proteins were better understood. To this end, we identified and characterized an interaction between CCT and the RNA-dependent RNA polymerase of SARS-CoV-2, the virus responsible for the deadly global pandemic that began in 2019. We showed that SARS-CoV-2 replication is impaired by loss of CCT and that the polymerase, designated Nsp12, interacts with CCT upon synthesis and quickly releases - the hallmark pattern of a CCT substrate. Furthermore, we solved a 3.3 Å cryo-EM structure of Nsp12 bound to open CCT showing Nsp12 binding between the two rings of CCT and extending up through of the folding chambers and out of CCT. At 107 kD, Nsp12 is the largest substrate ever visualized inside of CCT and answers a long-standing question in the field of how CCT could accommodate substrates larger than its 70 kD folding chamber. Given that CCT is known to fold proteins with WD40-repeat domains, we also investigated a potential relationship between CCT and RPE65. RPE65, which contains a WD40 domain, is the retinyl ester isomerase that converts all-trans retinyl esters into 11-cis retinol, a key step in the visual cycle, and its mutation is a common cause of hereditary retinal dystrophies. We showed that CCT interacts with RPE65 and that nascent RPE65 binds and releases from CCT albeit with relatively slow kinetics. However, RPE65 is not dependent on CCT for expression or activity. This suggests that the relationship between RPE65 and CCT may represent a novel CCT function distinct from the canonical obligate substrate dynamic.

A Esclerose Múltipla (EM) é uma doença inflamatória autoimune caracterizada por desmielinização e degeneração do sistema nervoso central (SNC) duas a três vezes mais frequente em mulheres. A etiologia da EM é dividida em dois subtipos principais: o tipo remitente-recorrente (EMRR) mais comum, caracterizado por dois ou mais episódios de agravamento dos sintomas que envolvem diferentes locais do SNC, separados por pelo menos 1 mês e remisão dos sintomas e o tipo progressivo é a forma menos comum, caracterizada por um contínuo agravamento dos sintomas desde o início geralmente sem recaídas claras ou remissões. A presente pesquisa teve por objetivo avaliar o comprometimento da visão de cores e de sua relação com perdas de atenção, memória visual imediata, memória visual tardia, memória operacional e funções executivas dos portadores de esclerose múltipla tipo remitente recorrente. Método: participaram 41 pacientes com esclerose múltipla de idades entre 20 e 58 anos (35,4±12,2) e 37 controles com idade (34,7±12,1) e escolaridade semelhante. A discriminação de cores foi avaliada com o Cambridge Colour Test (CCT) e a avaliação de funções neuropsicológicas foram utilizados subtestes da bateria Cambridge Neuropsychological Testing Automated Battery (CANTAB) - Pattern Recognition Memory, Spatial Span, Spatial Recognition Memory, Information Sampling Task, Stop Signal Task e Rapid Information Processing; Escalas de Depressão e Ansiedade de Beck e Escala de Determinação Funcional da Qualidade de vida (DEFU). Resultados: A visão de cores está prejudicada de forma difusa em 24% dos pacientes com esclerose múltipla, tanto em pacientes com histórico de neurite óptica quando em pacientes que não tiveram neurite óptica. Na parte cognitiva, os pacientes demonstraram lentidão em executar as tarefas, comprometimento na atenção, na memória visual de curto e longo prazo, na memória operacional, na memória visuoespacial, além de lentidão no processamento de informações. Há uma relação entre alterações na visão de cores e alterações de memória visual somente em testes em que os estímulos são complexos e com inúmeros detalhes / Multiple sclerosis (MS) is an autoimmune inflammatory disease characterized by demyelination and central nervous system (CNS) degeneration. It is two times more frequent in women. It can be presented in two subtypes: the relapsing-remitting (RR), most common type, characterized by two or more episodes of focal disorders in different sites of the CNS, and remission of the symptoms; and the progressive subtype, less common, characterized by a continuous worsening of the symptoms, since the beginning, usually with no remissions. The present research evaluated color vision impairment and its relationship to attention loss, short-term memory, long-term memory, working memory and executive function in patients with the relapsing-remitting MS type. Methods: 41 patients with multiple sclerosis (20 to 58 years, 35.4 ± 12.2 in average) and 37 controls matched to age (34.7 ± 12.1 in average) and years of education participated of the study. Color vision was tested using the Cambridge Colour Test (CCT) and the neuropsychological assessment was performed using tests of the Cambridge Neuropsychological Testing Automated Battery (CANTAB) - Pattern Recognition Memory, Spatial Span, Spatial Recognition Memory, Information Sampling Task, Stop Signal Task, Rapid Information Processing the Beck Depression and Anxiety Inventories and the Functional Assessment of Multiple Sclerosis quality of life instrument. Results: Color vision was diffusely impaired in 24% of the MS patients, with or with no history of optic neuritis. Patients also showed a delay during the tasks execution, impairment in attention, short and long-term visual memory and working memory, and slowness in the information processing. There was a relationship between color vision loss and visual memory impairment, but only in tests with complex and highly detailed stimuli

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The joining of metals is often associated to metallurgical problems. Specifically, metallurgical transformations in the HAZ (Heat Affected Zone) can occur, what lead to undesirable microestructural changes. The HAZ is a difficult region for studying due to its small dimensions and high thermal gradientes. Thus, in order to overcome this limitation of the HAZ study, there are in the literature different proposes of HAZ-simulator machines (physical simulation), which uses the Joule effect for heating specific coupons that cool down by conduction and convection. This approach intends to simulate the thermal cycle in a real HAZ obtained in a real welding. However, the use of traditional coupons with cylindrical geometry presents restrictions of portraying the real case, i.e., the welding. In the cylindrical geometry option, the obtained thermal cycles do not present temperature gradients closer to the ones in real weldments. Hence, to overcome this limitation, finite elements modeling was carried out and different coupon geometries were simulated. The objective is to reach thermal cycles as close as possible to the ones obtained in a real situation, for a subsequent physical simulation. This approach showed proper and the physical and numerical present coherent results. The next step is the physical simulation validation by comparing to real weldments. This would be the most intuitive way. However, it was proposed to conduct this validation by determining CCT (Continuous Cooling Transformation) diagrams. This approach has the advantage of reaching important technological results at the same time of validating the physical simulation, since CCD diagrams dedicated to welding are very difficult to find in literature. It is possible to concluded that the physical simulation does represent the HAZ and can be used to build up CCT diagrams / Freqüentemente a união de materiais metálicos está associada a problemas metalúrgicos. Especificamente, podem ocorrer modificações metalúrgicas na ZAC (zona afetada pelo calor), levando a alterações microestruturais não adequadas. Contudo esta é uma região de difícil estudo devido a sua pequena dimensão e grandes gradientes térmicos. Assim, a fim de superar esta limitação com relação ao estudo da ZAC, existem na literatura proposições de máquinas de simulação física, que fazem uso do Efeito Joule para o aquecimento de determinados corpos de prova, que se resfriam por condução e convecção, de forma a simular o ciclo térmico obtido por soldagem real. Entretanto, o uso de corpos de provas cilíndricos apresenta restrições em retratar o caso real. Nesta opção de geometria cilíndrica, os ciclos térmicos não apresentam gradientes de temperatura que sejam fiéis àqueles encontrados em uma soldagem. Desta forma, foram realizadas simulações numéricas em elementos finitos, variando-se a geometria dos corpos de prova, de forma a obter ciclos térmicos o mais próximos da realidade para uma posterior simulação física. Esta abordagem de variação da geometria do corpo de prova calculada via simulação numérica mostrou-se adequada, onde os resultados obtidos pela simulação física e numérica mostraram-se coerentes. Como forma de validar os resultados finais obtidos via simulação física, ao invés de se conduzir soldagens reais, o que seria mais intuitivo, optou-se pelo levantamento de Diagramas CCT (Transformação em Resfriamento Contínuo) dedicados à soldagem. Este direcionamento tem a vantagem de se, concomitantemente à validação da simulação física, atingir resultados tecnologicamente muito importantes que são as curvas CCT de difícil acesso na literatura. Conclui-se pela viabilidade da simulação física em retratar a ZAC e pela viabilidade em se conseguir Diagramas CCT para soldagem. / Mestre em Engenharia Mecânica

A Esclerose Múltipla (EM) é uma doença inflamatória autoimune caracterizada por desmielinização e degeneração do sistema nervoso central (SNC) duas a três vezes mais frequente em mulheres. A etiologia da EM é dividida em dois subtipos principais: o tipo remitente-recorrente (EMRR) mais comum, caracterizado por dois ou mais episódios de agravamento dos sintomas que envolvem diferentes locais do SNC, separados por pelo menos 1 mês e remisão dos sintomas e o tipo progressivo é a forma menos comum, caracterizada por um contínuo agravamento dos sintomas desde o início geralmente sem recaídas claras ou remissões. A presente pesquisa teve por objetivo avaliar o comprometimento da visão de cores e de sua relação com perdas de atenção, memória visual imediata, memória visual tardia, memória operacional e funções executivas dos portadores de esclerose múltipla tipo remitente recorrente. Método: participaram 41 pacientes com esclerose múltipla de idades entre 20 e 58 anos (35,4±12,2) e 37 controles com idade (34,7±12,1) e escolaridade semelhante. A discriminação de cores foi avaliada com o Cambridge Colour Test (CCT) e a avaliação de funções neuropsicológicas foram utilizados subtestes da bateria Cambridge Neuropsychological Testing Automated Battery (CANTAB) - Pattern Recognition Memory, Spatial Span, Spatial Recognition Memory, Information Sampling Task, Stop Signal Task e Rapid Information Processing; Escalas de Depressão e Ansiedade de Beck e Escala de Determinação Funcional da Qualidade de vida (DEFU). Resultados: A visão de cores está prejudicada de forma difusa em 24% dos pacientes com esclerose múltipla, tanto em pacientes com histórico de neurite óptica quando em pacientes que não tiveram neurite óptica. Na parte cognitiva, os pacientes demonstraram lentidão em executar as tarefas, comprometimento na atenção, na memória visual de curto e longo prazo, na memória operacional, na memória visuoespacial, além de lentidão no processamento de informações. Há uma relação entre alterações na visão de cores e alterações de memória visual somente em testes em que os estímulos são complexos e com inúmeros detalhes / Multiple sclerosis (MS) is an autoimmune inflammatory disease characterized by demyelination and central nervous system (CNS) degeneration. It is two times more frequent in women. It can be presented in two subtypes: the relapsing-remitting (RR), most common type, characterized by two or more episodes of focal disorders in different sites of the CNS, and remission of the symptoms; and the progressive subtype, less common, characterized by a continuous worsening of the symptoms, since the beginning, usually with no remissions. The present research evaluated color vision impairment and its relationship to attention loss, short-term memory, long-term memory, working memory and executive function in patients with the relapsing-remitting MS type. Methods: 41 patients with multiple sclerosis (20 to 58 years, 35.4 ± 12.2 in average) and 37 controls matched to age (34.7 ± 12.1 in average) and years of education participated of the study. Color vision was tested using the Cambridge Colour Test (CCT) and the neuropsychological assessment was performed using tests of the Cambridge Neuropsychological Testing Automated Battery (CANTAB) - Pattern Recognition Memory, Spatial Span, Spatial Recognition Memory, Information Sampling Task, Stop Signal Task, Rapid Information Processing the Beck Depression and Anxiety Inventories and the Functional Assessment of Multiple Sclerosis quality of life instrument. Results: Color vision was diffusely impaired in 24% of the MS patients, with or with no history of optic neuritis. Patients also showed a delay during the tasks execution, impairment in attention, short and long-term visual memory and working memory, and slowness in the information processing. There was a relationship between color vision loss and visual memory impairment, but only in tests with complex and highly detailed stimuli