Ekspertni sustavi primjenjuju se u različitim djelatnostima, s ciljem donošenja odluka na inteligentan i transparentan način. U poslovnim djelatnostima ekspertni sustavi usmjereni su k donošenju ...operativnih i ekonomskih odluka. U zaštiti okoliša, uvažavajući posebnosti različitih poslovnih djelatnosti, u izboru odluka koriste se i ostali aspekti odlučivanja, koji osim ekonomsko-operativnih razloga, vrednuju i druge razloge izbora odluka. Neizrazita logika je logički i matematički aparat, koji se preporuča za primjenu u takvim ekspertnim sustavima. U radu se prikazuju neke značajke i pravila konstrukcije ekspertnih sustava za primjenu u zaštiti okoliša u rudarstvu, a koji primjenjuju neizrazitu logiku.
S obzirom na to da ima brojne nepoželjne učinke koji uključuju smanjenje akti vnosti
, promijenjeno vrijeme poluživota u organizmu te povećanu imunogeničnost, agregacija proteina
predstavlja značajan ...problem kod administracije biofarmaceuti ka. Čimbenici koji uzrokuju
agregaciju proteina mogu biti strukturne karakteristi ke proteina (interni čimbenici) ili
okolišni uvjeti (eksterni čimbenici). Proteinska agregacija jedan je od najviše proučavanih faktora
imunogeničnosti terapeutskih proteina, ali usprkos tome do danas nije u potpunosti
razjašnjen mehanizam kojim agregati terapeutskih proteina izazivaju imuni odgovor. Pokazano
je, međuti m, kako ključnu ulogu u stvaranju imunološkog odgovora ima multi merno
predočavanje anti gena, kao i njegove konformacijske promjene. Kako bi se osigurao razvoj sigurnog
i efi kasnog proteinskog terapeuti ka u smislu nepoželjnih čimbenika koje izaziva proteinska
agregacija, potrebno je osigurati analiti čke metode za karakterizaciju i kvanti fi kaciju agregata.
Kvanti fi kacija agregata je nerijetko iznimno zahtjevna, što je povezano s činjenicom da se
agregati mogu pojavljivati u mnogim i različiti m oblicima. Za detekciju i kvanti fi kaciju agregata
uspješno se primjenuju brojne separacijske tehnike koje uključuju gel-fi ltracijsku kromatografiju,
gel-elektroforezu, razlučivanje protočnim poljem i analiti čko ultracentrifugiranje, kao i
spektroskopske detekcijske tehnike, od kojih se najčešće koriste UV/VIS, fl uorimetrija,
dinamičko raspršenje svjetlosti i višekutno raspršenje svjetlosti .
Ekstrakt biljke Ginkgo biloba može se naći kao sastojak brojnih dodataka prehrani te imamo još registrirane biljne lijekove na bazi Ginka. Ginko sadrži različite biološki aktivne tvari koje mogu ...djelovati na inhibiciju agregacije trombocita što može pozitivno ali i negativno djelovati na kardiovaskularni sustav u onkoloških bolesnika na kemoterapiji, a kod onkoloških operacija (naročito većih abdominalnih, ginekoloških, te operacija s rekonstrukcijom) povećan je rizik od krvarenja, te je kod takvih bolesnika potrebno perioperativno procijeniti utjecaj na agregaciju u slučaju korištenja pripravaka ginka. U ovom radu istražili smo učinak ginka i raznih suplemenata koji sadrže ginkgo na agregaciju trobocita. Ispitivanje je vršeno na 4 skupine po 6 muških štakora soja Y59, od kojih je jedna bila kontrola, a ostale tri su dobivale ginko i ginko s različitim biljnim dodacima. Agregacija trombocita mjerena je na Multiplate analizatoru, impedancijskom agregometru. Agregacija trombocita bila je produžena gdje smo u testiranju koristili ADP kao agonist u sve tri tretirane skupine u odnosu na kontrolu, skupini b ginkgo (P=0.025), c vulkan (P=0.004) i d GOI (P=0,035), dok agregacija s kolagenom kao agonistom nije bila statistički značajno produžena.
Probiotics are life microorganisms that, after administration in gastrointestinal (GI) tract, excert positive effects on host’s health. Host reaction to probiotic administration largely depands on ...ability of bacteria to closely interact with epithelial cells of GI mucosa. On the other hand, considering the unknown antigenic nature of administered bacteria, in immune-compromised host there is a risk of immune reaction of gut tissue which could lead to disturbance of mucosal barrier integrity and chronic inflammation in GI tract. The aim of the study was to examine the effects of the strain Lactobacillus fermentum BGHI14, which is alochtonous to GI tract of rats, after peroral administration in healthy rats and in rats with TNBS (trinitrobenzenesulfonate)-induced colitis. Methodologically, following parameters were examined in experiments: body masses of rats and expression of mRNA of inflammatory cytokines (IL-1β, TNFα, IL-17F), as well as markers of cellular stress (HSP70) and barrier integrity maintenanace (TJP1); next histological and biochemical analyses and microflora profiling were performed. Results of the study indicated immune-stimulating effects of BGHI14 in colon tissue after administration in GI tract of healthy rats in first 16 days of peroral treatment of rats. Although, histologically, with prolongation of BGHI14 treatment of rats for next 12 days, immune reaction in colon tissue had retreated, mRNA levels of proinflammatory IL-1β and TNFα cytokines remained elevated which points to the risk of uncontrolled inflammation in the case of subsequent colon epithelial barrier damage. In research it was shown that after immunostimulation of colon tissue with BGHI14, in the case of colitis induction, but with sequel of treatment with bacteria, transcription of Hsp70 gene was induced, in early (48 h) as well as late (7 days) acute phase of colitis. The increase of Hsp70 gene transcription, according to histological and inflammatory markers, as well as body mass changes during the experiment, was not followed by positive action of the strain. On the other hand, the cessation of treatment with BGHI14 after colitis induction had led to less pronounced body mass decrease compared to control colitic rats, but without concomitant reduction in inflammatory scores in colon tissue, at least in late acute phase of disease (seven days from induction). Based on the above, it could be concluded that mechanisms of adaptive immunity including memory T-cells might had been activated during interaction of BGHI14 with healthy colon tissue and those systems were induced after the injury in the case of continuous presence of same bacterial antigen. Contrary, therapeutic treatment with BGHI14 had brought about the protection in late acute colitis phase according to body mass changes as well as indicators of mucosal barrier disruption. Obtained results implicate that use of immune-stimulating probiotics immediately after colon tissue damage would represent effective approach in treatment of bowel inflammatory diseases, though more detailed research is needed in this area. Considering the importance of interaction of bacteria with gut mucosa in achieving its probiotic potential, in current research mechanisms for improvement of potential probiotic lactobacilli binding to rat’s GI mucosa were tested. In this respect the effects of mucinbinding domain containing protein (MbpL) from strain Lactococcus lactis subsp. lactis BGKP1-20 in ability of bacterial host to bind to ileal and colonic mucosa in vivo and ex vivo, as well as in assays in vitro with isolated mucin and mucin-producing HT29-MTX colonic cells were tested. Aside from using the homologous host, for functional tests we have used potential probiotic strain Lactobacillus salivarius BGHO1. Results have demonstrated that the expression of MbpL protein was not sufficient for binding of BGHO1 to gut mucosa, although it influenced positively the binding of BGKP1-20 to pig gastric mucin and HT29-MTX cells in culture. It is possible that in lactococcal strain BGKP1-20, phenotypic effects of MucBP protein expression are more pronounced compared to BGHO1 strain, because of presence of other potential adhesion factors synthesized in lactobacilli strains. Simultanously, utillizing the same methodology, the role of aggregation factor AggL from BGKP1-20 in binding to GI tract mucosal surfaces was tested. Expression of aggregation factor is commonly correlated to enhanced affinity of bacterial host to GI mucosa. Our results have demonstrated that, although, by increasing surface hydrophobicity, in lactococcal strains it could contribute to binding to colonic surface, the expression of aggregation factor generally aggravates binding of bacteria to surfaces exposed to dynamic peristaltic contractions such as those present in ileum. That way strains expressing aggregation factor could facilitate removal of pathogens but without directly affecting the host’s immune system.
Probiotici predstavljaju žive mikroorganizme koji, nakon unošenja u gastrointestinalni (GI) trakt, ostvaruju pozitivne efekte na zdravlje domaćina. Reakcija domaćina na unos probiotika u velikoj meri zavisi od sposobnosti bliske interakcije bakterija sa epitelnim ćelijama GI mukoze. S druge strane, s obzirom na nepoznatu antigenu prirodu unetih bakterija, kod imunokompromitovanog domaćina je prisutan rizik od imunske reakcije crevnog tkiva što može da dovede do narušavanja integriteta mukozne barijere i hronične inflamacije u GI traktu. Cilj istraživanja bio je ispitivanje efekta soja Lactobacillus fermentum BGHI14, alohtonog za GI trakt pacova, nakon peroralnog unosa u zdrave pacove i u pacove sa kolitisom indukovanim TNBS-om (trinitrobenzensulfonat). Metodološki, u eksperimentima su praćeni: telesna masa pacova i ekspresija iRNK za parametre inflamacije (IL-1β, TNFα, IL-17F), pokazatelje ćelijskog stresa (HSP70) i parametre uključene u održavanje integriteta mukoze (TJP1); zatim su rađene histološke i biohemijske analize i analiza sastava crevne mikroflore. Rezultati istraživanja su pokazali imunostimulišuće delovanje soja BGHI14 na nivou tkiva kolona nakon unosa u GI trakt zdravih pacova u prvih 16 dana peroralnog tretmana pacova. Iako se, histološki, sa produžetkom tretmana pacova sojem BGHI14 tokom narednih 12 dana, imunska reakcija u tkivu kolona povukla, nivoi iRNK za proinflamatorne IL-1β i TNFα citokine ostali su povišeni što ukazuje na rizik od nekontrolisane inflamacije u slučaju naknadnog oštećenja epitelne barijere kolona. U istraživanju je pokazano da je nakon imunostimulacije tkiva kolona sojem BGHI14, u slučaju indukcije kolitisa, ali uz nastavak tretmana sojem BGHI14 došlo do indukcije transkripcije iRNK za HSP70 protein, kako u ranoj (48 h) tako i u kasnoj (7 dana) akutnoj fazi kolitisa. Povećanje transkripcije Hsp70 gena, sudeći po histološkim i inflamatornim pokazateljima, kao i promenama telesnih masa pacova tokom eksperimenta, nije bilo praćeno pozitivnim delovanjem soja. S druge strane, prestanak tretmana sojem BGHI14 nakon indukcije kolitisa doveo je do manje izraženog pada telesnih masa životinja u poređenju sa kontrolnim pacovima sa kolitisom, iako bez pratećeg smanjenja inflamatorne reakcije u tkivu kolona, bar u kasnoj akutnoj fazi bolesti (sedam dana od indukcije). Na osnovu opisanog može se zaključiti da su se u interakciji soja BGHI14 sa zdravim tkivom kolona aktivirali mehanizmi adaptivne imunosti u vidu memorijskih T-limfocita koji su indukovani nakon povrede u slučaju kontinuiranog prisustva istog bakterijskog antigena. Nasuprot, terapijski tretman pacova sojem BGHI14 doveo je do pozitivnog efekta u kasnoj akutnoj fazi kolitisa ocenjujući po promenama telesnih masa životinja kao i na osnovu parametara oštećenja mukozne barijere kolona. Dobijeni rezultati ukazuju da bi primena imunostimulišućih probiotika neposredno po nastanku oštećenja crevnog tkiva predstavljala efikasan pristup u lečenju inflamatornih oboljenja crevnog trakta, iako su na ovu temu potrebna dodatna istraživanja. S obzirom na značaj koji interakcija bakterija sa crevnom mukozom ima u ostvarivanju njihovog probiotičkog potencijala, u istraživanju su testirani mehanizmi kojima se može poboljšati vezivanje laktobacila kao potencijalnih probiotika za GI mukozu pacova. U tom smislu praćeni su efekti proteina sa mucin-vezujućim domenom (MbpL) poreklom iz soja Lactococcus lactis subsp. lactis BGKP1-20 na sposobnost vezivanja bakterijskog domaćina za mukozu ileuma i kolona u uslovima ex vivo i in vivo, kao i u in vitro esejima sa izolovanim želudačnim mucinom i mucin-produkujućim HT29-MTX ćelijama kolona. Osim homolognog domaćina, za funkcionalna ispitivanja korišćen je potencijalni probiotički soj Lactobacillus salivarius BGHO1. Rezultati su pokazali da eksprimirani MbpL protein nije bio dovoljan za vezivanje soja BGHO1 za crevnu mukozu, iako je pozitivno uticao na vezivanje BGKP1-20 soja za svinjski želudačni mucin i HT20-MTX ćelije u kulturi. Moguće je da je fenotipski efekat ekspresije MucBP proteina kod laktokokalnog BGKP1-20 soja veći u odnosu na BGHO1 soj, s obzirom na prisustvo drugih potencijalnih adhezivnih faktora koji se sintetišu u laktobacilima. Koristeći istu metodologiju testirana je uloga koju u vezivanju za mukozne površine GI trakta ostvaruje agregacioni faktor AggL poreklom iz soja BGKP1-20. Agregacioni faktor eksprimiran na površini bakterijskih ćelija često se dovodi u vezu sa povećanim afinitetom bakterijskog domaćina za GI mukozu. Naši rezultati su pokazali da, iako bi, povećanjem površinske hidrofobnosti mogao doprineti vezivanju za površinu kolona kod laktokokalnih sojeva sa niskom ukupnom hidrofobnošću površine, eksprimirani agregacioni faktor generalno otežava vezivanje bakterija za površine izložene dinamičkim peristaltičkim kontrakcijama kakva je u najvećoj meri površina ileuma. Na taj način bi sojevi koji sintetišu faktor agregacije mogli olakšati uklanjanje patogena ali bez sposobnosti ostvarivan
The borate (C6 and C9,5) and citrate (C17 and C30) capped gold nanoparticles
of different average core sizes and properties, were characterized by
transmission electron microscopy (TEM), ...ultraviolet-visible spectroscopy
(UV-Vis), fluorescence spectroscopy, Fourier transform infrared spectroscopy
(FTIR), dynamic light scattering (DLS) and zeta potential measurements, in
the absence and presence of cyanine dye, sodium
(Z)-3-(5-chloro-2-((5-chloro-3-(3-sulfonatopropyl) benzothiazol-2(3H)-
ylidene)methyl)benzothiazol-3-ium-3-yl)propane-1-sulfonate (TC) in water
conditions, and the obtained results confirmed that TC was adsorbed on the
surface of these nanoparticles. The measurements of fluorescence of the
particle–dye assembly, corrected for inner filter effects, clearly indicated
that the fluorescence of TC was quenched by gold nanoparticles on the
concentration dependent manner. This process was found to be quantitatively
related to the surface coverage of the Au nanoparticle by TC dye molecules.
Significant increase of quenching efficiency was noticed when nanoparticle
size increased, keeping the concentration of nanoparticles of different size
constant. The experimental results of fluorescence quenching were compared
with those obtained by theoretical calculations, in which we assumed that the
maximum quenching of TC dye fluorescence was restricted to full monolayer
coverage of TC on the nanoparticle surface. In case of C17 and C30
nanoparticles, the experimentally obtained values for nanoparticle
concentration needed to completely quench TC dye fluorescence were lower than
the calculated ones, while higher experimental values in comparision with
calculated ones of quenched TC dye at constant nanoparticle concentration
were obtained. These findings leaded to the conclusion that the most probable
orientation of TC dye molecules on the surface of these nanoparticle was
slanted. On the other hand, in case of C6 and C9,5 nanoparticles, the
obtained calculated values for TC concentration needed to cover the
nanoparticle surface and be completely quenched, for all three possible
orientations of TC dye, were lower in comparision with the experimental
values. The experimentally obtained value of C9,5 concentration needed to
completely quench TC dye fluorescence most closely corresponds to vertical TC
dye orientation along the short side, while the experimentally obtained
results in case of C6 nanoparticles were much lower than theoretically
calculated values for all three possible TC dye orientations. These findings
indicated that on the surface of C6 nanoparticles the dye was accommodated in
more than one layer. In addition, from the fluorescence quenching
experiments, the equilibrium constants for the sorption of TC on the surface
of all mentioned nanoparticles were calculated. TC dye J-aggregate formation,
obtained only in the presence of C6 colloidal nanoparticles, as well as
kinetics and mechanism of this process were investigated. Thereby, the
kinetic and activation parameters of this reaction were determined, and the
influence of mono- (Li+, Na+, K+, Cs+), di- (Mg2+) i trivalent (Al3+) metal
cations on TC J-aggregation was estimated. Kinetic studies of this hybrid
dye/metal nanoparticle Jaggregate formation have shown a two-step process, a
fast initial formation of the surface layer, followed by the slower, growth
of J-aggregates on the initial TC layer. The adsorption of this negatively
charged dye on the surface of negatively charged gold nanoparticles is only
possible if the dye molecules are oriented toward the surface of
nanoparticles via thiazole sulfur, which carries partial positive charge.
This hypothesis is supported by the activation parameters estimated from
kinetic data. The presence of Jaggregates was noticed in the prescence of
metal ions in TC dye solution, and also after the addition of C6
nanoparticles and in the presence of all mentioned cations except Al3+. The
rate constants for faster and slower process of J-aggregate formation
decreased with increasing the size of monovalent cations. With increasing
temperature, in the presence of Cs+ ions, the values of rate constants for
both processes increased, while the obtained calculated lower values of
activation energies in comparison with values obtained without their presence
indicated that in this case the energy barrier was reduced and the
J-aggregate formation was promoted. Two more colloidal dispersions of gold
nanoparticles prepared by citrate reduction, namely C15 i C47, were
sintetized, and their cytotoxic properties were examined using two in vitro
human cells model systems: proliferating lymphocytes and connective tissue
fibroblasts. The obtained results indicated the concentration dependent
effects in both cases, as well as size dependence, although lymphocytes
turned to be more sensitive to gold nanoparticles. Treatment of lymphocyte
cultures with these gold nanoparticles caused cytotoxic effects as revealed
by significant enhancement of incidence of micronuclei but non significant
increase of cell proliferation potential when compared to the control, still
suggesting disturbed function of cell-cycle checkpoints. In a fibroblast cell
line, the same doses of gold nanoparticles induced the slightly higher level
of γ- H2AX foci than in a control.
Primenom više eksperimentalnih metoda (TEM, UV-Vis spektrofotometrija, fluorescentna spektroskopija, FTIR, DLS, merenja zeta potencijala) ispitane su nanočestice zlata različitih svojstava i veličina sa adsorbovanim boratnim (C6 i C9,5) i citratnim (C17 i C30) jonima na površini, u odsustvu i prisustvu TC boje (natrijum-(Z)-3- (5-hlor-2-((5-hlor-3-(3-sulfonatopropil)benzotiazol-2(3H)-iliden)metil)benzotiazol-3- ium-3-il)propan-1-sulfonata) u vodenoj sredini, i potvrđena je adsorpcija TC na površini ovih nanočestica. Merenje fluorescencije agregata čestica-boja, korigovane za efekat unutrašnjeg filtera, je ukazalo da nanočestice zlata, u zavisnosti od koncentracije, gase fluorescenciju TC boje. Nađeno je da je ovaj proces kvantitativno povezan sa stepenom pokrivenosti površine nanočestica zlata molekulima TC boje. Pritom je efikasnost gašenja rasla sa povećanjem veličine čestica, pri čemu je koncentracija nanočestica zlata u rastvoru održavana konstantnom. Uz pretpostavku da je maksimalno gašenje fluorescencije TC boje ograničeno na potpunu monoslojnu pokrivenost površine nanočestica TC bojom (zavisno od orijentacije boje), eksperimentalno dobijene vrednosti koncentracije nanočestica C17 i C30 potrebne za potpuno gašenje fluorescencije TC boje su bile niže od teorijski izračunatih, dok su za gašenje fluorecencije TC boje pri konstantnoj koncentraciji nanočestica dobijene više eksperimentalne vrednosti u poređenju sa teorijski izračunatim, iz čega je zaključeno da je najverovatnija orijentacija molekula TC boje na površini ovih nanočestica kosa. S druge strane, u slučaju nanočestica C6 i C9,5 teorijski dobijene vrednosti za koncentraciju TC boje potrebnu da pokrije površinu nanočestice i da fluorescencija boje bude potpuno ugašena, u slučaju sve tri orijentacije, su niže u poređenju sa dobijenim eksperimentalnim vrednostima. Koncentracija nanočestica C9,5, potrebna da bi došlo do potpunog gašenja fluorescencije TC boje, po vrednosti najpribližnije odgovara vertikalnoj orijentaciji boje duž kraće strane, dok je u slučaju C6 eksperimentalno dobijen rezultat dosta niži od teorijski izračunatih vrednosti za sve tri moguće orijentacije boje, iz čega je zaključeno da je na površini ovih nanočestica boja smeštena u više od jednog sloja. Iz eksperimenata u kojima je ispitivano gašenja fluorescencije, izračunate su i konstante ravnoteže za proces adsorpcije TC na površini svih ispitivanih nanočestica. Ispitano je formiranje J-agregata, dobijenih samo u prisustvu koloida C6, kao i kinetika i
mehanizam J-agregacije. Pritom su određeni kinetički i aktivacioni parametri ove
reakcije, kao i uticaj jednovalentnih (Li+, Na+, K+, Cs+), dvovalentnih (Mg2+) i
trovalentnih (Al3+) metalnih jona na reakciju J-agregacije. Kinetička ispitivanja formiranja
hibridnih J-agregata boje u prisustvu ovih nanočestica zlata su pokazala da se reakcija
odvija kroz dva koraka, brzo inicijalno formiranje površinskog sloja, koje je praćeno
sporijim rastom J-agregata na inicijalnom sloju J-agregata. Adsorpcija ove negativno
naelektrisane boje na površini negativno naelektrisanih nanočestica zlata je moguća kada
su molekuli boje prema površini nanočestica orijentisani preko tiazolnog sumpora, koji
nosi parcijalno pozitivno naelektrisanje, što je u skladu sa aktivacionim parametrima
određenim iz kinetičkih podataka. J-agregacija je primećena po dodatku metalnih jona u
rastvor boje, a nakon dodatka nanočestica u rastvor boje i u prisustvu svih navedenih
jona, osim Al3+, takođe je došlo do formiranja J-agregata. Pritom, sa porastom veličine
jednovalentnih katjona dolazi do smanjenja vrednosti konstante brzine i brzog i sporijeg
procesa J-agregacije. Sa povišenjem temperature, u prisustvu Cs+, vrednosti konstanti
brzina za oba procesa rastu, pri čemu su izračunate niže vrednosti aktivacionih energija u
poređenju sa vrednostima dobijenim bez njihovog prisustva ukazale da je u ovom slučaju
smanjena energetska barijera i pospešena reakcija J-agregacije.
Ispitana su citotoksična svojstva još dve različite koloidne disperzije zlata, C15 i C47,
sintetizovane sa citratom kao redukcionim sredstvom, korišćenjem dva in vitro model
sistema humanih ćelija: limfocita i fibroblasta. Dobijeni rezultati su pokazali da u oba
slučaja postoji koncentraciona zavisnost za različite efekte, kao i zavisnost od veličine
čestica, ali je u slučaju limfocita dobijena veća osetljivost na nanočestice zlata. Tretiranje
kultura limfocita sa nanočesticama zlata je izazvalo citotoksične efekte koji su pokazani
značajnim uvećanjem učestalosti mikronukleusa ali zanemarljivo malim povećanjem
ćelijskog proliferativnog potencijala u poređenju sa kontrolom, što ipak ukazuje na
The probiotic potential of bacteria depends on the surface characteristics of
bacterial cells. Keeping this in mind, in this work the surface components of
the cells responsible for the aggregation ...ability and the production of
exopolysaccharides (EPS) were analyzed from the natural isolates of
lactobacilli. Lactobacilli strains used in this work were isolated from
autochthonous cheeses produced in households according to the traditional
technology. Selected lactobacilli showing the autoaggregation ability
(BGAR75, BGGR2-68, BGGR2- 82, BGDP9-85, BGDP1-84, BGNJ1-3, BGNJ1-61,
BGNJ1-70) as well as two selected strains which do not form aggregates
(BGAR76 and BGGR2-20) according to the comparison of their 16S rRNA gene
sequence to the NCBI database were classified in the group Lactobacillus
casei. The strain BGDU4-71 using the 16S rRNA gene sequence was determined as
Lactobacillus delbrueckii subsp. bulgaricus and the strain BGCG11, the
producer of the exopolysaccharide (EPS-CG11), was determined by AFLP
methodology as Lactobacillus paraplantarum. In order to characterize factors
involved in the aggregation process the kinetics and the type of
autoaggregation were analyzed. The kinetics (spectrofotometrically
determined) as well as the shape of the aggregates was variable among the
strains. Strains with fastest and the largest aggregates were BGSJ2-8, BGDP1-
84 and BGNJ1-6. The characterization of the nature of factors involved in
autoaggregation of selected strains of lactobacilli was performed by
exhaustive washing of the strains in distilled water and in PBS solution. It
was noticed that the autoaggregation ability was lost after exhaustive
washing in distilled water in all tested strains except BGDP1-84, which led
to the conclusion that the presence of some ions was necessary for the
formation of the aggregates. Besides, it was shown that some of the factors
promoting autoaggregation were of proteinaceous nature, since the ability was
lost after the proteinase K treatment. Lb. paracasei subsp. paracasei BGSJ2-8
was able to form coaggregates with Listeria innocua ATCC33090, Escherichia
coli ATCC25922 or with Salmonella enterica ser. Typhimurium TR251, while its
derivative BGSJ2-81 that was not able to autoaggregate, did not show
coaggregation. Spectrofotometrical measurements of the interaction showed
that the coaggregation was disturbed after proteinase K treatment, which
indicated that coaggregation, as well as the autoaggregation, involve some
factors with proteinaceous nature. The analysis of the surface
characteristics of strains showing autoaggregation by hexadecane adhesion
method showed that all strains with the autoaggregation ability were highly
hydrophobic, while the ones that do not aggregate (as well as for the
derivative BGSJ2-81) have hydrophilic cell surface . The other part of the
work was including the analysis of the probiotic potential of the strain Lb.
paraplantarum BGCG11, which produces exopolysaccharide EPS-CG11, in in vitro
model systems. In this part of the work, the Muc derivatives from the
strain BGCG11 (NB1, NB4, NB16), obtained by novobiocin plasmid curing, were
included in analyses. The derivatives produce significantly lower amount of
EPS that differs from EPS-CG11 in composition. The analysis of survival in
simulated transit trough gastrointestinal tract (GIT), as well as the
adhesion to three cell lines (Caco-2, HT29, HT29-MTX) were performed by
counting of bacterial colonies before and after the treatment/interaction and
the percentage of survival/adhesion was calculated referring to the number of
bacteria before the incubation with cell lines. BGCG11 and Muc derivatives
(NB1, NB4 and NB16), resuspended in 10% skimmed milk, survived (1- 2%) the
simulated GIT transit. The EPS-CG11 was isolated and purified from the
supernatant of the liquid BGCG11 culture, cultivated in minimal media with
glucose as the only sugar source. HPLC method with MALLS detector was used to
monitor the change in molar mass and the amount of EPS-CG11 polymer after
simulated transit trough gastric and intestinal digestion. Obtained results
showed that the molar mass and the amount of EPSCG11 remained unchanged,
indicating that the enzymes in GIT did not affect the stability of the
purified EPS-CG11. Testing of the adhesion ability of BGCG11, as well as the
control probiotic strain Lb. rhamnosus GG, showed similar percentage of
adhesion for these two lactobacilli, while Mucderivatives NB1, NB4 and
NB16 expressed statistically significant higher percentage of adhesion to all
three cell lines. All tested strains showed very low percentage of adhesion
to HT29-MTX cell line, what is the most likely due to the presence of the
mucus barrier (HT29-MTX cell line, produces mucins, while Caco-2 cells do
not). The probiotic potential of the strain BGCG11 was also monitored by in
vitro analysis of the induction of the immune response in the presence of
UV-inactivated bacteria (BGCG11, Mucderivatives) and purified EPS-CG11, in
human peripheral blood lymphocytes (PBLs) isolated from healthy volunteers.
The immune response was monitored by the proliferation of PBLs (by using kit
for the proliferation measurement) and the cytokine production (IFNγ, TNFα,
IL-12, IL-10, IL-1β and IL-17) by flow cytometry. It was noticed that PBLs
proliferated in the presence of lactobacilli (BGCG11 and Mucderivatives
NB1, NB4 and NB16), while EPS-CG11 did not affect to the proliferation of
PBLs, which led to the conclusion that some other molecules, rather than EPS
were involved in inducing of the proliferation. Generally analyzing the type
of the immune response, the presence of BGCG11 and the EPS-CG11 (in
concentration of 100 g/ml) induced anti-inflammatory and a pro-Th17
response, while Muc derivatives NB1, NB4 and NB16 induced inflammatory
response in PBLs. Furthermore, the detailed analysis of the influence of
potential pathogenic microorganisms, alone or in coincubation with BGCG11,
derivative NB1 and EPS-CG11, on HT29-MTX cell line was performed. The
cytotoxic effect in HT29-MTX cell line (performed by measurement of the
lactate dehydrogenase activity) and the analysis of the production IL-8 (by
ELISA method) was performed. Obtained results were compared to the effects of
Lb. rhamnosus GG and its isolated and purified EPS-GG in the same
experimental conditions. From all tested pathogens, only Listeria
monocytogenes LMG13305 induced significant cell lyses in HT29-MTX cells,
which was reduced after coincubation of L. monocytogenes LMG13305 with
EPS-CG11. The level of the IL-8 production in HT29-MTX cells after
coincubation of pathogens with lactobacilli (BGCG11, Mucderivative NB1 and
Lb. rhamnosus GG) or with EPS molecules (EPS-CG11 or EPS-GG) was different in
comparison to levels obtained after incubation only with pathogenic strains.
BGCG11 and NB1 induced significantly higher level of IL-8 in the presence of
Salmonella enterica ser. Thyphymurium LMG15660, Shigella sonnei LMG10473 and
Yersinia enterocolitica LMG7899. In coincubation with L. monocytogenes
LMG13305, only BGCG11 increased the level of IL-8 production. EPS-CG11
decreased the level of this cytokine in coincubation with L. monocytogenes
LMG13305, while both type of EPS molecules (EPS-CG11 and EPS-GG)
significantly decreased the IL-8 production in coincubation with Clostridium
difficile LMG21717. The conclusion is that different molecules from the cell
surface of pathogens, and lactobacilli modify nonspecific immune response of
HT29-MTX cell line, while the presence of purified EPS molecules showed
silencing of this response. Hence, it is important to see whether the effects
of EPS-CG11 and EPS-GG are different, since the difference was seen when Lb.
rhamnosus GG and Lb. paraplantarum BGCG11 strains were. At the end of this
work, the potential EPS-CG11 operon was localized and characterized. Using
techniques of molecular genetics (cloning, sequencing, DNA-DNA hybridization)
26463 bp of the sequence of large plasmid pCG1, lacking in Muc derivatives,
were obtained. In this plasmid the potential operon EPS-CG11 was localized:
the region of 15 kb with 15 open reading frames (ORFs). Based on the amino
acid sequence homology these ORFs were identified as: the priming
glycosyltransferase (ORF 1), glycosyltransferases (ORF 2, 3, 4, 5 and ORF 8),
polysaccharide polymerases (ORF 6 and 7), chain length determinators (ORF 9,
10 and 11) and the EPS biosynthesis regulators (ORF 10 and 11). Downstream
from these ORFs was the transposase, while ORF 13, 14, 15 and 16 showed
homology at the amino acid level with proteins involved in synthesis of
dTDP-rhamnose (rfbACBD genes) that is a part of the EPS-CG11 composition. In
this way, the new and original structure of the EPS-CG11 gene operon was for
the first time characterized in Lactobacillus paraplantarum species.
Moreover, this operon is also specific by its plasmid localization.
Probiotički potencijal bakterija u velikoj meri zavisi od površinskih
karakteristika bakterijske ćelije. Stoga su u ovom radu analizirane
površinske komponente ćelija odgovorne za agregacione sposobnosti i
produkciju egzopolisaharida (EPS) prirodnih izolata laktobacila. Sojevi
laktobacila korišćeni u ovom radu su izolovani iz autohtonih sireva
proizvedenih u domaćinstvima prema tradicionalnoj tehnologiji. Odabrani
laktobacili koji su ispoljavali autoagregaciju (BGAR75, BGGR2-68, BGGR2-82,
BGDP9-85, BGDP1-84, BGNJ1-3, BGNJ1-61, BGNJ1-70), kao i dva odabrana soja
koja ne agregiraju (BGAR76 i BGGR2-20), su klasifikovani na osnovu poređenja
nukleotidnih sekvenci gena za 16S rRNK sa NCBI bazom podataka i svrstani u
grupu Lactobacillus casei. Soj BGDU4-71 je determinisan sekvenciranjem 16S
rDNK kao Lactobacillus delbrueckii subsp. bulgaricus, a soj BGCG11,
proizvođač egzopolisaharida (EPS-CG11), je determinisan AFLP metodom kao
Lactobacillus paraplantarum. U cilju karakterizacije faktora uključenih u
proces agregacije u ovom radu su analizirani brzina
Kadulja (Salvia sp.) je rod mediteranskih biljaka koji se koristi za aromatiziranje hrane. U ovom su radu ispitani ukupni udjel polifenola, antioksidacijska svojstva i antitrombocitni učinak ...kratkozupčaste kadulje (Salvia brachyodon Vandas), te su uspoređeni s najčešće korištenom vrstom kadulje (S. officinalis, tj. dalmatinska kadulja). Udjel ukupnih flavonoida iznosio je 0,08-0,23 %, a fenolnih kiselina 0,47-3,04 %. Antioksidacijska aktivnost kratkozupčaste kadulje, ispitana pomoću DPPH testa, bila je izraženija (29-36 mg/mL ekvivalenata galne kiseline) od aktivnosti dalmatinske kadulje. U funkcionalnom testu primarne hemostaze, ekstrakt kratkozupčaste kadulje nanomolarne koncentracije od 21 nM spriječio je agregaciju trombocita, čime je potvrđeno da se može upotrijebiti za prevenciju tromboze, i to kao funkcionalna hrana ili nadomjestak prehrani. Antitrombocitna aktivnost povezana je s antioksidacijskim kapacitetom (r=0,7014, p=0,0352), što upućuje na zaključak da na prevenciju agregacije ne utječe samo jedan spoj, već je to rezultat sinergističkog učinka polifenola. Dobiveni rezultati su preliminarni i zahtijevaju detaljnije ispitivanje moguće primjene navedenih biljnih vrsta u svrhu prevencije kardiovaskularnih bolesti i agregacije trombocita.
Točna etiologija proturječnih hemostatskih poremećaja u terminalnom stadiju bubrežne bolesti, tj. tromboze i sklonosti krvarenju, nije u potpunosti razjašnjena. Poremećena funkcija trombocita u ...bolesnika s bubrežnim zatajenjem nije uzrokovana povišenom koncentracijom ureje, premda prisutnost fragmenata fibrinogena može spriječiti vezivanje normalnog fibrinogena, odnosno stvaranje agregata tromobocita. Na poremećaj hemostaze kod bolesnika s bubrežnim zatajenjem može utjecati i izbor nadomjesnog bubrežnog liječenja. Bolesnici na hemodijalizi imaju povećani rizik tromboze prvenstveno zbog oslobađanja tromboksana A2 i ADP -a u cirkulaciju, kao i zbog degranulacije trombocita. U stanovitoj mjeri trombociti se aktiviraju i prolaskom krvi kroz sustav crpki, dok moguću ulogu imaju i mikromjehurići. Transplantacija bubrega je metoda izbora u liječenju bolesnika s bubrežnim zatajenjem. Imunosupresivna terapija je povezana s povećanim rizikom razvoja trombembolijskih komplikacija. Imajući u vidu dugotrajnu potrebu za imunosupresivnim liječenjem kod bolesnika s transplantiranim bubregom potrebna su daljnja istraživanja radi utvrđivanja mogućeg povoljnog učinka različitih imunosupresiva u odnosu na agregaciju trombocita.
Opskrbni lanci su vrlo rijetko u jednostavnom generičkom obliku, već uključuju različite sudionike, koji neovisno provode prognozu potražnje u svojem djelokrugu. Prognoziranje potražnje temeljem ...podataka o narudžbama umjesto podataka o potražnji krajnjeg kupca (korisnika) u lancu, na sljedećim višim razinama kumulativno generira sve veća odstupanja. Svaki od sudionika opskrbnog lanca tako dobiva drugačije podatke o potražnji što je uzrokovano tim kumulativnim djelovanjem, poznatim pod nazivom “efekt biča”. U cilju smanjivanja ovih nepravilnosti, proizvođači obvezuju distributere na dostavljanje podataka o prognozi potražnji na svojem tržištu. Distributeri su pritom suočeni s problemom prikupljanja i obrade heterogenih uzoraka potražnje od ostalih sudionika na nižim razinama. U radu je prikazana analiza agregacije uzoraka koji su korišteni za prognoziranje potražnje primjenom različitih metoda prognoziranja.
Ponavljajući moždani udar je najčešći uzrok pobola i smrtnosti među preživjelima nakon moždanog udara. Uz veće preživljavanje nakon prvog ishemijskog moždanog udara, ponavljajući moždani udari ...zauzimaju velik dio nadolazećih godišnjih izdataka vezanih za zbrinjavanje moždanog udara. Usprkos napretku strategija za prevenciju i liječenje moždanog udara ponovni moždani udar je najopasnija prijetnja bilo kome tko je preživio moždani udar. Ovaj pregledni članak posvećen je antitrombotskoj terapiji u sekundarnoj prevenciji moždanog udara, gdje ima najznačajniji utjecaj u kliničkoj praksi.
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