Differential response and withdrawal profile of glucocorticoid-treated human trabecular meshwork cells

The goal of the study was to examine secreted protein response and withdrawal profiles from cultured human trabecular meshwork (HTM) cells following short- and long-term glucocorticoid treatment. Primary cultures of five human HTM cell strains isolated from 5 different individual donor eyes were tested. Confluent HTM cells were differentiated in culture media containing 1% FBS for at least one week, and then treated with Dexamethasone (Dex, 100 nM) 3 times/week for 1 or 4 weeks. Cell culture supernatant was collected 3 times per week for 8 weeks. Secretion profiles of myocilin (MYOC), matrix metalloproteinase-2 (MMP2) and fibronectin (FN) were determined by Western blot analysis and MMP2 activity by zymography. Dex treatment reduced MMP2 expression and activity, returning to normal levels shortly after Dex withdrawal in 5 HTM cell strains. All five cell strains significantly upregulated MYOC in response to Dex treatment by an average of 17-fold, but recovery to basal levels after Dex withdrawal took vastly different periods of time depending on cell strain and treatment duration. Dex treatment significantly increased FN secretion in all strains but one, which decreased FN secretion in the presence of Dex. Interestingly, secretion of FN and MYOC negatively correlated during a 4 week recovery period following 4 weeks of Dex treatment. Taken together, the time course and magnitude of response and recovery for three different secreted, extracellular matrix-associated proteins varied greatly between HTM cell strains, which may underlie susceptibility to glucocorticoid-induced ocular hypertension

Enhancement of outflow facility in the murine eye by targeting selected tight-junctions of Schlemm's canal endothelia

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system

Altered mechanobiology of Schlemm’s canal endothelial cells in glaucoma

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm’s canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell—likely accounting for increased outflow resistance—that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness—and its biomechanical effects on pore formation—as a therapeutic target in glaucoma

Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma

Improvement Of Relationship Between Consultants And Clients

Magistro studijų baigiamasis darbas: darbą sudaro 71 puslapis, darbe yra 15 paveikslų, 7 lentelės, 53 literatūros šaltiniai, 3 priedai, lietuvių kalba. RAKTINIAI ŽODŽIAI: konsultavimas, konsultantų ir klientų santykiai, klasifikavimas, veiksniai, tyrimas, santykių efektyvumas, santykių tobulinimas. Tyrimo objektas – klientų ir konsultantų santykiai. Tyrimo tikslas – įvertinti konsultantų ir klientų santykius sąlygojančius veiksnius bei numatyti jų tobulinimo galimybes. Tyrimo uždaviniai: 1. Apibendrinti konsultantų ir klientų santykių sampratą. 2. Nustatyti konsultantų ir klientų santykius sąlygojančius veiksnius. 3. Ištirti konsultantų ir klientų santykius konsultavimo įmonėse. 4. Pateikti konsultantų ir klientų santykių tobulinimo galimybes. Tyrimo metodai: mokslinės literatūros analizė bei sintezė, lyginamoji analizė, loginė analizė, anketinė apklausa, grafinis vaizdavimas, statistinė duomenų analizė, apibendrinimo metodas. Darbas susideda iš trijų dalių: teorinės, metodinės ir praktinės. Teorinėje dalyje analizuojama konsultantų ir klientų samprata, šių santykių ypatumai. Daug dėmesio skiriama siekiant ištirti veiksnius, sąlygojančius konsultantų ir klientų santykius bei jų efektyvumą. Metodinėje dalyje pagrįstas tyrimo metodo pasirinkimas, aprašytas tyrimo instrumentarijus. Praktinėje dalyje anketavimo būdu atliktas tyrimas, kuriuo siekiama išsiaiškinti, kaip ir kokie veiksniai lemia konsultantų ir klientų santykių efektyvumą.Final work of the Master’s studies: 71 pages, 15 pictures, 7 tables, 53 literature sources, 3 appendices, in Lithuanian language. KEY WORDS: improvement of clients, relation between consultants and clients, classifications, factors, research, efficiency of relationship, development of relationship. Research object: relationship between consultants and clients. Purpose of the research: to analyse the aspects of relations and factors which determine efficiency of relationship among consultants and clients and foresee possibility of improvement. Tasks: 1) to determine conseption of relationship between consultants and clients; 2) to define factors which determine the relation of consultants and clients; 3) to research relationship between consultants and clients; 4) to offer suggestions for improvement of consultants and clients relationsip. Research methods – scientific literature analysis and synthesis, comparison, analysis and synthesis, relative values, graphical description, data collation, questionnaire inquiry, method of generalization. The work consists of three parts: theoretical, methodological and practical. The theoretical part contains an analysis of consultants and clients relationship. Much attention is paid to investigate the factors leading to the consultants and client relationships, and their effectiveness. In methodological part of this work is based choice of research method and described instrumentation of the research. In the practical part is done analysis, in order to ascertain how and what factors affect consultants and client relations.Žemės ūkio akademijaVytauto Didžiojo universiteta

Elements of communication of consultants and clients in agricultural consulting companies

The relation between consultants and their clients are miscellaneous and very complex during the consulting process. Factors that make an influence on business relationships can be evaluated by looking at the objects of business communication. The main aspect of such relationship is transfer of information and its assimilation. Peculiarities of interpersonal communication show the intellectual or emotional distance. The feeling of comfort during the communication process and efficiency of consultation depends on them. This article research concentrates on influence of main factors to interpersonal and business relations between consultants and clients. Efficiency of relationships between consultants and clients and success of consulting depends from different factors: from education of consultant and client, from their knowledge and other skills, also from consultants' experience

Konsultantų ir klientų santykių ypatumai konsultavimo įmonėse

Dabartiniu metu vis daugiau asmenų kreipiasi į konsultantus, tikėdamiesi patarimų ir pagalbos plėtojant savo verslą bei vystant asmeninę profesinę karjerą. Konsultantas savo ruožtu stengiasi suteikti kuo kokybiškesnes konsultavimo paslaugas. Siame darbe analizuojami konsultantų ir klientų santykiai, apibrėžiami bendravimo dalykiniai ir tarpasmeniniai aspektaiVytauto Didžiojo universitetasŽemės ūkio akademij

Elements of communication of consultants and clients in agricultural consulting companies

Dabartiniu metu vis daugiau asmenų kreipiasi į konsultantus, tikėdamiesi patarimų ir pagalbos plėtojant savo verslą bei vystant asmeninę profesinę karjerą. Konsultantas savo ruožtu stengiasi suteikti kuo kokybiškesnes konsultavimo paslaugas. Darbo tikslas: išanalizavus konsultantų ir klientų bendravimo veiksnius žemės ūkio konsultavimo įmonėse, pateikti konsultantų ir klientų bendravimo tobulinimo galimybes. Šiame darbe analizuojami konsultantų ir klientų bendravimo veiksniai, apibrėžiami bendravimo dalykiniai ir tarpasmeniniai aspektai. Nustatyta, kad klientams atrodo svarbesni tie veiksniai, kurie, jų nuomone, tiesiogiai lemia konsultacijos efektyvumą, t. y. konsultanto gebėjimas nustatyti problemą, perduoti informaciją, konsultanto kompetencija. Ugdant konsultantą, kaip savo srities specialistą, reikėtų kurti kompetencijos kėlimo ir žinių atnaujinimo sistemą, kuri ne tik suteiktų naujausią informaciją konsultantui, bet ir motyvuotų konsultantą tobulėti. Konsultantai turėtų skatinti grįžtamąjį ryšį, atidžiai išklausyti ir pagarbiai reaguoti į klientų pateiktus pasiūlymus ir nuomones. Darbe taikyti metodai: mokslinės literatūros analizė bei sintezė, lyginamoji analizė, loginė analizė, anketinė apklausa, grafinis vaizdavimas, statistinė duomenų analizė, apibendrinimo metodasThe relation between consultants and their clients are miscellaneous and very complex during the consulting process. Factors that make an influence on business relationships can be evaluated by looking at the objects of business communication. The main aspect of such relationship is transfer of information and its assimilation. Peculiarities of interpersonal communication show the intellectual or emotional distance. The feeling of comfort during the communication process and efficiency of consultation depends on them. This article research concentrates on influence of main factors to interpersonal and business relations between consultants and clients. Efficiency of relationships between consultants and clients and success of consulting depends from different factors: from education of consultant and client, from their knowledge and other skills, also from consultants’ experienceVytauto Didžiojo universitetasŽemės ūkio akademij

Klientų ir konsultantų santykių aspektai žemės ūkio konsultavimo įmonėse

Dabartiniu metu vis daugiau asmenų kreipiasi j konsultantus, tikėdamiesi patarimų ir pagalbos plėtojant savo verslą bei vystant asmeninę profesinę karjerą. Konsultantas savo ruožtu stengiasi suteikti kuo kokybiškesnes konsultavimo paslaugas. Šiame darbe analizuojami konsultantų ir klientų santykiai, apibrėžiami bendravimo dalykiniai ir tarpasmeniniai aspektaiVytauto Didžiojo universitetasŽemės ūkio akademij