1,721,596 research outputs found
cGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-dependent manner
No full textChromosomal instability (CIN) in cancer cells has been reported to activate the cGAS-STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, converse effects of the cGAS/STING pathway per se on CIN have not yet been investigated. I addressed this issue using knockdown and add-back of each component of the cGAS/STING/TBK1/IRF3 pathway, and monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in an increased micronucleus phenotype and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK binding kinase-1), or IRF3 (interferon regulatory factor-3) also increased micronuclei formation. Moreover, transfection of cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1- and IRF3-knockdown cells in association with precocious G2/M transition and an enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated precocious G2/M transition, indicating that the decrease in p21 and subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN by defective cGAS/STING signaling.I. INTRODUCTION 1
II. MATERIALS AND METHODS 14
A. Antibodies 14
B. Cell culture 14
C. Synchronization and drug treatment 15
D. Plasmids and transfection experiments 16
E. Knock-down experiments 16
F. Immunoblotting 17
G. Immunocytochemistry 18
H. Time-lapse analysis 18
I. Mitotic index 19
J. Statistical analysis 19
III. RESULTS 20
A. Investigating cGAS/STING pathway to choose best suited cancer cell-line for my experimental setting 20
B. cGAS add-back can rescue cells from cGAS-depletion induced CIN 22
C. cGAS down-regulation augments chromosomal missegregations 26
D. Micronuclei formation is regulated by enzymatic activity of cGAS 29
E. STING plays mediating role to maintain chromosomal stability 32
F. Depletion of TBK1 or IRF3 also enhanced CIN phenotypes 35
G. p21 expression levels are dependent on cGAS/STING pathway 37
H. p21 as a principal regulator in maintaining chromosomal integrity via cGAS/STING pathway 42
IV. DISCUSSION 47
V. CONCLUSION 52
REFERENCES 53Maste
A Novel DNA-PK/PLK1/STING/IRF3 Axis Regulates Normal Mitotic Progression
No full textDoctor1. INTRODUCTION 1
2. MATERIALS AND METHODS 10
2.1. Cell culture 10
2.2. Antibodies 10
2.3. Synchronization and drug treatment 11
2.4. Plasmids and transfection experiments 12
2.5. Knock-down experiments 12
2.6. Immunoblotting 14
2.7. cGAMP ELISA 15
2.8. Native-PAGE 15
2.9. Time-lapse analysis 16
2.10. 2D Blue Native-PAGE 16
2.11. Immunocytochemistry 17
2.12. Immunoprecipitation 17
2.13. In-vitro kinase assay 18
2.14. Quantitative RT-PCR 19
2.15. Mitotic index 19
2.16. Statistical analysis 20
3. RESULTS 21
3.1. IRF3 is phosphorylated during mitosis in a cGAS-independent but STING-dependent manner 21
3.2. Phosphorylation of IRF3 is required for normal mitotic progression 28
3.3. STING and IRF3 dimerization is a pre-requisite during mitosis 35
3.4. DNA-PKcs activity is required for STING and IRF3 phosphorylation during mitosis 39
3.5. PLK1 is the major mitotic kinase responsible for IRF3 phosphorylation during mitosis 47
3.6. STING, IRF3, DNA-PK and PLK1 form a complex during mitosis where STING acts as a mediator 53
3.7. Recruitment of PLK1 and IRF3 to the DNA-PK/STING complex requires DNA-PK activity 62
3.8. Ku70 and Ku80 may play different roles in the activation of STING/IRF3 axis in a cell-dependent manner 66
4. DISCUSSION 71
5. REFERENCES 7
Gender Classification Using Smartphone Sensors and Machine Learning Approaches
No full textGait analysis is typically associated with the pattern of the human walk. Determining it with computational means can be helpful in many ways-from identifying individual humans to detecting gait-related diseases. In comparison to the expensive approaches and devices, which are limited to laboratories, smart- phones with motion sensors are low-cost solutions through which we can analyze mobility and gait patterns. Thus, in this work, we present the usage of smartphone sensors for data acquisition followed by machine learning-based gender classification, which is a baseline for different gait-related tasks. In this regard, we collected data from 14 persons in different tracks, paces, and movement styles; after adequate normalization, iterative feature elimination, and Monte-Carlo experiment-based ML training, we found the Decision Tree is the most optimal algorithm with attaining 90.6 % balanced-accuracy
Synthesis of diclofenac-cyclodextrin conjugates for colon delivery
No full textTese de doutoramento em Farmácia (Pré-Bolonha), Especialidade de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraAs doenças inflamatórias, nomeadamente a artrite, são doenças que afectam a população po
todo o mundo. A ausência de dor é considerada um factor determinante para a qualidade de
vida. Os anti-inflamatórios, esteróides ou não esteróides são a classe de fármacos usada no
controlo deste tipo de doenças. No entanto, estes estão associados a diversos efeitos adversos
e a variabilidade inter-individual também afecta em termos eficácia. Diferentes estratégias
foram exploradas com o objectivo de aumentar a eficácia dos anti-inflamatórios e, ao mesmo
tempo, diminuir os seus efeitos adversos. Entre as várias estratégias, destaca-se a que envolve
a libertação específica destes fármacos ao nível do cólon. Para este efeito, o desenvolvimento
de profármacos constitui uma solução promissora. As ciclodextrinas podem ser usadas como
transportadoras para o design de profármacos, denominados conjugados, para libertação
específica no cólon, estabelecendo uma ligação covalente ao respectivo fármaco. Esta tese
resulta de um trabalho desenvolvido com base neste conceito tendo como objectivo sintetisar
um profármaco do diclofenac para libertação específica no colon, usando ciclodextrinas como
transportadores.
O primeiro desafio consistiu em explorar diferentes estratégias para a síntese do conjugado
entre o diclofenac e a β-ciclodextrina. Das diversas tentativas, apenas a substituição
nucleofílica do 6-mono-tosil-β-cyclodextrina sob radiação de microondas, permitiu a síntese
com sucesso. A aplicação da mesma estratégia para a síntese de outros conjugados,
nomeadamente com γ- e α ciclodextrina foi explorada, contudo, inúmeras dificuldades foram
encontradas. Deste modo, a nossa investigação focou-se no estudo do conjugado diclofenac-
β-cyclodextrina. Este novo conjugado foi caracterizado por diversas técnicas:
Ionização/Dessorção de Matriz Assistida por Laser (MALDI), espectroscopia de infravermelho
(IV), ressonância magnética nuclear de protão (1H-RMN) e espectroscopia
bidimensional do efeito nuclear de Overhauser (ROESY). A sua pureza foi avaliada por
cromatografia de alta pressão (HPLC) tendo-se posteriormente realizado ensaios de
estabilidade química.
Efectuaram-se estudos in vitro e ex vivo no sentido de investigar a estabilidade do diclofenac-
β-cyclodextrina ao longo do tracto gastrointestinal. Foram realizados ensaios num sistema de
fermentação com fezes humanas, de forma a descobrir a capacidade do conjugado libertar o
diclofenac no intestino grosso. Estes estudos de estabilidade foram executados em
comparação com um profármaco conhecido, a sulfasalazina. De forma a encontrar um sistema
viii
enzimático que mimetize o metabolismo deste conjugado ao nível do cólon, realizaram-se
estudos com enzimas capazes de degradar açúcares e enzimas com capacidade de hidrolisar
ligações ester. Posteriormente, foi estudada a estabilidade ao nível do tracto gastrointestinal
superior, utilizando simulados gástricos e intestinais descritos na Farmacopeia. O conjugado
permitiu a libertação do diclofenac ao nível do intestino grosso e exibiu uma boa estabilidade
no tracto gastrointestinal superior. Consequentemente, foram realizados estudos ex vivo em
fluídos recolhidos de diferentes espécies de animais (porco, coelho e rato), de forma a
identificar o animal adequado para a realização dos estudos in vivo. Os resultados obtidos no
rato foram os que mais se assemelharam ao comportamento do conjugado obtido no sistema
de fezes humanas. Por este motivo, o rato foi o animal escolhido para realizar os estudos
subsequentes.
Com o objectivo de escolher o regime alimentar mais adequado para concretizar os estudos de
biodisponibilidade, foi investigada a influência do regime de alimentação nas condições
fisiológicas do tracto gastrointestinal e na degradação do conjugado versus sulfasalazina no
intestino grosso. Os resultados mostraram que o regime de alimentação influencia os
conteúdos (massa e pH), mais especificamente a nível do estomago e do intestino grosso, e
também afecta a velocidade de metabolismo do diclofenac-β-cyclodextrina, mas não da
sulfasalazina. O jejum permite uma libertação mais rápida do conjugado, possivelmente
devido a falta de competição (ausência de alimento) para as enzimas microbianas.
Como prova do conceito, realizaram-se estudos in vivo em ratos em jejum, através da
administração oral de uma suspensão de conjugado com sulfasalazina a um grupo de animais.
Em paralelo, foi efectuada a administração de diclofenac de sódio e sulfapiridina a um grupo
controlo. Observou-se um atraso entre a administração oral dos profármaco e o aparecimento
do respectivo fármaco, contrariamente ao que se verificou com a administração dos fármacos
livres, cujo aparecimento no plasma foi imediato. Estes resultados confirmaram a capacidade
do conjugado em libertar o diclofenac especificamente no cólon. Uma redução de 50% na
biodisponibilidade do diclofenac ocorre aquando da administração do conjugado
comparativamente à administração do fármaco livre.
Poderemos concluir que o diclofenac-β-cyclodextrina constitui um profármaco promissor a
ser explorado no combate a doenças inflamatórias, tais como a artrite, ultrapassando
simultaneamente os efeitos adversos associados ao diclofenac
Towards the prediction of the effect of food on orally administered medicines using preclinical in vivo models and machine learning technologies
Full text linkThe intake of food and drinks with orally administered medicines can significantly impact the therapeutic efficacy or adverse side effects of a drug, posing barriers to effective therapeutic treatment in patient populations. There are unmet pharmaceutical and clinical needs to improve the prediction of the food effect in drug product development. This research has focused on in vivo and in silico tools that can be used in early drug development to predict the food effect. The overall aims of this research were to: explore the food-mediated changes to intestinal efflux transporter expression in rodent animal models, and leverage machine learning tools to predict the food effect.
Our understanding of the effects of the fed state on clinically relevant transporters in preclinical rodent animal models has been enhanced. P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) expression were altered to different extents between the prandial states, sexes, and strains. A non-nutritive fibre meal increased the acute expression of intestinal P-gp, BCRP, and MRP2. Significant changes were seen in male rats, when comparing the fibre meal and the standard housing meal, but not in female rats.
The repertoire of computational tools to predict the food effect was expanded. Here, classification and regression machine learning technologies were tested to predict the food effect on large datasets of >300 drugs using key drug physicochemical properties.
In summary, this work has uncovered that the rodent animal model shows food, sex, and strain differences for the expression of key intestinal efflux transporters. Furthermore, machine learning technologies were harnessed to predict the food effect from the drug structure. While more work is needed to further understand the mechanisms of the food effect and to build more accurate machine learning tools, these findings offer insights to guide early drug development
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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