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Challenges in preclinical research of Echinacea species
No full textBiljne vrste roda Echinacea bilježe višestoljetnu primjenu u prirodnoj
herbalnoj medicini stoga su i danas zanimljive za istraživanja. Posjeduju
brojna korisna svojstva koja primarno djeluju na imunološki sustav jedinke
kroz takozvane imunomodulatorne učinke koji utječu na brojne stanične
puteve i same stanice. Djelovanje se očituje aktivacijom fagocitoze,
stimulacijom fibroblasta i pojačanjem respiratorne aktivnosti te
djelovanjem stanica kao što su makrofagi, leukociti, limfociti, granulociti,
polimorfonuklearni leukociti (PMN) te stanice prirodni ubojice (NK stanice).
Ovim putevima dolazi do lučenja brojnih citokina, kemokina i upalnih
odnosno protuupalnih medijatora poput NF‑κB, TNF‑α, IL-1, IL-1α, IL-1β,
IL-6, IL-8, IL-10, adhezijske molekule i COX‑2, komplementa C3b. Kako bi
sve ovo moglo djelovati postoje i receptori koji prepoznaju te signale i šalju
nove, neki od kojih su kanabinoidni receptori tipa 2 (CB2) i brojna obitelj
TLR receptora. Uz učinke na imunološki sustav biljne vrste roda Echinacea
odnosno biljne aktivne tvari takozvane fitokemikalije pronađene u njima
posjeduju i protuupalne, psihoaktivne, citotoksične te antioksidativne,
antibakterijske, antifungalne i antivirusne učinke. Neke od važnijih
fitokemikalija su alkamidi, derivati kavene kiseline, polisaharidi i
glikoproteini. Ne bi li se ovi učinci dokazali, korišteni su brojni testovi
svojstveni pretkliničkim istraživanjima. In vitro istraživanja provođena su
na raznim staničnim kulturama poput različitih vrsta mišjih i štakorskih
makrofaga i limfocita, dendritičkih stanica, TPH-1 te Jurkat stanicama dok
su in vivo istraživanja provedena testovima kao što su metoda uklanjanja
ugljika ili test induciranja edema šape izazvanog karagenanom. Iako svi ovi
testovi pokušavaju biti što točniji problem lipopolisaharidne kontaminacije i
dalje stvara lažno pozitivne slike učinaka ove biljke. Stoga treba nastojati
otkrivati nove, detaljnije mehanizme kako djelovanja vrsta roda Echinacea
na organizam tako i same metode detekcije njihove korisnosti u organizmuEchinacea species have been used in natural herbal medicine for centuries,
so interest for their research stands even up today. They possess numerous
useful properties that primarily affect the immune system through so-called
immunomodulatory effects through numerous cellular pathways and the
cells themselves. These effects are manifested by the activation of
phagocytosis, the stimulation of fibroblasts and respiratory burst, and cells
such as macrophages, leukocytes, lymphocytes, granulocytes,
polymorphonuclear leukocytes (PMN) and natural killer cells (NK cells).
These pathways lead to the secretion of numerous cytokines, chemokines
and inflammatory or anti-inflammatory mediators such as NF-κB, TNF-α,
IL-1, IL-1α, IL-1β, IL-6, IL-8, IL-10, adhesion molecules and COX‑2,
complement C3b. In order for all of this to work, there are receptors that
recognize these signals and send new ones, some of which are cannabinoid
receptors type 2 (CB2) and a large family of TLR receptors. In addition to
the immune-effect of the phytochemicals found in Echinacea species also
have anti-inflammatory, psychoactive, cytotoxic and antioxidant,
antibacterial, antifungal and antiviral effects. Some of the more important
phytochemicals are alkamides, caffeic acid derivatives, polysaccharides and
glycoproteins. In order to prove these effects, numerous tests characteristic
of preclinical research were conducted. In vitro research was conducted on
various cell cultures such as different types of mouse and rat macrophages
and lymphocytes, dendritic cells, TPH-1 and Jurkat cells, while in vivo
research was conducted with tests such as the carbon clearance method or
carrageenan induced paw edema. Although all these tests try to be as
accurate as possible the problem of lipopolysaccharide contamination still
creates false positive images of the effects of this herb. Therefore, efforts
should be made to discover new, more detailed mechanisms of the effects
of Echinacea species on the body, as well as methods of detecting their
usefulness in the body
Characterisation of neuropathological features of a new Niemann-Pick type C disease murin model with genetically depleted BACE1
No full textBolest Niemann-Pick tipa C (NPC) je rijetka monogenska neurodegenerativna bolest i
poremećaj nakupljanja lizosoma. Bolest NPC uzrokovana je mutacijom u genima NPC1 ili
NPC2 koji kodiraju za prijenosnike kolesterola. Zbog nedostatka ili promijenjene funkcije
proteina NPC1 ili NPC2, u kasnim endosomima i lizosomima dolazi do nakupljanja
slobodnog kolesterola, što uzrokuje poremećaj funkcije endolizosomalnog puta, snažnu
neuroinflamaciju (aktivaciju astrocita i mikroglija) i neurodegeneraciju prvenstveno
Purkinjeovih neurona u malom mozgu. Klinički, bolest NPC karakterizira heterogeni fenotip
koji uključuje brojne neurovisceralne poremećaje i ranu smrt. Unatoč etiološkoj razlici
između bolesti NPC i kompleksne Alzheimerove bolesti (AB), ovi poremećaji dijele nekoliko
patoloških značajki, poput promijenjenog metabolizma kolesterola, neurodegeneracije,
neuroinflamacije te nakupljanja hiperfosforiliranog proteina tau i peptida amiloida-β u
mozgu. Povećano stvaranje amiloida-β uslijed povećanog proteolitičkog cijepanja proteina
APP enzimom BACE1 smatra se glavnim mehanizmom patogeneze AB. Zanimljivo,
povećana proteolitička aktivnost enzima BACE1 zabilježena je i u bolesti NPC. S obzirom
da je enzim BACE1 potencijalna farmakološka meta za liječenje AB, cilj ovog doktorskog
rada bio je ispitati učinak genetičke modulacije razine enzima BACE1 na razinu i progresiju
neuropatoloških značajki bolesti NPC. Za ovo istraživanje korišten je mišji model knock-in
koji nosi najčešću mutaciju u ljudi, NPC1-I1061T, te je križan s modelom knock-out BACE1.
Neuropatološke značajke (uključujući gubitak Purkinjeovih neurona, poremećaj funkcije
endolizosomalnog puta, aktivaciju astrocita i mikroglija te hiperfosforilaciju proteina tau)
ispitane su u malom mozgu, kori velikog mozga i hipokampusu metodama
imunofluorescencije i Western blot. Delecija oba alela gena BACE1 u jedinki knock-in NPC1-
I1061T (N-KI) uzrokovala je ranu smrt uz povećanu progresiju svih neuropatoloških značajki
(izuzev odumiranja Purkinjeovih neurona) već u asimptomatskoj fazi bolesti. Delecijom
jednog alela gena BACE1 u jedinki N-KI ublaženo je nakupljanje ranih endosoma i lizosoma,
aktivacija astrocita te odumiranje Purkinjeovih neurona u simptomatskoj fazi bolesti.
Međutim, u završnoj fazi bolesti, delecijom jednog alela gena BACE1 u jedinki N-KI
povećana je aktivacija mikroglija te ubrzano odumiranje Purkinjeovih neurona, što je
uzrokovalo raniju smrt u odnosu na jedinke N-KI koje sadrže divlji tip gena BACE1.
Zaključno, genetičko smanjenje razine enzima BACE1 ima i povoljne i štetne učinke na
neuropatološke značajke bolesti NPC u mišjem modelu knock-in NPC1-I1061T, čime je
istaknuta važnost uloge enzima BACE1 i njegovih supstrata za funkciju mozga. Daljnja
istraživanja trebala bi razjasniti koji supstrati enzima BACE1 te koji tipovi stanica doprinose
patobiologiji bolesti NPC. Dobiveni rezultati doprinose boljem razumijevanju bolesti NPC,
kao i terapijskog potencijala enzima BACE1 u liječenju bolesti NPC i drugih
neurodegenerativnih poremećaja sličnog mehanizma nastankaNiemann-Pick type C disease (NPC) is a rare monogenic neurodegenerative disease and
lysosomal storage disorder. NPC is caused by mutation in NPC1 or NPC2 genes which
code for cholesterol transporters. Upon lack or dysfunction of NPC1 or NPC2 proteins,
free cholesterol accumulates in late endosomes and lysosomes causing disruption of the
endolysosomal pathway, profound neuroinflammation (activation of astrocytes and
microglia) and neurodegeneration of primarily Purkinje cells in the cerebellum.
Clinically, NPC is characterized by heterogenous phenotype including numerous
neurovisceral pathologies and early death. Despite etiological differences between NPC
and complex Alzheimer’s disease (AD), these disorders share several pathological
features, such as altered cholesterol metabolism, neurodegeneration, neuroinflammation
and accumulation of hyperphosphorylated tau and amyloid-β peptides in the brain.
Increased generation of amyloid-β due to increased BACE1-mediated proteolysis of APP
is considered a potential mechanism of AD pathogenesis. Interestingly, increased
BACE1-cleavage is observed in NPC as well. Since BACE1 is a potential
pharmacological target against AD, the goal of this doctoral thesis was to analyse the
effect of BACE1-genetic depletion on the levels and progression of neuropathological
features of NPC. Therefore, we used a knock-in murine model carrying the most common
human mutation, NPC1-I1061T, and crossed it with BACE1 knock-out mice.
Neuropathological features (including Purkinje cell loss, endolysosomal dysfunction,
activation of astrocytes and microglia, and hyperphosphorylation of tau) were analysed
in the cerebellum, cortex and hippocampus using immunofluorescence and Western blot.
Deletion of both BACE1 alleles in NPC1-I1061T knock-in (N-KI) mice caused early death
with increased progression of all neuropathological features analysed (except Purkinje
cell loss) already at the presymptomatic disease stage. Deletion of one BACE1 allele in
N-KI mice attenuated early endosome and lysosome accumulation, astrocyte activation,
and Purkinje cell loss at the symptomatic disease stage. However, at the terminal disease
stage, deletion of one BACE1 allele in N-KI mice enhanced microglial activation and
Purkinje cell loss, resulting in earlier death compared to N-KI mice containing wild-type
BACE1 gene. In conclusion, BACE1-genetic depletion has both beneficial and
detrimental effects on neuropathological features of NPC in NPC1-I1061T knock-in
murine model, which highlights the importance of BACE1 and its substrates in brain
function. Future studies should aim to determine which BACE1 substrates and which cell
types contribute to NPC pathobiology. These findings contribute to a better understanding
of NPC disease and the potential of BACE1-targeted therapies against NPC and other
neurodegenerative disorders with similar underlying pathologies
Synthesis and photochemistry of oligopeptides and their potential application in diagnostic and therapy
No full textPeptidi su obećavajući kandidati za lijekove te ih se sve više istražuje zbog farmaceutskih i
medicinskih primjena. Međutim, poznato je da su peptidi skloni enzimskoj razgradnji unutar
stanice, što predstavlja velik problem za njihovu primjenu, a jedan od pristupa je priređivanje
neprirodnih derivata koje enzimi ne mogu prepoznati. U okviru doktorske disertacije
pripremljena je neprirodna aminokiselina, derivat tirozina, koja u fotokemijskim reakcijama
stvara reaktivne intermedijere kinon-metide. Novopripremljena neprirodna aminokiselina
ugrađena je u oligopeptide koji kao jedinicu za prepoznavanje polinukleotida sadrže dva
triptofana, fenantridin ili piren. Za pripravu oligopeptida korištena je sinteza peptida u otopini
uz standardne aktivacijske protokole i Boc zaštitnu skupinu. Novim dipeptidima i tripeptidima
ispitana je fotokemijska reaktivnost, a kinon-metidi su detektirani laserskom pulsnom
fotolizom. Peptidima su ispitana i fotofizička svojstva (molarni apsoprcijski koeficijent, kvatni
prinos fluorescencije, vrijeme života singletnog pobuđenog stanja), što je važno za potencijalnu
primjenu u dijagnostici kao i za ispitivanje nekovalentnog vezanja na polinukleotide. Konstante
stabilnosti kompleksa s polinukleotidima određene su fluorescencijskim titracijama, a ispitan
je i utjecaj spojeva na termičku denaturaciju dok je način vezanja utvrđen korištenjem CD
spektroskopije. Kovalento vezanje novopripremljenih oligopeptida na oligonukleotide i
polinukleotide ispitano je ozračivanjem njihovih kompleksa te različitim spektroskopskim i
kromatografskim analizama.Peptides are promising drug candidates and their further development and research is important
for pharmaceutical and medicinal applications. However, peptides have some limitations due
to short half-life in vivo and fast clearance from the circulation by the liver and kidneys. One
way/possibility to sovle these obstacles is by incorporation of unnatural amino acids, which
enzymes cannot recognize. This PhD thesis describes the preparation of an unnatural amino
acid, a tyrosine derivative, which in the photochemical reaction generates reactive intermediate
quinone methide. The unnatural amino acid was incorporated into oligopeptides containing bistryptophane unit, phenanthridine or pyrene. The synthetic protocol for the preparation of
oligopeptides was based on the standard peptide activation protocols in solution and Boc
protective groups. The photochemical reactivity of the oligopeptides was investigated, and the
quinone methides were detected by laser flash photolysis. The photophysical properties (molar
absorption coefficient, quantum yield of fluorescence and singlet excited state lifetime) of the
oligopeptides were investigated, which is important for their potential diagnostic applications,
as well as for the investigation of noncovalent binding to polynucleotides. The stability
constants of the peptide-polynucleotide complexes were determined by fluorescence titrations,
and the effect of the compounds on the thermal denaturation was investigated. The binding
modes to polynucleotides were probed by CD spectroscopy. The ability of the peptides to
covalently bind to oligonucleotides and polynucleotides was examined by irradiating their
complexes and detecting adducts by various spectroscopic and chromatographic techniques
Kratki linearni i ciklicki katalitički peptidi inspirirani esterazom
No full textIn this dissertation, two key approaches for the design of catalytic peptides are explored: (i) the incorporation of amino acids from the catalytic triad and (ii) an approach
involving metal ions. To identify these main design approaches, a comprehensive dataset
based on the collection of published data about purely peptidic catalysts for ester hydrolysis was gathered and analyzed including both active and inactive sequences tested
towards the p-NPA and p-NPP substrates providing an insight into the currently developed
catalytic mechanisms. Additionally, this open-access dataset also includes the SMILESbased representation of peptides and it can be used for training of machine learning-based
models. Moreover, the standard p-NPA method was optimized. This extensively employed assay for determining catalytic activity, initially designed for enzymatic reactions,
is not directly applicable to peptides due to variations in reaction parameters, such as
the concentrations of catalysts and reaction times, when comparing peptides to enzymes.
Consequently, standardizing conditions, including temperature, pH, buffer choice, and
concentrations of both substrate and peptide, became essential to ensure accurate and
reliable results in peptide reactions. Furthermore, the synthesis of a set of histidine-rich
linear and cyclic peptides allowed us to understand the impact of amino acid arrangement,
cyclization, and inclusion of D-amino acids on their self-assembly, coordination of Zn2+
ions and ability to hydrolyze p-NPA. Investigating structural changes crucial to the functionality of short peptides contributes to the development of a new generation of catalytic
peptides and advances our understanding of the potential of self-assembly in improving
catalytic efficiency. Additionally, we investigated the feasibility of isolating the active
site of an enzyme to develop a minimalist catalyst, with a specific focus on recognizing
cysteine as a crucial component within the catalytic triad, essential for the effectiveness
of the peptide catalyst. The synthesis of a set of cysteine-rich peptides showing tunable
activity resulting from the sensitivity of the thiol groups to the redox environment could
open promising opportunities for tailored enzymatic catalysis. The overall goal of this
research is to gain a deeper insight into the relationship between the sequence and function of catalytic peptides, leading to the development of innovative peptide catalysts with
potential applications in various fields, including biocatalysis, pharmaceutical synthesis,
and other industrial processes.U ovoj disertaciji predstavljena su dva kljucna pristupa za dizajn katalitičkih peptida: (i) peptidne sekvence bazirane na aminokiselinama iz kataliticke trijade i (ii) pristup koji se temelji na interakciji peptida i metalnih iona. U svrhu identifikacije ovih glavnih pristupa, prikupljen je i analiziran sveobuhvatan skup podataka na temelju objavljenih katalitičkih peptida za hidrolizu estera, uključujući aktivne i neaktivne sekvence testirane na supstratima p-NPA i p-NPP, pružajuci uvid u trenutno razvijene katalitičke mehanizme. Ovaj skup podataka s otvorenim pristupom ukljucuje i računalnu reprezentaciju peptida u formatu SMILES koja se može koristiti za treniranje i razvoj modela zasnovanih na strojnom ucenju. Nadalje, optimizirana je standardna metoda p-NPA za određivanje katalitičke aktivnosti. Ovaj često korišteni esej, prvotno dizajniran za enzime, nije
izravno primjenjiv na peptide zbog varijacija u parametrima katalitiče reakcije, kao što su koncentracija katalizatora i trajanje reakcije u usporedbi peptida s enzimima. Stoga je bilo potrebno standardizirati uvjete, uključujući temperaturu, pH, izbor pufera i koncentracije supstrata i peptida, kako bi se osigurali tocni i pouzdani rezultati. Nadalje, sinteza skupa linearnih i ciklickih peptida bogatih histidinima omogućila nam je razumijevanje utjecaja rasporeda aminokiselina, ciklizacije i uključivanja D-aminokiselina na njihovu sposobnost samo-sastavljanja, koordinacije iona Zn2+ i sposobnost hidrolize pNPA. Istraživanje strukturnih promjena kljucnih za funkcionalnost kratkih peptida doprinosi razvoju nove generacije katalitičkih peptida i unaprjeđuje naše razumijevanje potencijala samo-sastavljanja u poboljšanju katalitičke učinkovitosti. Dodatno, ispitana je mogućnost izdvajanja aktivnog mjesta enzima radi razvoja minimalističkog katalitičkog peptida, s posebnim naglaskom na cistein kao ključnoj komponenti unutar katalitičke trijade, neophodnoj za učinkovitost takvih peptida. Sinteza seta cisteinom bogatih peptida koji pokazuju prilagodljivu aktivnost kao rezultat osjetljivosti tiolnih skupina na redoks okolinu, otvara nove mogućnosti za prilagodljivu peptidom posredovanu katalizu. Glavni cilj ovog istraživanja je razumjeti odnos izmedu sekvence i funkcije katalitičkih peptida, čime bi se omogućio razvoj inovativnih katalitičkih peptida primjenjivih u različitim područjima, uključujući biokatalizu, sintezu lijekova i industrijske procese
Lipophilicity determination of macrocyclic compounds by high-performance liquid chromatography
No full textMakrociklički spojevi omogućuju moduliranje kompleksnih farmakoloških meta, pritom
zadržavajući dobra farmakokinetička svojstva. Lipofilnost je jedno od najvažnijih fizikalno
kemijskih svojstava te je izuzetno važno u ranim faza istraživanja imati pouzdanu metodu
za lipofilno profiliranje novih makrocikličkih spojeva. U okviru ove doktorske disertacije
istražena je primjenjivost literaturne CHI metode koja koristi tekućinsku kromatografiju
visoke djelotvornosti za određivanje lipofilnosti makrocikala. Analizirana je početna skupina
makrocikličkih spojeva pri čemu je utvrđeno da je ova metoda manje precizna pri
analizama makrocikala u usporedbi s malim molekulama zbog širenja i asimetričnosti
kromatografskih pikova.
Proučavanjem utjecaja promjena eksperimentalnih kromatografskih uvjeta na vremena
zadržavanja makrocikala utvrđeno je da temperatura kromatografske kolone, brzina
primjerenog gradijentnog programa te protok pokretne faze nemaju značajan utjecaj na
relativne odnose u vremenima zadržavanja makrocikličkih spojeva. Promjena
kromatografske kolone unutar iste USP klasifikacije ne utječe na relativni odnos vremena
zadržavanja istraživanih spojeva, ali poboljšava učinkovitost analiza. Značajan utjecaj na
analize makrocikličkih spojeva primijećen je smanjenjem koncentracije amonijevog acetata
korištenog kao vodeni dio pokretne faze, kao i zamjena amonijevog acetata s amonijevim
hidrogenkarbonatom. Također, upotreba metanola umjesto acetonitrila kao organskog
otapala u pokretnoj fazi značajno mijenja relativni odnos vremena zadržavanja istraživanih
spojeva.
Na temelju slaganja pokazatelja lipofilnosti dobivenih primjenom istraživanih
kromatografskih uvjeta s vrijednostima dobivenim metodom izmućkivanja upotrebom
CAMDIS metode, odabrani su najprikladniji kromatografski uvjeti za lipofilno profiliranje
spojeva iz klase makrocikala. Vrijednosti lipofilnosti dobivene metodom izmućkivanja za
proširenu skupinu makrocikličkih spojeva uspoređene su s onima dobivenima CHI
metodom, optimiziranom metodom i in silico izračunatim vrijednostima. Najbolja korelacija
dobivena je između vrijednosti dobivenih metodom izmućkivanja i metodom za lipofilno
profiliranje makrocikličkih spojeva razvijenom u ovom radu.Macrocyclic compounds give an opportunity to modulate difficult targets while maintaining
good pharmacokinetic properties. Lipophilicity is considered to be one of the most
important physio-chemical properties so it is of utmost importance to have a reliable and
precise method for lipophilic profiling of new macrocyclic compounds in the early phases
of drug discovery process. In this dissertation, the applicability of the literature CHI method
that uses high-performance liquid chromatography for lipophilicity determination was
investigated. An initial set of macrocyclic compounds was analyzed and it was discovered
that the method is less precise for lipophilicity determination of macrocycles due to
chromatographic peak broadening and severe tailing.
Investigation of the influence of experimental chromatographic changes showed that the
chromatographic column temperature, gradient speed and mobile phase flow rate do not
influence the relative relationships between retention times of the macrocyclic compounds.
The use of a different chromatographic column in the same USP classification does not
change the relative relationships between the retention times of analytes but results in
enhanced efficiency of analyses. Significant influence on the analyses was observed when
the concentration of ammonium acetate used as the aqueous part of the mobile phase was
decreased, but also when it was replaced with ammonium hydrogencarbonate. Also,
switching from acetonitrile to methanol as the organic part of the mobile phase resulted in
different elution order of macrocyclic compounds.
Based on the correlation of lipophilicity parameters obtained with investigated
chromatographic conditions with the values obtained with the shake-flask method, the
most suitable chromatographic conditions for lipophilic profiling of macrocycles were
selected. The lipophilicity values obtained with the shake-flask method were compared with
the values obtained with the CHI method, the optimized method as well as in silico
calculated lipophilicity. The best correlation was obtained between the shake-flask method
and the chromatographic method developed in this thesis
The use of monoclonal antibodies in the treatment of Alzheimer's disease
No full textCilj ovog rada je pojasniti patologiju Alzheimerove bolesti (AZ), opisati mehanizme njenog nastanka, te opisati proteine povezane s patologijom bolesti. Rad se posebno fokusira na upotrebu monoklonskih protutijela u terapiji bolesti, s naglaskom na ADUCANUMAB. Rad detaljno opisuje načine proizvodnje protutijela, njihovu terapijsku osnovu, mehanizam djelovanja i učinkovitost u liječenju Alzheimerove bolesti. Alzheimerova bolest je teška neurodegenerativna bolest koja se manifestira kroz niz teško uočljivih simptoma, simptomi mogu biti raznovrsni kao što su na primjer gubitak kratkoročnog pamćenja pa sve do halucinacija i deluzija. Patogeneza uključuje primarno akumulaciju amiloid-β peptida (Aβ) u mozgu i promjene u citoskeletu uzrokovane hiperfosforilacijom Tau proteina. Istraživanja su usmjerena na razumijevanje mehanizama nastanka bolesti, uključujući genetičke čimbenike, kao što su APP, PSEN1, PSEN2 i APOE-e4, i okolišne utjecaje. Metode uključuju pregled literarnih izvora o patogenezi, simptomima i liječenju Alzheimerove bolesti, te laboratorijske tehnike za proizvodnju i testiranje učinkovitosti monoklonskih protutijela. Monoklonska protutijela, uključujući ADUCANUMAB, pokazala su smanjenje amiloidnih plakova u mozgu i poboljšanje kognitivnih funkcija u predkliničkim i kliničkim ispitivanjima. ADUCANUMAB se veže selektivno za agregirane oblike Aβ, aktivirajući makrofage koji razgrađuju plakove bez značajnih nuspojava. Klinička ispitivanja ovog lijeka pokazuju smanjenje Aβ plakova i usporavanje kognitivnog pada iako nisu riješeni svi klinički problemi. Monoklonska protutijela predstavljaju stoga perspektivnu opciju za razvoj liječenja AZ bolesti, ali zahtijevaju dodatna temeljna istraživanja i klinička ispitivanja.The aim of this paper is to clarify the pathology of Alzheimer's disease (AD), describe the mechanisms of its onset, and outline the proteins associated with the disease's pathology. It particularly focuses on the use of monoclonal antibodies in therapy of the disease, with an emphasis on ADUCANUMAB. The paper provides a detailed description of the methods of antibody production, their therapeutic basis, mechanisms of action, and effectiveness in treating Alzheimer's disease. Alzheimer's disease is a severe neurodegenerative condition that manifests through a range of subtle symptoms. Symptoms can vary widely, from short-term memory loss to hallucinations and delusions. Pathogenesis involves the accumulation of amyloid-β peptides (Aβ) in the brain and cytoskeletal changes caused by hyperphosphorylation of Tau proteins. Research is focused on understanding the mechanisms of disease onset, including genetic factors such as APP, PSEN1, PSEN2, and APOE-e4, and environmental influences. Methods include a review of the literature on the pathogenesis, symptoms, and treatment of Alzheimer's disease, as well as laboratory techniques for the production and testing of the effectiveness of monoclonal antibodies. Monoclonal antibodies, including ADUCANUMAB, have shown a reduction in amyloid plaques in the brain and improvement in cognitive functions in preclinical and clinical trials. ADUCANUMAB selectively binds to aggregated forms of Aβ, activating macrophages that degrade the plaques without significant side effects. Phase I, II, and III clinical trials demonstrate a reduction in Aβ plaques and slowing of cognitive decline, although not all clinically significant results have been achieved. Monoclonal antibodies represent a promising option for future treatment but require further research and clinical trials
The effects of the herbal preparation Ilex paraguriensis in the prevention of experimental type 1 diabetes
No full textDijabetes tipa 1 (T1D), autoimuna je bolest posredovana T stanicama koja
napada beta stanice gušterače čija je uloga u proizvodnji inzulina. Trenutno
na tržištu ne postoji učinkovita imunoterapija koja bi prevenirala njegovo
napredovanje. Yerba mate (YM) (Ilex paraguariensis) prirodan je biljni
preparat za koji se pokazalo da ima protuupalna i hipoglikemijska svojstva.
Ova studija je imala cilj ispitati učinke YM u odgodi početka T1D u kontekstu
djelovanja na T limfocite u višestrukim niskim dozama streptozotocina (VNDSTZ)-induciranom eksperimentalnom C57BL/6 mišjem modelu. Korištene su
koncentracije od 4% i 2% YM (w/v) ad libitum tijekom 6 tjedana.
Administracija YM značajno je snizila incidenciju T1D i hiperglikemiju, uz
povećanje tjelesne težine tretiranih miševa. Također, pokazan je trend
smanjenja pojavnosti T1D i razine glukoze u krvi, ali uz pad tjelesne težine,
primjenom 4% YM u pilot eksperimentu na modelu spontano razvijajućeg
T1D u (non-obese diabetic (NOD)) miševa. Ex vivo analiza T-stanica slezene
izoliranih iz VND-STZ-C57BL/6 miševa pokazala je da YM primijenjena u obje
koncentracije nije kompromitirala vijabilnost stanica, uz značajno smanjenje
proliferacije T-stanica, prolazne promjene u imunofenotipovima T-stanica
(samo sa tretmanom 2% YM) i reduciranje proizvodnje proupalnih i
protuupalnih citokina, osobito u tretmanu 4% YM. T-stanice izravno izložene
YM in vitro, pokazale su smanjenju proliferaciju ovisno o dozi, te povećanu
proizvodnju IL-2 uz smanjenje IFN-λ. Dobiveni rezultati sugeriraju
preventivni potencijal YM u eksperimentalnom T1D i u skladu su s drugim
studijama, potvrđujući hipoglikemijsko i imunomodulativno djelovanje YM na
T-stanice.Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells that
attack the insulin-producing beta cells of the pancreas. Currently, there is no
effective immunotherapy on the market that would reverse the progression
of the disease. Yerba mate (YM) or Ilex paraguariensis is a natural herbal
supplement that has been shown to have anti-inflammatory and
hypoglycemic properties. This study aimed to examine the effects of YM in
delaying the onset of T1D in the context of T lymphocytes in multiple low
dose streptozotocin (MLD-STZ)-induced experimental C57BL/6 mouse
model. Concentrations of 4% and 2% YM (w/v) were used ad libitum for 6
weeks. Administration of YM significantly reduced the incidence of T1D and
hyperglycemia, while increasing the body weight of treated mice. Also, a
trend of decreasing the incidence of T1D and decreasing the blood glucose
level, but with a decrease in body weight, was demonstrated by the
application of 4% YM in a pilot experiment on a model of spontaneously
developing T1D non-obese diabetic NOD mice. Ex vivo analysis of splenic Tcells isolated from MLDSTZ-C57BL/6 mice showed that YM administered at
both concentrations did not compromise cell viability, with a significant
reduction in T-cell proliferation, transient changes in T-cell
immunophenotypes (only with 2b% YM treatment) and reduction of the
production of pro-inflammatory and anti-inflammatory cytokines, especially
in the 4% YM treatment. T-cells directly exposed to YM in vitro showed a
dose-dependent decrease in proliferation, an increased production of IL-2
with a decrease of IFN-λ. The obtained results suggest the preventative
potential of YM in the experimental T1D and are consistent with other
studies, confirming the hypoglycemic and immunomodulatory effects of YM
on T-cells
Contamination, cross-contamination and cleaning validation in pharmaceutical industry
No full textPrema zahtjevima dobre proizvođačke prakse, farmaceutska industrija dužna je provoditi validaciju čišćenja kako bi se osigurala kvaliteta i sigurnost proizvoda. Validacija čišćenja je dokumentirani dokaz da su postupkom čišćenja primjereno i dosljedno uklonjeni svi ostaci prethodnog proizvoda, nusproizvodi, mikroorganizmi i ostaci sredstva za čišćenje. Njena svrha je pravovremena identifikacija i ispravljanje mogućih problema. Kako bi se ocijenio postupak čišćenja, potrebno je odrediti kriterij prihvatljivosti – prihvatljivu razinu ostataka. Cilj ovog rada dijeli se na dva dijela. Prvi je analiza i eksperimentalno ispitivanje problema učinkovitosti čišćenja proizvoda na specifičnom mješaču. Laboratorijski su se stimulirali uvjeti čišćenja, analizirajući različite metode pranja. Cilj je utvrditi koji od šest mogućih proizvoda ostavlja zaostatke nakon čišćenja te koja od ispitanih metoda osigurava najbolju razinu čistoće. Ovaj eksperiment je obuhvatio vaganje pločica, nanošenje proizvoda, sušenje te primjenu različitih metoda pranja – pročišćena voda, otopine COSA CIP92 i CIP72 deterdženta i ClearKlens Mega deterdženta. Drugi cilj je usmjeren na validaciju učinkovitosti postojeće metode čišćenja, kako bi se osiguralo da nema ostataka aktivnog sastojka i deterdženta na opremi. Kako bi se potvrdilo da nema zaostataka korištene su metoda HPLC-a i TOC-a. Rezultati eksperimenta ukazuju na to da su primijenjeni deterdženti učinkovitiji od pročišćene vode. Najveće probleme su stvarali proizvodi 2 i proizvod 4 te su zahtijevali specifične pristupe. Na kraju se za proizvod 4 hladna 1%-tna otopina CIP92 deterdženta pokazala učinkovita, dok je za proizvod 2 još uvijek neodgovoreno to pitanje te su potrebne dodatne analize. Validacijom čišćenja potvrđena je učinkovitost postojeće metode. Pokazalo se da su svi uzorci bili unutar prihvatljivih granica.According to the requirements of Good Manufacturing Practice, the pharmaceutical industry is obligated to perform cleaning validation to ensure the quality and safety of products. Cleaning validation is documented evidence that the cleaning process appropriately and consistently removes all residues of previous product, by-product, microorganisms and cleaning agents. Its purpose is the timely identification and correction of potential issues. To evaluate the cleaning process, it is necessary to determine the acceptance criteria - Acceptable Residue Level (ARL). The aim of this thesis is divided into two parts. Firstly, the analysis and experimental examination of the problem of cleaning efficiency of products on a specific mixer. The cleaning conditions were stimulated in the laboratory, analyzing different washing methods. The aim is to determine which of the six possible products leaves residues after cleaning and which of the tested methods ensures the best level of cleanliness. The experiment included weighing the plates, applying the product, drying and applying washing methods – purified water, COSA CIP92 and CIP72 detergent solution and ClearKlens Mega detergent. The second aim is focused on validating the effectiveness of the existing cleaning method to ensure that there are no residues of the active ingredient and detergent on the equipment. The HPLC and TOC methods were used to confirm the absence of residues. The experimental results indicate that the applied detergents are more effective than purified water. The biggest problems were caused by product 2 and product 4, which required specific approaches. In the end, a cold 1%-CIP92 detergent solution was effective for product 4, while the issue with product 4 remains unresolved and requires further analysis. The cleaning validation confirmed the effectiveness of the existing method. All samples were found to be within acceptable limits
Effect of methamphetamine exposure on phenotypic changes in offspring
No full textMetamfetamin, kao i ostali psihostimulansi, uzrokuju mnoge fiziološke
promjene povezane sa misregulacijom dopaminskog signalizacijskog puta u
ljudi. Produljeno korištenje dovodi do promjena u signalizaciji mozga kao
posljedica svojstva neuroplastičnosti, te se određenim epigenetskim
modifikacijama koje utječu na gensku ekspresiju prenosi na potomke te
stvara novi fenotip, unatoč neizloženosti stimulansu.
Cilj ovog rada bio je ispitati kako metamfetamin u formi aerosola
(vMETH) utječe na stvaranje fenotipa s povišenom lokomotornom aktivnosti
u F1 generaciji Drosophile melanogaster. vMETH je administriran metodom
FlyBonga grupi mužjaka, od kojih je 23% izdvojeno s obzirom na najveću
lokomotornu aktivnost te spareno s djevicama koje nisu tretirane vMETHom. Utjecaj izloženost mužjaka je zatim praćen u F1 generaciji mjerenjem lokomotorne aktivnosti.
Rezultati su pokazali statistički veću lokomotornu aktivnost između
tretirane F0 i F1 generacije, no pokazalo se da nema statistički značajne
razlike između kontrolne i tretirane grupe. Rezultati bi mogli ukazivati na
postojanje novog fenotipa koji bi bilo potrebno ispitati kroz više generacija.Methamphetamine and other psychostimulants can instigate many
physiological changes that lead to the misregulation of the dopamine
signaling pathway in humans. Extended use can cause permanent changes
in this signaling pathway due to neuroplasticity, and those changes are
carried into the next generation by certain epigenetic modifications that
affect gene expression. As a result, a phenotype with an increased
locomotor activity is created and persists even without the initial stimuli.
The purpose of this thesis was to investigate how volazilized
methamphetamine (vMETH) affects the creation of the new phenotype in
the F1 generation of Drosophile melanogaster. vMETH is administered using
the FlyBong method to a group of male flies and 23% of them were selected
based on locomotor activity. They were then bred with untreated virgin flies.
The effects of the methamphetamine dose wer observed in the F1
generation by measuring locomotor activity.
Results have shown a statistically higher activity in the F1 treated
group, but there was no statistically significant difference between the
control and the treated group. This could point to the existance of the
mentioned phenotype, which should be investigated through several
generations
DARTS method in the researching of the biological targets of erlotinib
No full textProteomika je jedna od omics metoda koja istražuje funkcije, strukture i ekspresiju svih proteina u biološkom sustavu. Ključna je za razumijevanje mehanizama bolesti, a okuplja neke inovativne tehnike poput masene spektrometrije, Western blottinga i elektroforeze. DARTS metoda (eng. Drug affinity responsive target stability) naziv je za skup tehnika proteomike koje se koriste za istraživanje bioloških meta lijekova. Princip njezinog rada počiva na činjenici da se proteini bolje odupiru digestiji proteolitičkih enzima ukoliko je za njih vezan ligand (lijek). Posebnu ulogu ima u istraživanju nepoznatih bioloških meta malih molekula koje imaju sposobnost ciljanja specifičnih proteina važnih za reguliranje unutarstaničnih signalnih puteva. Uzorci staničnih lizata se tretiraju malom molekulom, izlažu digestiji proteaze te se potom kvalitativno i kvantitativno analiziraju. Posebna prednost DARTS-a je ta što koristi lijek u svom nativnom obliku, bez potrebe modificiranja. Ova metoda koristi se za različite vrste istraživanja, a najviše za istraživanje poznatih i nepoznatih meta lijekova, mehanizama djelovanja lijekova i mehanizama rezistencije.
U ovom radu odabrana mala molekula je erlotinib (Tarceva) čija je poznata biološka meta receptor epidermalnog faktora rasta (EGFR). Erlotinib je inhibitor tirozin-kinaza te vezanjem zaustavlja proliferaciju tumorske stanice i izbjegavanje apoptoze. Uzorke za DARTS metodu, pripremili smo pomoću lizata HeLa i A431 stanica koje pretjerano izražavaju EGFR. Kao proteazni enzim kojim je provođena digestija, korišten je enzim pronaza, a proteini su razdvajani pomoću gradijentne SDS-PAGE elektroforeze, prema molekulskoj masi. Western blottingom potvrđena je specifičnost upotrebom protutijela te je povećana je osjetljivost metode što je rezultiralo boljom vizualizacijom promjene intenziteta EGFR-a za kojeg je vezan erlotinib. Potvrđeno je da je EGFR direktna meta za vezanje erlotiniba te da je njegov mehanizam rada zaustavljanje autofosforilacije i prekid signalnog puta za proliferaciju stanice. Također su istražene i nepoznate mete erlotiniba (tzv. off-tagets) koje pokazuju značajnu promjenu u intenzitetu u njegovoj prisutnosti, no one nisu identificirane. Zaključeno je da je DARTS višenamjenska, brza i jednostavna biofizikalna metoda, koja ne zahtjeva kemijske modifikacije istraživanog lijeka, te je korisna u brojnim aspektima istraživanja i razvoja lijekova.Proteomics one of the omics methods that investigates the functions, structures and expression of all proteins in a biological system. It is important for understanding the disease mechanisms and brings together some innovative techniques such as mass spectrometry, Western blotting and electrophoresis. The DARTS method (Drug affinity responsive target stability) is the name of a set of proteomics techniques used to investigate the biological targets of drugs. The principle of its work is based on the fact that proteins better resist the digestion of proteolytic enzymes if a ligand (drug) is attached to them. It has a special role in the research of unknown targets of small molecules that have the ability to aim at specific proteins important for regulating intracellular signaling pathways. Samples of cell lysates are treated with a small molecule, subjected to protease digestion and then analyzed qualitatively and quantitatively. A special advantage of DARTS is that it uses the drug in its native form, without any modification. This method is used for different types of research, and mostly for research into known and unknown targets of drugs, the mechanisms of action of drugs, but also the mechanisms of resistance.
The small molecule chosen in this thesis is erlotinib (Tarceva), whose known biological target is the Epidermal growth factor receptor (EGFR). Erlotinib is a tyrosine-kinase inhibitor which stops the proliferation of tumor cells and the avoidance of apoptosis when bound. Samples for the DARTS method were prepared using lysates of HeLa and A431 cells that overexpress EGFR. Pronase was the protease enzyme used for digestion, and proteins were separated using gradient SDS-PAGE electrophoresis, according to molecular weight. Western blotting confirmed the specificity using antibodies and increased the sensitivity of the method, which resulted in a better visualization of the change in the intensity of EGFR, to which erlotinib is bound. It was confirmed that EGFR is a direct target of erlotinib, but also that its mechanism of action is stopping the autophosphorylation that interrupts the signaling pathway for cell proliferation. Unknown targets of erlotinib (off-targets), that show a significant change in intensity in its presence, were also investigated, but not identified. It was concluded that DARTS is a multipurpose, fast and simple biophysical method that does not require chemical modifications of the drug and is useful in many aspects of drug research and development