Fabricação de estruturas bio-hibridas contendo ácido fólico para a absorção controlada em células cancerígenas

Mestrado em Ciência e Engenharia de MateriaisIn this work, biohybrid structures (BHS) based on streptavidin and pentavalent biotinylated dendritic glycopolymer were fabricated through non-covalent streptavidin-biotin conjugation and further functionalized with the folic acid for the selective targeting to tumor cells. Characterization of the hydrodynamic size and zeta potential of the structures together with the cytotoxicity studies were performed to assess the physical properties and biocompatibility of the structures. It was shown that stable BHS with defined sizes can be obtained due to a controlled polyassociation reaction. Furthermore, BHS are biocompatible and do not cause toxicity to the cells under the given experimental conditions. Next, the effect of the folic acid ligand on the targeting function towards folate receptor expressing cancer cells was studied in flow cytometry experiments. For this purpose the amount of folic acid, the length of the PEG-chain / spacer, and the type of protein used were evaluated, as well as the impact of purification strategies. Additionally, the cell uptake mechanism was also briefly looked into.Neste trabalho foram fabricadas estruturas bio-híbridas (BHS) à base de glicopolímeros dendríticos biotinilados e estreptavidina as quais foram posteriormente funcionalizadas com o ácido fólico com vista ao reconhecimento preferencial por células tumorais. A estratégia de fabricação recorreu, em ambos os casos, a interações não-covalentes. A caracterização das estruturas no que respeita ao seu tamanho hidrodinâmico e potencial zeta, em conjunto com os estudos de citotoxicidade, foram realizados no sentido de avaliar as propriedades físicas, assim como a biocompatibilidade das estruturas. Os resultados obtidos demonstraram que BHS estáveis, com tamanhos definidos, podem ser obtidas através de poliassociação controlada. Além disso, nas condições experimentais consideradas, as BHS apresentam biocompatibilidade e não apresentam toxicidade para as células. Seguidamente, avaliou-se a influência do ligando do ácido fólico sobre a segmentação de células cancerígenas ricas em receptores de folato por citometria de fluxo. Para tal, foi avaliado o efeito da quantidade de ácido fólico, do comprimento da cadeia de PEG (espaçador) e da concentração de BHS, tal como o impacto das estratégias de purificação. Adicionalmente foi ainda estudado o mecanismo de absorção celular

Focal adhesion regulation of cell behavior

AbstractFocal adhesions lie at the convergence of integrin adhesion, signaling and the actin cytoskeleton. Cells modify focal adhesions in response to changes in the molecular composition, two-dimensional (2D) vs. three-dimensional (3D) structure, and physical forces present in their extracellular matrix environment. We consider here how cells use focal adhesions to regulate signaling complexes and integrin function. Furthermore, we examine how this regulation controls complex cellular behaviors in response to matrices of diverse physical and biochemical properties. One event regulated by the physical structure of the ECM is phosphorylation of focal adhesion kinase (FAK) at Y397, which couples FAK to several signaling pathways that regulate cell proliferation, survival, migration, and invasion

Stres oksydacyjny indukowany hiperglikemią w cukrzycy ciążowej (GDM)

An imbalance between production of reactive oxygen species (ROS) and their clearance by antioxidant defence mechanisms results in the development of oxidative stress. Biological consequences of this state involve oxidative damage of key cellular components such as nucleic acids, lipids, or proteins and, in turn, impairment of cell and tissue function. Evidence from clinical and experimental studies supports the notion that oxidative stress is one of pathologic factors associated with gestational diabetes mellitus (GDM), metabolic disorder defined as any degree of glucose intolerance with onset or first recognition during pregnancy. It has been established that high blood glucose concentrations in diabetic pregnancy induce oxidative stress by several mechanisms, including an enhanced ROS production in mitochondria, the polyol pathway and the hexosamine pathway, as well as protein kinase C (PKC) activation and an advanced glycation end-products (AGEs) formation, and changes in biomarkers of free radical-induced damage and antioxidant defences have been detected in maternal diabetes. Moreover, hyperglycaemia-induced oxidative stress is related with some congenital anomalies in diabetic pregnancy. The current article provides an overview how oxidative stress is related to GDM, with special emphasis on the involvement of the hyperglycaemia-induced mechanisms in ROS overproduction, followed by discussion of indicators of oxidative stress. In addition, the relationship between oxidative stress and congenital malformations in diabetic pregnancy is described.Brak równowagi między produkcją reaktywnych form tlenu (ROS) a ich usunięciem przez antyoksydacyjne mechanizmy obronne prowadzi do rozwoju stresu ok­sydacyjnego. Biologicznymi konsekwencjami tego stanu są oksydacyjne uszkodzenia kluczowych składników komórkowych, takich jak kwasy nukleinowe, białka lub lipidy, które prowadzą do upośledzenia funkcji komórek i tkanek. Dowody pochodzące z badań klinicznych i eksperymentalnych popierają koncepcję, że stres oksydacyjny jest jednym z patologicznych czynników związanych z cukrzycą ciążową (GDM), definiowaną jako różny stopień zaburzeń tolerancji węglowodanów po raz pierwszy rozpoznany lub rozwijający się podczas ciąży. Ustalono, że wysokie stężenia glukozy we krwi kobiet z GDM indukują stres oksydacyjny poprzez kilka mechanizmów, w tym zwiększoną produkcję ROS w mitochondriach, szlak poliolowy, szlak heksozoaminy, aktywację kinazy białkowej C (PKC) oraz tworzenie końcowych produktów zaawansowanej glikacji (AGEs). Zmiany zarówno w biomarkerach uszkodzeń oksydacyjnych, jak i antyoksydacyjnym systemie obronnym były wykryte u pacjentek z GDM. Ponadto stres oksydacyjny indukowany hiperglikemią w GDM jest związany z roz­wojem wad wrodzonych płodu. Niniejszy artykuł stanowi podsumowanie wiedzy o związku stresu oksydacyjnego z cukrzycą ciążową, ze szczególnym podkreśleniem udziału mechanizmów indukowanych hiperglikemią, które prowadzą do nadprodukcji ROS, oraz omówieniem wskaźników stresu oksydacyjnego. Opisano także związek między stresem oksydacyjnym a występowaniem wad wrodzonych płodu podczas cukrzycy w ciąży

Adenosine receptors expression is elevated in leukocytes of gestational diabetes mellitus (GDM) subjects — a preliminary study

Introduction: Adenosine receptors (ARs), belonging to the G-protein-coupled receptors (GPCRs), are present in the majority of human cells and tissues. Depending on their biochemical and pharmacologic properties, four subtypes of ARs (i.e. A1, A2A, A2B, and A3) have been distinguished. Currently, these receptors are attractive molecular targets for pharmacological interventions in various diseases, including diabetes. The literature published to date has shown an altered expression of ARs in several types of cells under diabetic conditions. However, there has been no publication devoted to the investigation of ARs expression in leukocytes of subjects with gestational diabetes mellitus (GDM). Therefore, this study was aimed to determine the expression level of AR subtypes in leukocytes of GDM patients and its relationship to anthropometric and biochemical parameters. Material and methods: Gene expression of four AR subtypes in leukocytes of both healthy (n = 34) and GDM (n = 67) subjects in the third trimester of pregnancy (from 24 to 33 weeks) was investigated. Multiple regression analyses were used to assess the association between the expression level of ARs and both anthropometric and biochemical parameters. Results: Statistically significant (p < 0.05) higher levels of A2A and A2B mRNAs were observed in leukocytes of the GDM subjects compared to the control group. There was a positive correlation of A2B mRNA level with glucose concentration at 120 min of oral glucose tolerance test (OGTT) (r = 0.24, p = 0.041). Conclusions: Overexpression of A2BAR in leukocytes of the GDM subjects and, additionally, the existence of a relationship between its elevated expression level in these cells and abnormal values of glucose concentration at 120 min of OGTT for GDM, suggest that this subtype might be involved in the pathogenesis of GDM. (Pol J Endocrinol 2012; 63 (2): 110&#8211;114)Wstęp: Receptory adenozynowe (ARs) należą do błonowych receptorów sprzężonych z białkami G, które są obecne w większości ludzkich komórek i tkanek. Na podstawie ich biochemicznych i farmakologicznych właściwości wyróżniono cztery podtypy ARs (tj. A1, A2A, A2B, A3). Obecnie receptory te stanowią atrakcyjne molekularne cele dla farmakologicznych interwencji w różnych chorobach, w tym cukrzycy. W dostępnym piśmiennictwie istnieją dane wskazujące, że poziom ekspresji receptorów może ulegać zmianom w niektórych typach komórek podczas cukrzycy, natomiast nie ma publikacji dotyczących badania ekspresji ARs w leukocytach pacjentek z cukrzycą ciążową (GDM). Dlatego celem pracy było określenie poziomu ekspresji czterech podtypów ARs w leukocytach kobiet z GDN i ich korelacja z wybranymi parametrami antropometrycznymi i metabolicznymi. Materiał i metody: Poziom ekspresji czterech podtypów ARs badano w leukocytach kobiet w ciąży, zarówno zdrowych (n = 34; grupa kontrolna), jak i ze zdiagnozowaną GDM (n = 67). Poziomy ekspresji korelowano z oznaczonymi parametrami antropometrycznymi i biochemicznymi. Wyniki: W grupie GDM zaobserwowano istotny statystycznie (p < 0,05) wzrost poziomu mRNA receptorów A2A and A2B w porównaniu z grupą kontrolną. Zaznaczyła się również dodatnia korelacja między poziomem ekspresji receptora A2B a stężeniem glukozy w 2. godzinie testu doustnego obciążenia 75 g glukozy (r = 0,24; p = 0,041). Wnioski: Zarówno istotnie podwyższony poziom ekspresji receptora A2B w leukocytach kobiet z GDM, jak i istnienie dodatniej korelacji między poziomem ekspresji receptora A2B w tych komórkach a stężeniem glukozy wskazują na jego potencjalny udział w patogenezie GDM. (Endokrynol Pol 2012; 63 (2): 110&#8211;114

Ocena leukocytarnej ekspresji SIRT1 u kobiet ze zdiagnozowaną cukrzycą ciążową (GDM) w trzecim trymestrze ciąży

Background. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, has been implicated as a key regulator of glucose/lipid metabolism, insulin secretion as well as adiponectin production and inflammation in metabolic disorders, including type 2 diabetes mellitus (T2DM). However, its role in gestational diabetes mellitus (GDM) remains widely unknown. Since GDM is associated with inflammation, the aim of this study was to determine whether leukocyte SIRT1 mRNA expression is alteredin GDM women in the third trimester of pregnancy, and whether this change is correlated with clinical characteristics of patients. Methods. Leukocytes were isolated from the blood of GDM (n = 135) and normal glucose tolerant (NGT;n = 52) pregnant women. After extracting RNA from leukocytes, a quantitative real-time polymerase chain reaction (qRT-PCR) approach was performed to assess SIRT1 gene expression in these cells. Univariate regression analyses were applied to investigate correlations between SIRT1 expression and clinic parameters of patients. Results. Leukocyte SIRT1 mRNA was increased 1.7-foldin the GDM vs. NGT subjects (p = 0.001) and it positively correlated with 2 h glucose concentration during oral glucose tolerance test (OGTT) in the whole studygroup and negatively correlated with pregnancy agein the GDM and NGT groups. The positive association was also observed between SIRT1 mRNA and plasma high density lipoprotein (HDL) cholesterol level in the NGT subjects. Conclusions. GDM is accompanied by leukocyte SIRT1 mRNA over expression associated with hyperglycemia. Additionally, there is a close and beneficial relationship between enhanced leukocyte SIRT1 expression and increased plasma HDL-cholesterol level during normal pregnancy.Wstęp. Sirtuina 1 (SIRT1) jest NAD+-zależną deacetylazą, która odgrywa istotną rolę w regulacji metabolizmu węglowodanów i lipidów, sekrecji insuliny, produkcji adiponektyny oraz stanu zapalnego w chorobach metabolicznych, w tym cukrzycy typu 2 (T2DM). Do tej pory brak jest informacji o związku leukocytarnej SIRT1 z cukrzycą ciążową (GDM). Zważywszy związek SIRT1 ze stanem zapalnym, celem badania było określenie zmian w poziomie ekspresji SIRT1 mRNA w leukocytach kobiet z GDM w trzecim trymestrze ciąży i skorelowanie ich z klinicznymi parametrami pacjentek. Materiały i metody. Leukocyty wyizolowano z krwi pobranej od kobiet ciężarnych z GDM (n = 135) oraz kobiet ciężarnych z prawidłową gospodarką węglowodanową (NGT; n = 52). Po ekstrakcji RNA z leukocytów poziom ekspresji SIRT1 mRNA w tych komórkach określono metodą ilościowego qRT-PCR. Korelacje między ekspresją SIRT1 a klinicznymi parametrami pacjentek analizowano z wykorzystaniem regresji jednokrotnych. Wyniki. Ekspresja SIRT1 była 1,7-krotnie wyższa w leukocytach kobiet z GDM niż w grupie kobiet z NGT (p = 0,001) i dodatnio korelowała ze stężeniem glukozy w 2. godzinie testu doustnego obciążenia glukozą (OGTT) w całej badanej populacji pacjentek oraz ujemnie korelowała z wiekiem ciąży kobiet zarówno w grupie GDM, jak i NGT (p &lt; 0,05). Stwierdzono również istotną statystycznie dodatnią korelację międzyekspresją SIRT1 a stężeniem cholesterolu frakcji HDL w grupie NGT (p &lt; 0,05). Wnioski. GDM towarzyszy podwyższona ekspresja SIRT1 w leukocytach, która jest związana z hiperglikemią. Ponadto zaobserwowano istnienie bliskiego i korzystnego związku między podwyższonymi poziomami ekspresji SIRT1 a cholesterolem frakcji HDLu kobiet z prawidłowym przebiegiem ciąży

DNA Damage/Repair and Polymorphism of the hOGG1 Gene in Lymphocytes of AMD Patients

Oxidative stress is thought to play a role in the pathogenesis of age-related macular degeneration (AMD). We determined the extent of oxidative DNA damage and the kinetics of its removal as well as the genotypes of the Ser326Cys polymorphism of the hOGG1 gene in lymphocytes of 30 wet AMD patients and 30 controls. Oxidative DNA damage induced by hydrogen peroxide and its repair were evaluated by the comet assay and DNA repair enzymes. We observed a higher extent of endogenous oxidative DNA damage and a lower efficacy of its repair in AMD patients as compared with the controls. We did not find any correlation between the extent of DNA damage and efficacy of DNA repair with genotypes of the Ser326Cys polymorphism. The results obtained suggest that oxidative DNA damage and inefficient DNA repair can be associated with AMD and the variability of the hOOG1 gene may not contribute to this association

The feasibility of the CD271+ and CD271– mesenchymal stromal cell enrichment toward nucleus pulposus-like cells

Introduction. Factors promoting nerve cell ingrowth are considered responsible for chronic back pain resulting from the intervertebral disc degeneration (IDD). One of the recent exploratory IDD treatments is stem cell transplantation therapy. The CD271 (low-affinity nerve growth factor receptor) has been identified as a mark­er of the most homogeneous mesenchymal stem cell (MSC) subset. It is capable of promoting differentiation along adipogenic, osteogenic and chondrogenic lineages and producing significantly higher levels of cytokines as compared to the total population of plastic adherence-mesenchymal stem cells (PA-MSCs). We investigated the ability of CD271+ MSCs to differentiate into chondrocyte-like cells of the nucleus pulposus (NP) of intervertebral disc. We also examined CD271– MSCs, using PA-MSCs as a control cell population. Material and methods. Bone marrow derived PA-MSCs and its two subsets, CD271– MSCs and CD271+ MSCs, were seeded in collagen scaffolds. After two weeks of growth in NP-differentiation medium, RNA was isolated from cells-scaffold constructs and was analyzed by q-PCR for expression of NP markers. Glycosaminoglycans were analyzed biochemically directly in cells-scaffold constructs. Results. Expression of NP markers — extracellular matrix components such as aggrecan, collagen type II and glycosaminoglycans on both RNA and the protein levels — was significantly higher in CD271– MSCs compared to the CD271+ MSCs and PA-MSCs cell populations. Conclusions. CD271– MSCs may be superior candidates for NP restorative treatment compared to CD271+ MSCs and PA-MSCs due to their ability of expressing NP-supporting extracellular matrix components at levels higher than the other two studied MSC subsets

Is copeptin a new potential biomarker of insulin resistance in polycystic ovary syndrome?

Objectives: Copeptin has been reported to play an important role in metabolic response in women with PCOS. However, the optimal cut-off value for detecting subjects with insulin resistance (IR) remains undetermined. We investigated whether copeptin can serve as an indicator of IR and tried to determine the optimal cut-off value of plasma copeptin concentration in detecting subjects with PCOS and IR.  Material and methods: We carried out a case-control study on 158 women with PCOS and HOMA-IR &lt; 2.5, 96 women with PCOS with HOMA-IR ≥ 2.5, and 70 healthy volunteers. Plasma copeptin, as well as hormonal, biochemical, metabolic, and IR parameters, were measured. To investigate whether copeptin allows IR to be predicted in PCOS, we used logistic regression models and ROC curve analysis.  Results: Median plasma copeptin concentration was the highest in the women with PCOS and HOMA-IR ≥ 2.5. Logistic regression analysis revealed that copeptin was the strongest predictor of HOMA ≥ 2.5 (OR: 53.34 CI 7.94–358.23, p &lt; 0.01). Analysis of ROC curves indicated that the cut-off value above 4 pmol/L of plasma copeptin concentration had high (99%) specificity but very low (21%) sensitivity in diagnosing of IR (AUC 0.607 (95% CI 0.53–0.68.  Conclusions: Our findings suggest that copeptin is associated with IR in PCOS patients, but due to low sensitivity should not be considered as a marker of IR.