Repository of the University of Rijeka, Department of Biotechnology
Not a member yet
715 research outputs found
Sort by
Galectins as markers of carcinogenesis and goals of oncology therapy
No full textGalektini (Gal) su proteini koji se neenzinskim načinom vežu na ugljikohidrate,
pokazujući visoku specifičnost za njihove šećerne skupine, točnije βgalaktozide. Ova obitelj proteina broji 15 članova (galektini 1-15), od kojih je
utvrđeno 12 postojećih gena u ljudi. Na staničnoj razini, uloga i važnost Gal se
temelji na posredovanju adhezije i vezanja molekula na ciljne stanice te
uključenost u signalne puteve u stanici te modulacije funkcija i uloge unutar
organizma.
Brojnim znanstvenim istraživanjima je dokazano da su Gal najviše izraženi u
stanicama imunološkog sustava gdje imaju važnu ulogu u nastanku i razvoju
zaraznih, a posebice nezaraznih bolesti, posebice onkoloških bolesti. Do sada
je uspješno utvrđena korelacija ekspresije Gal-1, Gal-2, Gal-3, Gal-4, Gal-7 i
Gal-8 s razvojem određenih tipova raka u kojima je zabilježena povišena
ekspresija pa se ovi proteini mogu smatrati specifičnim pokazateljima
pojedinih tipova i podtipova raka. Nadalje, istraživanja u kojima je utvrđena
ekspresija ili supresija pojedinog Gal tijekom njegovog napredovanja pokazala
su karakteristiku invazivnosti i daljnjeg rasta.
Stoga se smatra da će se nastale promijene u ekspresiji Gal omogućiti novi
pristup terapiji određenim tipova i podtipova raka, odnosno, omogućiti će nove
terapije veće djelotvornosti od postojećih. Obzirom na ulogu Gal u
imunološkom odgovoru, novi pristup terapiji će biti moguć i u ostalih
nezaraznih bolesti.Galectins (Gal) are proteins that bind to carbohydrates in a non-enzymatic
manner and have high specificity for their sugar groups, especially βgalactosides. This protein family includes 15 members (galectins 1-15), of
which 12 genes have been identified in humans. At the cellular level, the role
and importance of Gal is based on mediating adhesion and binding of
molecules to target cells and participation in signaling pathways within the cell,
as well as modulation of functions and tasks within the organism.
Numerous scientific studies have shown that Gal is mainly expressed in the
cells of the immune system, where it plays an important role in the
development and progression of infectious and especially non-infectious
diseases, particularly oncological diseases. To date, a correlation has been
demonstrated between the expression of Gal-1, Gal-2, Gal-3, Gal-4, Gal-7,
and Gal-8 and the development of certain cancers, where increased expression
has been detected, and thus these proteins can be considered as specific
indicators of certain cancers and subtypes. In addition, studies demonstrating
expression or suppression of a specific Gal during progression have shown the
property of invasiveness and further growth.
Therefore, the resulting changes in Gal expression are expected to provide a
new approach to the therapy of certain cancers and subtypes, i.e., they will
enable new therapies with greater efficacy than those currently available.
Given the role of Gal in the immune response, a new therapeutic approach will
also be possible for other non-infectious diseases
Stres kao faktor utjecaja na agregaciju NPAS3 i ostalih proteina prisutnih u mentalnim bolestima
No full textChronic mental illness are some of the leading cause of disability worldwide.
They include major depressive disorder, bipolar disorder and schizophrenia.
Schizophrenia has the most severe symptoms, with an estimated
prevalence of 24 million people in the general population. Chronic mental
illness are characterized by both genetic and non-genetic risk factors,
however, their underlying biological cause remains relatively unknown.
Several candidates have been implicated in protein aggregation pathology
of chronic mental illness, including TRIOBP-1, DISC1, CRMP1 and NPAS3.
In this thesis, we investigate the effect of environmental stress factors on
aggregation of these proteins, when into cultured cells. Initially we were
focusing on treatment protocol optimization and settled on stress treatment
for 3 hours. The results suggest that TRIOBP-1, DISC1 and NPAS3 show
signs of aggregation when following application of stress. However, NPAS3
showed the most interesting results, where it clearly mislocalized from
nuclei to the cytoplasm forming insoluble aggregates. The tests were
repeated, showing aggregation of NPAS3 after it was treated with sodium
arsenide, iron (II) chloride or zinc acetate, as well as potential aggregation
when treated with calcium chloride and an MG132 proteasome inhibitor.
Environmental stress factors potentially do affect protein aggregation,
which can be used as a new approach to investigate the effect on chronic
mental illness. Further cell culture research could provide us with initial
results and could be used as a model for observing stress in chronic mental
illness based on these findings. Further research should be focused on
stress treatment protocol modification, as well as the implication of other
stress factors, such as heat shock, as the high temperature resulted in a
quantitative test failure, where most of the transfected cells showed signs
of NPAS3 aggregation.Kronične mentalne bolesti jedan su od vodećih uzroka smetnji širom svijeta.
One uključuju depresivni poremećaj, bipolarni poremećaj i shizofreniju.
Shizofreniju karakteriziraju nateži simptomi, te je procijenjena prevalencija
kod 24 miljuna ljudi u populaciji. Kronične mentalne bolesti karakteriziraju
genski faktori i faktori koji nemaju genski uzrok, međutim, njihov temeljni
biološki uzrok ostaje relativno nepoznat. Nekoliko kandidata povezanih s
patologijom agregacije proteina kod mentalnih bolesti uključuju TRIOBP-1,
DISC1, CRMP1 i NPAS3. U ovom diplomskom radu istražujemo učinak
stresnih faktora okoliša na agregaciju ovih proteina u staničnim kulturama.
U početku smo se usredotočili na optimizaciju protokola za tretiranje stanica
stres faktorima i odlučili smo se na tretiranje u trajanju od 3 sata. Rezultati
ukazuju da TRIOBP-1, DISC1 i NPAS3 agregiraju nakon tretiranja stres
faktorima. Međutim, NPAS3 je pokazao najzanimljivije rezultate, gdje se
jasno vidi premještaj iz jezgre stanice u citoplazmu, stvarajući netopljive
agregate. Ponovljena testiranja potvrđuju NPAS3 aggregaciju nakon
tretiranja s natrijevim arsenidom, željezovim (II) kloridom i cink acetatom,
kao i potencijalnu agregaciju nakon tretiranja kalcijevim kloridom i
inhibitorom proteasoma MG132. Okolišni stres faktori potencijalno utječu
na agregaciju proteina, te se mogu koristiti kao novi pristup istraživanja
učinka stres faktora na kronične mentalne bolesti. Daljnja istraživanja na
staničnim kulturama mogla bi nam dati početne rezultate i koristiti se kao
model za promatranje učinka stresa kod kroničnih mentalnih bolesti na
temelju ovih otkrića. Daljenja istraživanja bi trebala biti usmjerena na
modifikaciju protokola za tretiranje stanica, kao i korištenje drugih faktora
stresa, primjerice toplinski šok, obzirom da je visoka temperatura
rezultirala neuspješnim kvantitativnim eksperimentom, gdje je većina
transficiranih stanica pokazivala znakove NPAS3 agregacije
Effects of volatilized methamphetamine on Drosophila melanogaster development cycle
No full textMehanizmi obiteljskog nasljeđivanja različitih fenotipova, uzrokovanih roditeljskom konzumacijom droga, odnose se na kombinacije genetskih i okolišnih čimbenika. Epigenetski faktori mogu upravljati aktivacijom i utišavanjem genske ekspresije, a povezani su s dugotrajnim neuroplastičnim promjenama povezanih s uzimanjem droga u ljudskim i životinjskim modelima. Takve promjene mogu perzistirati unutar više generacija, iako je prekinuto izlaganje izvornom podražaju. Stoga epigenetsko transgeneracijsko nasljeđe sugerira mehanizam utjecaja roditeljske upotrebe droga na nekoliko generacija potomaka.
Cilj ovog istraživanja bio je ispitati kako volatirizirani metamfetamin (vMETH) utječe na razvojni ciklus Drosophile melanogaster. Pomoću FlyBong metode izvršili smo administraciju vMETH-a velikoj grupi mužjaka, iz koje smo potom izdvojili 10% mužjaka koji su pokazali najveći lokomotorni odgovor i parili ih s djevicama koje nisu bile izložene vMETH-u. Praćen je utjecaj izloženosti F0 očeva na brzinu razvoja i broj potomaka F1 i F2 generacije.
Rezultati su pokazali trend povećanog broja čahura te povećani broj muških i ženskih potomaka F1 generacije u skupini koja je bila izložena vMETH-u. Dok je u F2 generaciji vidljiv trend povećanog broja čahura i ženskih potomaka, taj trend nije prisutan kod muških potomaka. Stoga rezultati sugeriraju da postoji mogući utjecaj vMETH-a na razvojni ciklus Drosophile melanogaster te da takav efekt ima potencijalne transgeneracijske karakteristike.
Kako bi se ovakve hipoteze potvrdile, ove preliminarne rezultate potrebno je potvrditi preciznijim istraživanjima utjecaja vMETH-a na razvojni ciklus. Potrebno je utvrditi konkretne mehanizme i nasljedna svojstva ovog efekta.The familial inheritance mechanism of different phenotypes caused by parental drug use consists of genetic and environmental factors. Epigenetic factors can govern the activation and silencing of gene expression and have been associated with long-term neuroplastic changes related to drug use in humans and animal models. Such changes may persist over several generations even though exposure to the original stimulus has been discontinued. Therefore, epigenetic transgenerational inheritance suggests a mechanism for the influence of parental drug use on several generations of offspring.
This research aimed to examine how volatilized methamphetamine (vMETH) affects the developmental cycle of Drosophila melanogaster. Using the FlyBong method, we administered vMETH to a large group of males, from which we then selected 10% that showed the highest locomotor response and mated them with virgins that were not exposed to vMETH. The impact of exposure of F0 fathers on the development speed and the number of F1 and F2 generation offspring was monitored.
In the F1 generation, the results showed a trend of an increased number of pupae and both male and female offspring in the group exposed to vMETH. While in the F2 generation, there is a visible trend of an increased number of pupae and female offspring, this trend is not present in male offspring. Therefore, the results suggest a possible influence of vMETH on the developmental cycle of Drosophila melanogaster, and such an effect has potential transgenerational characteristics.
In order to confirm these hypotheses, these preliminary results need to be validated by more detailed studies of the influence of vMETH on the development cycle. It is necessary to determine this effect's specific mechanisms and hereditary properties
Evaluation of the influence of chemically functionalized single-walled carbon nanotubes on the levels of oxidative stress and inflammation parameters in astrocytes exposed to in vitro traumatic injury
No full textSvake godine traumatska ozljeda mozga (engl. traumatic brain injury, TBI) pogađa gotovo 15 milijuna ljudi uz visoku stopu smrtnosti i invalidnosti te predstavlja ozbiljan globalni javnozdravstveni i socioekonomski problem. Astrociti su ključne stanice središnjeg živčanog sustava (SŽS) u odgovoru na traumu te dosadašnja istraživanja upućuju na njihovu potencijalnu ulogu u oporavku SŽS-a nakon ozljede. Manjak učinkovite farmakološke terapije potaknuo je ideju za korištenjem nanomaterijala kao potencijalnim rješenjem u liječenju TBI. U ovom radu opisani su neki učinci primjene jednozidnih ugljikovih nanocjevčica (engl. single walled carbon nanotubes, SWCNTs), kemijski funkcionaliziranih s poli-m-aminobenzen sulfonskom kiselinom (PABS) na primarne mišje astrocite u in vitro modelu teške traumatske ozljede mozga (engl. severe traumatic brain injury, sTBI).
Ispitan je utjecaj PABS-SWCNTs na razine oksidativnog oštećenja proteina nakon ozljede astrocita brzim rastezanjem te nije zamijećena značajna razlika u razini sadržaja karboniliranih proteina u ozlijeđenih stanica, netretiranih ili nakon dodavanja PABS-SWCNTs u odnosu na kontrolnu skupinu. Primjena PABS-SWCNTs povećala je razinu izražaja kiselog fibrilarnog proteina glije (engl. glial fibrillary acidic protein, GFAP) nakon sTBI u odnosu razine zabilježene u uzrocima stanica kontrolne skupine, ali i ozlijeđenih, netretiranih stanica. Primjena PABS-SWCNTs na ozlijeđene stanice ne utječe na razinu ekspresije iNOS, markera oksidativnog stresa, te EAAT1, membranskog prijenosnika ekscitotoksičnih aminokiselina.
Zaključno, rezultati ovog rada upućuju da PABS-SWCNTs ne utječu na razinu oksidativnog stresa u ozlijeđenim astrocitima. Također pokazan je utjecaj PABS-SWCNTs na povećanu ekspresiju GFAP-a. Međutim, potrebno je provesti daljnja kako bi se utvrdilo jesu su PABS-SWCNTs učinkovite, ali i sigurne za primjenu u liječenju ove bolesti.Each year, traumatic brain injury (TBI) affects nearly 15 million people with high mortality and disability rates and is a serious global public health and socioeconomic problem. Astrocytes are key cells of the central nervous system (CNS) in response to trauma, and research to date suggests their potential role in CNS recovery after injury. The lack of effective pharmacological therapy has prompted the idea of using nanomaterials as a potential solution in the treatment of TBI. This thesis reports some effects of the application of single-walled carbon nanotubes (SWCNTs) chemically functionalized with poly-m-aminobenzene sulfonic acid (PABS) on primary mouse astrocytes in an in vitro model of severe traumatic brain injury (severe traumatic brain injury, sTBI).
The effect of PABS-SWCNTs on levels of oxidative protein damage after astrocyte injury by rapid stretching was examined and no significant difference was observed in the level of carbonylated proteins in injured cells, untreated or after the addition of PABS-SWCNTs compared to the control group. The use of PABS-SWCNTs increased the level of glial fibrillary acidic protein (GFAP) expression after sTBI in relation to the level recorded in the control cells but also injured, untreated cells. Application of PABS-SWCNTs to injured cells does not affect the expression level of iNOS, a marker of oxidative stress, and EAAT1, a membrane transporter of excitotoxic amino acids.
In conclusion, the results of this study suggest that PABS-SWCNTs do not affect the level of oxidative stress in injured astrocytes. The effect of PABS-SWCNTs on increased GFAP expression has also been shown. However, further implementation is needed to determine if PABS-SWCNTs are effective, but also safe for use in the treatment of this disease
The influence of different antimicrobial substances on the growth curve of Enterococcus faecalis
No full textBakterije roda Enterococcus su Gram – pozitivni, anaerobni patogeni koji se prirodno nalaze u gastrointestinalnom traktu čovjeka, a najzastupljenija vrsta je pritom Enterococcus faecalis. Iako se smatraju komenzalnim bakterijama, vrlo često uzrokuju urinarne infekcije – infekcije prisutne u bilo kojem dijelu mokraćnog sustava. Urinarne infekcije jedne su od najčešćih bakterijskih infekcija te su jedan od najučestalijih razloga posjeta liječniku. Uz urinarne infekcije, enetrokoki su i nerijetko uzročnici endokarditisa, intraabdominalnih infekcija, neonatalne sepse, meningitisa te nozokomijalne bakterijemije.
Uslijed povećane rezistencije enterokoka na antimikrobna sredstva, poželjno je istražiti druge alternativne pristupe koji bi omogućili efikasnu terapiju i profilaksu. Zbog toga je u ovom istraživanju ispitan utjecaj različitih antimikrobnih tvari na krivulju rasta dva soja E. faecalis. Ispitivao se utjecaj dva antibiotika: nitroksolina, efikasnog antibiotika u borbi protiv infekcija biofilmom čije se antimikrobno djelovanje temelji na keliranju te gentamicina, aminoglikozidnog antibiotika sa širokim rasponom baktericidnog djelovanja. Ispitao se i utjecaj hidrokinona koji je aromatski, fenolni organski spoj nastao razgradnjom arbutina u enterocitima crijevnih resica.
Cilj istraživanja bio je ispitati kako različite antimikrobne tvari u različitim koncentracijama djeluju i pojedinačno i u kombinacijama na krivulje rasta E. faecalis soja 36 i soja ATCC29212. Također, na temelju minimalne inhibitorne koncentracije (MIK) i frakcijske inhibitorne koncentracije (FIC) određene su interakcije između kombinacija antimikrobnih tvari. Rezultati su pokazali veliki potencijal između različitih koncentracija kombinacije nitroksolina i hidrokinona pa je tu kombinaciju spojeva potrebno dodatno istražiti.Bacteria of the genus Enterococcus are Gram-positive, anaerobic pathogens that are naturally found in the gastrointestinal tract of humans, and the most common species is Enterococcus faecalis. Although they are considered commensal bacteria, they very often cause urinary infections - infections present in any part of the urinary tract. Urinary tract infections are one of the most common bacterial infections and are one of the most common reasons for visiting a doctor. In addition to urinary infections, enetrococci are often the cause of endocarditis, intra-abdominal infections, neonatal sepsis, meningitis and nosocomial bacteremia.
Due to the increased resistance of enterococci to antimicrobial agents, it is advisable to investigate other alternative approaches that would enable efficient therapy and prophylaxis. Therefore, in this study, we were examining the influence of different antimicrobial substances on the growth curve of two strains of E. faecalis. The impact of two antibiotics was tested: nitroxolin, an effective antibiotic in the fight against biofilm infections whose antimicrobial action is based on chelation, and gentamicin, an aminoglycoside antibiotic with a wide range of bactericidal action. The influence of hydroquinone, which is an aromatic, phenolic organic compound created by the breakdown of arbutin in the enterocytes of the intestinal villi, was also tested.
The aim of the research was to examine how different antimicrobial substances in different concentrations act both individually and in combinations on the growth curves of E. faecalis strain 36 and strain ATCC29212. Also, on the basis of minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC), interactions between combinations of antimicrobial substances were determined. The results showed great potential between different concentrations of the combination of nitroxoline and hydroquinone, so this combination of compounds needs to be further investigated
The potential connection between molecular changes and biomarkers related to ALS and the development and regeneration of CNS
No full textNeurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis
TRIOBP-1 Protein Aggregation Exists in Both Major Depressive Disorder and Schizophrenia, and Can Occur through Two Distinct Regions of the Protein
No full textThe presence of proteinopathy, the accumulation of specific proteins as aggregates in neurons, is an emerging aspect of the pathology of schizophrenia and other major mental illnesses. Among the initial proteins implicated in forming such aggregates in these conditions is Trio and F-actin Binding Protein isoform 1 (TRIOBP-1), a ubiquitously expressed protein involved in the stabilization of the actin cytoskeleton. Here we investigate the insolubility of TRIOBP-1, as an indicator of aggregation, in brain samples from 25 schizophrenia patients, 25 major depressive disorder patients and 50 control individuals (anterior cingulate cortex, BA23). Strikingly, insoluble TRIOBP-1 is considerably more prevalent in both of these conditions than in controls, further implicating TRIOBP-1 aggregation in schizophrenia and indicating a role in major depressive disorder. These results were only seen using a high stringency insolubility assay (previously used to study DISC1 and other proteins), but not a lower stringency assay that would be expected to also detect functional, actin-bound TRIOBP-1. Previously, we have also determined that a region of 25 amino acids in the center of this protein is critical for its ability to form aggregates. Here we attempt to refine this further, through the expression of various truncated mutant TRIOBP-1 vectors in neuroblastoma cells and examining their aggregation. In this way, it was possible to narrow down the aggregation-critical region of TRIOBP-1 to just 8 amino acids (333–340 of the 652 amino acid-long TRIOBP-1). Surprisingly our results suggested that a second section of TRIOBP-1 is also capable of independently inducing aggregation: the optionally expressed 59 amino acids at the extreme N-terminus of the protein. As a result, the 597 amino acid long version of TRIOBP-1 (also referred to as “Tara” or “TAP68”) has reduced potential to form aggregates. The presence of insoluble TRIOBP-1 in brain samples from patients, combined with insight into the mechanism of aggregation of TRIOBP-1 and generation of an aggregation-resistant mutant TRIOBP-1 that lacks both these regions, will be of significant use in further investigating the mechanism and consequences of TRIOBP-1 aggregation in major mental illness
Synthesis of the amphiphilic derivates of methyl red indicator and the physiochemical characterization of its supramolecular aggregates
No full textSamonakupljanje molekula spontan je proces gdje je nastajanje supramolekularnih struktura rezultat djelovanja slabih nekovalentnih interakcija. Posljednjih godina se sve više pojavljuju studije usredotočene na interakcije sintetiziranih azo boja s različitim vrstama površinski aktivnih tvari, za koje promjene u UV-VIS spektru ukazuju na ostvarivanje međumolekulskih interakcija koje dovode do stvaranja agregata. Među njima veliku pažnju zauzimaju amfifilne boje koje djeluju kao kiselinsko-bazni indikatori, a primjenu pronalaze ne samo u industrijskim procesima, već i u biomedicinskim istraživanjima kao potencionalni agensi u prijenosu lijekova.
U ovom radu sintetizirani su amfifilni derivati indikatora metil crveno, supstitucijom alkilnog lanca sa 6 i 12 C atoma. Optimiziran je sintetski put promjenom parametara reakcije kao i otapala pri pročišćavanju te su istražena svojstva novih spojeva u vodenom mediju u odnosu na koncentraciju i pH. Fotofizikalna svojstva spojeva i njihovih agregata u vodenom mediju istražena su mjerenjem UV-VIS spektara, a proces samonakupljanja tehnikama dinamičkog raspršenja svjetlosti i tenziometrije s visećom kapi. Potencijalna primjena novih spojeva ispitana je eksperimentom vezanja na površinu hidrofobiziranog stakalca.
Pomaci u apsorpcijskim spektrima ukazali su na prisutnost agregata u vodenome mediju kao i nepostojanje ovisnosti apsorpcije o pH pripremljenih suspenzija. Mjerenjem veličine agregata i usporedbom vrijednosti hidrodinamičkih promjera uočeno je da sintetizirani spojevi agregiraju u vezikule čija veličina ne ovisi značajno o koncentraciji. Također veličine vezikula izmjerene unutar duljeg vremenskog perioda ukazuju na razliku u njihovoj stabilnosti, pri čemu vezikule sačinjene od spoja s kraćim lancem, pokazuju uspostavu termodinamičke ravnoteže u kraćem vremenskom periodu. Mjerenjem površinske napetosti i potenciometrijskom titracijom, utvrđen je neionski karakter sintetiziranih spojeva, koji nikako (spoj 1) ili minimalno (spoj 2) smanjuju površinsku napetost na granici faza voda/zrak. Rezultati potenciometrijske titracije, kao i UV/VIS spektrofotometrije, pobijaju hipotezu da bi novi derivati mogli djelovati kao pH indikatori. Ipak, sintetizirani spojevi pojačano se vežu na hidorofobno staklo u odnosu na metil crveno, što pokazuje smjer za razvoj sličnih spojeva s mogućom primjenom u području funkcionalnih površina.Self-assembly of molecules is a spontaneous process where the formation of supramolecular structures is the result of weak non-covalent interactions. In recent years, there has been an increase in the number of studies focusing on the interactions of synthesized azo dyes with different types of surfactants, for which changes in the UV-VIS spectrum indicate the existence of intermolecular interactions that lead to the formation of supramolecular assemblies (aggregates). Among those previously mentioned, amphiphilic dyes that act as acid-base indicators are quite prominent while their application is found not only in industrial processes, but also in biomedical research as potential agents in the transfer of drugs.
In this work, amphiphilic derivatives of the methyl red indicator were synthesized by substitution of the alkyl chain with 6 and 12 C atoms. The synthetic route was optimized by changing the parameters of the reaction and the solvent used in the purification. The properties of the new compounds were also tested in the aqueous medium with respect to concentration and pH. The photophysical properties of the compounds were investigated by measuring the UV-VIS spectrum, while the process of self-assembly was characterized by dynamic light scattering technique and pendant drop tensiometry. Potential applications of new compounds were tested by their binding at the surface of hydrophobized glass slides.
Shifts in the absorption spectra indicated the presence of aggregates in the aqueous solution, as well as the lack of pH-dependence of absorption by the prepared suspensions. By measuring the size of the aggregates and comparing the values of their hydrodynamic diameters, it was evidenced that the synthesized compounds aggregate into vesicles whose size does not significantly depend on the concentration. The size of the vesicles formed by the two synthesized compounds, measured within a prolonged time period, point to a difference in the stability, whereby the vesicles formed by the compound with a shorter hydrocarbon chain, exhibit an establishment of a thermodynamic equilibrium in a shorter period of time. From the surface tension and potentiometric titration measurements, a non-ionic character of the compounds was established, whereby these compounds only minimally (compound 2) or not at all (compound 1) reduce the surface tension at the air/water interface. The results of the potentiometric titration and the UV/VIS spectrophotometry denounce the hypothesis that the new derivatives could act as pH indicators. Nevertheless, the observed increased binding of these compounds at the surface of hydrophobized glass, point to the perspective of development of similar compounds with an application in the field of functional surfaces
Ispitivanje utjecaja inhibicije proteasoma na agregaciju TRIOBP-1 u odnosu na istraživanje shizofrenije
No full textSchizophrenia is a chronic mental illness, which a great number of patients around the world struggle with daily. The pathology of schizophrenia is still largely unknown, and the purpose of this thesis is to attempt to solve a piece
of the schizophrenia puzzle. The role of TRIO and F-actin-binding protein 1 (TRIOBP-1) is the unsolved problem that we are seeking to resolve and, by doing so, contribute to the broader study of schizophrenia. More specifically, the formation of insoluble TRIOBP-1 aggregates is the main point of interest in my research. TRIOBP-1 is a protein that scientists have previously studied in an attempt to discover its role in the proteinopathy of schizophrenia. What this thesis is seeking to accomplish is expand upon the knowledge revolving around this protein which has already accumulated over the years. Specific data must be obtained on the mechanism of protein aggregation and the involvement of the proteasome in the protein's behaviour. My research involved two TRIOBP-1 constructs: a full-length wild type gene with no alterations and a mutant gene with a deletion between amino acids 333 and 340 as well as a deletion of the N-Terminal section (1-59). The two constructs were used to do a comparison against each other and to test how the two constructs behave when placed in different cell lines. The results showed that the mutant variant of the TRIOBP-1 with the aforementioned deletions did not aggregate into any of the cell lines that it was transfected in which proves that those are the regions responsible for aggregation of the full-length TRIOBP-1 plasmid. The impact of inhibiting the proteasome did not impact the number of insoluble protein aggregates which opened the door for further research on how TRIOBP-1 behaves when a combination of stressors is applied. These findings bring us closer to establishing which exact amino acids are prone to aggregation in TRIOBP-1 and open a new chapter in the investigation of the impact of TRIOBP-1 in the broader study of schizophrenia.Shizofrenija je kronična mentalna bolest s kojom se svakodevno bori velik broj oboljelih diljem svijeta. Patologija shizofrenije još je uvelike nepoznata, a svrha ovog rada je pokušati riješiti dio te slagalice. Uloga TRIO i F-aktinvezujućeg proteina 1 (TRIOBP-1) misterij je koji nastojimo razriješiti i na taj način pridonijeti širem proučavanju shizofrenije. Konretno, formiranje netopivih TRIOBP-1 proteinskih agregata je glavna točka interesa u mom istraživanju. TRIOBP-1 je protein kojeg su znanstvenici prethodno izučavali pokušavajući otkriti njegovu ulogu u proteinopatiji shizofrenije. Ono što ova teza želi postići jest proširiti znanje o ovom proteinu koje se već akumuliralo tijekom godina. Mora se doći do specifičnih podataka o mehanizmu agregacije proteina i uključenosti proteasoma u ponašanje proteina. Moje istraživanje uključivalo je dva konstrukta TRIOBP-1: prvi je full-length wild type gen bez ikakvih alteracija, a drugi mutirani gen sa delecijama između
aminokiselina 333-340, kao i delecije N-terminalnog dijela koji odgovara aminokiselinama (1-59). Dva navedena konstrukta su korištena za međusobnu usporedbu te se testiralo kako se ta dva konstrukta ponašaju u različitim staničnim linijama. Rezultati su pokazali da mutirana varijanta TRIOBP-1 s gore navedenim delecijama nije agregirala ni u jednoj staničnoj liniji u koju je transficirana, što dokazuje da su to regije odgovorne za agregaciju TRIOBP-1 plazmida full-length. Utjecaj inhibicije proteasoma nije utjecao na broj netopivih proteinskih agregata što je otvorilo vrata za daljnja istraživanja o tome kako se TRIOBP-1 ponaša kada se primijeni kombinacija stresora. Ova otkrića nas približavaju utvrđivanju koje su točno aminokiseline sklone agregaciji u TRIOBP-1 i otvaraju novo poglavlje u istraživanju utjecaja TRIOBP-1 te u širem kontekstu istraživanja shizofrenije
New Spectroscopically and Photochemically Active Small Molecules that Target DNA and RNA
No full textU sklopu doktorskog rada sintetizirani su novi diariletenski (DAE) derivati s pirenskim
fluoroforima i referentni sustavi u svrhu dobivanja potencijalnih molekulskih fotoprekidača.
Setom spektroskopskih metoda u potpunosti su opisana njihova spektroskopska svojstva i
fotokemijska reaktivnost u MeOH i vodenoj otopini, kao i njihove nekovalentne interakcije s
polinukleotidima različitih sekundarnih struktura, te proteinom albuminom iz goveđeg seruma
(BSA). Svi DAE derivati pokazali su sposobnost reverzibilne fotociklizacije središnje DAE
jezgre u MeOH i vodi, osim bis-pirenskog analoga koji se u vodenoj otopini mogao
fotokemijski zatvoriti (ciklizirati), ali ne i reverzibilno otvoriti (deciklizirati). Također, samo je
otvoreni (neciklizirani) bis-pirenski DAE derivat ostvario interakcije s DNA/RNA fleksibilnim
umetanjem u određene utore polinukleotida, dok se zatvoreni oblik DAE nije vezao na DNA i
RNA. Također, fleksibilni otvoreni oblik DAE vezao se na protein BSA na najmanje tri različita
vezna mjesta, za razliku od zatvorenog oblika koji je u dominantmoj interakciji samo s jednim
veznim mjestom. Preliminarna ispitivanja antiproliferalne aktivnosti na humanoj staničnoj liniji
karcinoma pluća A549, pokazala su da novi DAE derivati nisu toksični. Međutim, fluorescentni
bis-pirenski analog uspješno je ušao u stanicu i lokalizirao se u citoplazmi, pri čemu je njegovo
osvjetljavanje UV svjetlom (315-400 nm) uzrokovalo snažan fotoinducirani citotoksični
učinak, pripisan sposobnosti pirena da proizvodi reaktivne kisikove vrste (ROS) pod utjecajem
zračenja. U drugom dijelu doktorata istraživani su novi derivati asimetričnih cijaninskih boja koje se
međusobno razlikuju u vrsti terminalnog heterocikla, duljini metinskog mosta, vrsti
supstituenata na heterociklu i količini naboja. Primijenjene su spektroskopske i kalorimetrijske
metode kako bi se boje ispitale u prisutnosti biopolimera te ocijenila njihova sposobnost
biološkog oslikavanja. Monokationske, trimetinske AK-3 boje vezale su se za DNA/RNA u mikromolarnom afinitetu
(Log Ks = 5,4-6,5). Vrlo selektivan fluorescentni odgovor samo za AT-DNA u uvjetima suviška
DNA nad bojom, u kombinaciji s podjednako selektivnim AT-DNA ICD odgovorom u
uvjetima suviška boje nad DNA (pripisan stvaranju dimera), omogućuje određivanje AT-DNA
i pri submikromolarnim koncentracijama. Nadalje, alifatska boja s n-butilnim supstituentom za
razliku od boje s benzilnim supstituentom, pokazala je ICD signal vezanjem u veći AU-RNA
utor i AT sekvence u mješovitim sekvencama ct-DNA.
Dikationske, benzooksazolne i benzotiazolne monometinske AK-A boje s tioesterskom
skupinom, vezale su se za ds-DNA/RNA u mikromolarnom afinitetu (Log Ks = 5,0-6,2) snažno
stabilizirajući ds-uzvojnicu. Dominantan način vezanja AK-A boja za ds-DNA je interkalacija,
dok je glavno vezno mjesto boja za RNA njezin veći utor unutar kojega tvore agregate. U
vodenoj otopini spojevi ne pokazuju intrinzičnu fluorescenciju, dok se vezanjem na dsDNA/RNA značajno povećava intenzitet fluorescencije. U biološkim ispitivanjima spojevi su
pokazali zanemariv učinak na proliferaciju stanica i visoko selektivnu lokalizaciju u
mitohondrijima.
Višestruko pozitivno nabijene cijaninske boje opremljene 1,4-diazobiciklo[2,2,2]oktanskom
(DABCO) skupinom, također su pokazale mikromolarni afinitet (Log Ks = 5,9-6,9) prema dsDNA/RNA. Neke od predstavljenih boja imaju zanemarivi antiproliferativni učinak prema
humanim tumorskim stanicama, no vrlo učinkovito ulaze u stanicu, uz visoko selektivnu
lokalizaciju u mitohondrijima, što ih čini potencijalno korisnim novim fluorescentnim bojama
za mitohondrije u živim stanicama.As part of the doctoral dissertation, new diarylethene (DAE) derivates with pyrene fluorophores and reference systems were synthesized for the purpose of obtaining potential molecular photoswitches. A set of spectroscopic methods fully described their spectroscopic and
photochemical properties in MeOH and aqueous solution, as well as their noncovalent
interactions with polynucleotides of different secondary structures and with the bovine serum
albumin (BSA). All DAE derivatives showed reversible photocyclization reactivity of the
central DAE nucleus in MeOH and water, except for the bis-pyrene analog, which could be
photochemically closed but not reversibly opened due to strong intramolecular interactions of
pyrene overlap with the closed DAE unit. Also, only the open bis-pyrene DAE derivative
interacted with DNA/RNA by flexible insertion into specific polynucleotide grooves, while the
self-folded closed form of DAE did not bind to DNA and RNA. For the same steric reasons,
flexible open DAE-bis-pyrene form was bound to at least three different binding sites in BSA,
while rigid, self-stacked closed-form interacted dominantly with only one BSA site.
Preliminary studies of antiproliferative activity on the human lung cancer cell line A549 have
shown that the new DAE derivatives are non-toxic. However, the fluorescent bis-pyrene analog
successfully entered cells and localized in the cytoplasm, whereby its illumination with UV
light (315-400 nm) caused a strong photoinduced cytotoxic effect, typical for pyrene-related
singlet oxygen species production. In the second part of the doctoral dissertation, new derivatives of asymmetric cyanine dyes were investigated, which differ from each other in the type of terminal heterocycle, the length of the
methine bridge, the type of substituents on the heterocycle, and the amount of charge.
Spectroscopic and calorimetric methods were applied to examine the dyes in the presence of
biopolymers and to evaluate their ability to bioimage. Monocationic, trimethyne AK-3 dyes bound to DNA/RNA at micromolar affinity (Log Ks =
5,0-6,9). A highly selective fluorescent response > 650 nm only for AT-DNA sequences at
excess of DNA over dye, combined with equally AT-DNA selective ICD response at dye/DNA
crowded conditions (attributed to dye-dimer formation) allows for determination of AT-DNA
at submicromolar concentrations. Furthermore, a comparison of aliphatic- and benzyl- dye
showed that only aliphatic- derivative revealed ICD band upon binding to AU-RNA major
groove and short AT-sequences in mixed sequence (ct-)DNA.
Dicationic, benzoxazole and benzothiazole monomethine AK-A dyes with a thioester group
bound to ds-DNA/RNA at micromolar affinity and strongly stabilized the ds-helix. The
dominant way of binding AK-A dyes to ds-DNA is intercalation, while the main binding site
in RNA is its major groove within which they form aggregates. In an aqueous solution, the
compounds do not show intrinsic fluorescence, while binding to ds-DNA/RNA significantly
increases the fluorescence intensity. In biological studies, the compounds showed a negligible
effect on cell proliferation and highly selective localization in mitochondria.
Multiple positively charged cyanine dyes substituted with the 1,4-diazobicyclo [2,2,2] octane
(DABCO) group showed a high affinity for ds-DNA/RNA. Some of the presented dyes combine
low antiproliferative effects against human tumor cells, with efficient cellular uptake, high
affinity towards ds-DNA/RNA, and remarkable fluorescent staining of mitochondria