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Utjecaj poremećaja u raspadu mRNA na strukturu RNA 3 kraja na razini genoma
No full textUridylation is a common widespread 3'-end modification in eukaryotes;
however, its impact on mRNA fate is not clear. The mRNA decay mechanism
in S. pombe is quite similar to that in humans, where the first step usually
involves uridylation of the 3´-end of mRNA by terminal uridyltransferases
(TUTases). The aim of this project was to investigate the effects on mRNA
decay and mRNA tails when gene expression levels of the factors involved
in mRNA decay are manipulated. We decided to delete the enhancer of
mRNA decapping gene, edc1, and the exonuclease gene xrn1, while
overexpressing the terminal uridyltransferases genes, cid1 and cid16. The
transformations were based on the cellular intake of a linear cassette and
homologous recombination of the same cassette. The transformed strains
were verified using colony-PCR, and overexpression strains were
additionally verified using western blotting. Subsequently, mRNA was
isolated and purified, and NGS libraries were prepared using a custom GW3´RACE (TAIL-seq) protocol. The samples were then sent for paired-end
Illumina sequencing. The sequencing data was analyzed using bioinformatic
tools, and included read trimming, filtering and genome alignment.
Although the libraries for strains overexpressing cid1 and cid16 did not
work, the cid16 expression dependent strain showed a growth defect. Other
main findings were as follows: deletion of edc1 or xrn1 resulted in the
shortening of mRNA tail length, increased mRNA uridylation, and
accumulation of non-tailed mRNAs. In other words, the deletion of edc1 or
xrn1 produced the same results, namely the accumulation of mRNA decay
intermediaries. Additionally, the differential gene expression analysis
revealed six commonly up-regulated genes between the mutants, with the
most up-regulated gene being ubi4, which encodes for ubiquitin.Uridilacija je česta modifikacija 3'-kraja u eukariotima; međutim, njen
utjecaj na sudbinu mRNA nije jasan. Mehanizam raspada mRNA kod S.
pombe prilično je sličan onom kod ljudi, gdje prvi korak obično uključuje
uridilaciju 3´-kraja mRNA terminalnim uridiltranferazama (TUTazama). Cilj
ovog projekta bio je istražiti učinke na raspad mRNA i repove mRNA kada
se manipuliraju ekspresije gena faktora uključenih u raspad mRNA. Odlučili
smo izbrisati gen edc1, koji je pojačavač dekapiranja mRNA, i gen xrn1,
koji kodira egzonukleazu, dok smo prekomjerno izražavali gene terminalnih
uridiltranferaza, cid1 i cid16. Transformacije su se temeljile na staničnom
unosu linearnog kasetnog elementa i homologne rekombinacije iste kasete.
Transformirani sojevi su potvrđeni korištenjem colony-PCR-a, a sojevi s
prekomjernim izražavanjem su dodatno potvrđeni korištenjem western blot
analize. Nakon toga je mRNA izolirana i pročišćena, a NGS libraries su
pripremljene korištenjem prilagođenog GW-3´RACE (TAIL-seq) protokola.
Uzorci su zatim poslani na paired-end sekvenciranje Illumina tehnologijom.
Podaci sekvenciranja su analizirani korištenjem bioinformatičkih alata,
uključujući podrezivanje očitanja, filtriranje i poravnanje s referentnim
genomom. Iako biblioteke za sojeve s prekomjernim izražavanjem cid1 i
cid16 nisu funkcionirale, soj s prekomjernim izražavanjem cid16 je pokazao
defekt rasta. Ostali glavni rezultati su sljedeći: brisanje edc1 ili xrn1
rezultiralo je skraćivanjem duljine repova mRNA, povećanom uridilacijom
mRNA i nakupljanjem mRNA bez repova. Drugim riječima, brisanje edc1 ili
xrn1 proizvelo je iste rezultate, odnosno nakupljanje intermediarnih
produkata mRNA raspada. Također, analiza diferencijalnog izraza gena je
otkrila šest zajednički up-reguliranih gena između mutanata, pri čemu je
najviše up-regulirani gen bio ubi4, koji kodira ubiquitin
Monodelphis domestica: a new source of mammalian primary neurons in vitro
No full textMammalian central nervous system (CNS) in vitro cell models are mostly derived from the late embryonic or early postnatal mice and rats. Other mammalian species have been less used, meaning that inter-species diversity is not sufficiently investigated and that the additional comparative analyses are required to avoid misinterpretations in translating the knowledge to humans.
We have recently established and extensively characterized the long-term primary dissociated cortical neuronal cultures derived from the neonatal grey South American short-tailed opossums [Monodelphis (M.) domestica] that we propose as a new source of mammalian CNS cells for in vitro developmental and regeneration studies. These cultures are unique for the possibility to obtain embryonic-like CNS cells from the postnatal animals, at wide range of postnatal days
Polimorfizmi s jednim nukleotidom u GABRG2, GRIN2B i ODC1 genima i slovenske žrtve samoubojstva
No full textAs one of the leading causes of death worldwide, suicide is a complex event affected by many factors (such as environment, genetics, and the presence of mental disorder). Differences in genetic background of individuals need to be studied for better understanding of genetics’ part as a risk factor in suicide. The most commonly studied genetic variants are single nucleotide polymorphisms (SNPs). They are single base changes on a specific DNA position, usually with the possibility of being one of two different nucleotides for each position. The majority of researched SNPs in the context of suicide are those in genes of the serotonin signalling pathway, but more recent studies are focusing on genes of other neurotransmitter systems. For example, γ-aminobutyric acid (GABA) as the major inhibitory system with function of maintaining neurochemical balance in a neuronal network. Furthermore, the glutamate signalling pathway showed an important role in synaptic plasticity and excitatory transmission throughout N-methyl-D-aspartate (NMDA) receptors which are in direct contact with polyamine stress response systems.
The suicide rate differs between countries all over the world, with Slovenia being among the ones with a very high rate (19.8 suicides per 100 000 citizens according to 2019 data). In this thesis we analysed polymorphisms of genes involved in mentioned systems: in the GABA neurotransmitter system, the gene GABRG2 (rs424740), in the glutamate pathway, the gene GRIN2B (rs2268115 and rs220557), and in the polyamine system, the gene ODC1 (rs1049500 and rs2302614) to determine which alleles are overrepresented in the group of suicide victims in comparison to the control group. After DNA isolation, alleles of each sample were determined with RT-qPCR. As a result of statistical analysis with the PLINK program, all five polymorphisms showed that there are no statistically significant differences in genotype/allele frequency distribution between controls and completed suicide. Only rs220557 showed statistically significant difference in genotype frequency distribution between suicide cases and control, as well as between suicide case done with violent methods and controls, while rs2268115 showed a difference in genotype frequency distribution between suicides done with non-violent methods and controls. Additionally, rs1049500 showed significant difference in genotype frequency distribution between suicide victims that had grave somatic illness and controls. Furthermore, there was no difference in genotype/allele frequency distribution between polymorphisms and suicide subgroups (males, females, violent, non-violent method, addicted, psychiatric patients, and patients with grave somatic illness) in other SNPs. Although the results were negative, they could still contribute to the understanding of molecular genetics in Slovenian suicide victims.Kao jedan od vodećih uzroka smrti u svijetu, samoubojstvo je kompleksni događaj s mnogo faktora (npr. utjecaj okoline, genetika i prisutnost mentalnog poremećaja). Potrebno je istraživati razlike u genetičkoj pozadini individualaca kako bi se bolje razumio genetički utjecaj kao faktor rizika u samoubojstvu. Najčešće istraživane genetske varijacije su polimorfizmi jednog nukleotida (engl. Single nucleotide polymorphisms – SNPs). To su promjene jednog nukleotida na određenom položaju DNA te je najčešće da postoje dvije mogućnosti različitih nukleotida za jedan par baza DNA. Većina istraživanih SNP-a su oni na genima signalnog puta serotonina, no, nedavna istraživanja posvetila su se genima drugih neurotransmiterskih sistema. Na primjer, γ-aminomaslačna kiselina (GABA) kao glavni inhibitorski sistem s funkcijom održavanja neurokemijske ravnoteže u živčanoj mreži. Nadalje, signalni put glutamata je pokazao da ima važnu ulogu u sinatičkoj plastičnosti i ekcitatorskom prijenosu pomoću N-metil-D-aspartat (NMDA) receptora koji su u direktnom kontaktu s poliaminskim sustavom odgovora na stres.
Stopa samoubojstva se razlikuje među državama diljem svijeta, a Slovenija je među onima koji imaju vrlo visoku stopu (19,8 na 100 000 stanovništva prema podacima iz 2019. godine). U ovom radu analizirali smo polimorfizme gena koji utječu na spomenute sisteme: u GABA neurotransmiterskom sistemu, gen GABRG2 (rs424740), u signalnom putu glutamata, gen GRIN2B (rs2268115 i rs220557) te u poliaminskom sistemu, gen ODC1 (rs1049500 i rs23022614) kako bi se odredilo koji aleli su reprezentativni u grupi žrtava samoubojstva u usporedbi s kontrolnom grupom. Nakon DNA izolacije, aleli svakog uzorka su određeni s RT-qPCR. Potom, kao rezultat statističke analize s PLINK programom, utvrđeno je da svih pet polimorfizma nemaju statistički značajnu razliku u distribuciji frekvencija genotipa/alela između samoubojstva i kontrola. Iznimka je rs220557 koji je pokazao statistički značajnu razliku u distribuciji frekvencija genotipa između slučajeva samoubojstva i kontrole te između slučajeva samoubojstva počinjenih s nasilnim metodama i kontrola, dok rs2268115 je pokazao razliku u distribuciji frekvencija genotipa između slučajeva samoubojstva počinjenih s nenasilnim metodama i kontrola. Također, rs1049500 je pokazao značajnu razliku u distribuciji frekvencija genotipa između žrtva samoubojstva koje su imale tešku somatsku bolest i kontrola. Nadalje, nije utvrđena povezanost između polimorfizama i podgrupa (muškarci, žene, nasilne i ne-nasilne metode, ovisnici, psihijatriski bolesnici te bolesnici s teškim somatskim bolestima) u drugim SNPs. Ovi rezultate pridonose razumijevanju molekularne genetike kod slovenskih žrtva samoubojstva
Funkcionalizacija tinjca 3-aminopropiltrietoksisilanom: utjecaj na vizualizaciju izvanstaničnih vezikula iz cerebrospinalne tekućine ljudi mikroskopijom atomskih sila
No full textExtracellular vesicles (EV) are nanoparticles that are present in biofluids, including human cerebrospinal fluid (CSF). Their visualisation is performed by Electronic Microscopy (EM) and atomic force microscopy (AFM). AFM is advanced nanotechnology that enables the determination of morphology in nanometer resolution. The most common substrate used for visualisation is mica, however, its functionalisation is needed with the aim of stronger binding of EVs, especially in the AFM air mode. (3-Aminopropyl)triethoxysilane (APTES) is an amino silane linker that is often used in the functionalisation of surfaces, whose adsorption on the surface increases the number of positively charged sites for EV binding. Functionalisation improves the electrostatic interaction between the negatively charged EV cell membrane and the positively charged surface of the mica. The aim of the work is to establish a protocol for the functionalisation of mica with APTES and to investigate the effect of binding on visualisation in the air mode of AFM by applying different methods for dehydration and drying of the sample. Treatment of EVs before their visualisation included: fixation with paraformaldehyde (PFA, 3 %) and glutaraldehyde (GA, 1.5 %), dehydration with ethanol or 2,2-dimethoxypropane (2,2-DMP) and drying at either critical point of CO2 (CPD) or with hexamethyldisilazane (HMDS). The most optimal sample preparation protocol for visualisation is the one that involves functionalisation of mica by APTES in vapour, dehydration with 2,2-DMP and HMDS drying. This protocol showed the best overlap in total detected EVs according to diameter and height, highest diameter median and mean and lowest number of outliers out of all other protocols. The detected shapes of EVs were round, “cup-shaped” and multilobed. Future research should focus on methods that can differentiate EVs from other particles of the same outward appearance, like immuno-based interaction of EV antigens with antibodies.Izvanstanične vezikule (IV) su nanočestice koje su prisutne u tjelesnim tekućinama uključujući i cerebrospinalnu tekućinu ljudi. Njihova vizualizacija se provodi elektronskom mikroskopijom i mikroskopijom atomskih sila, engl. Atomic Force Microscopy (AFM). AFM je napredna nano-tehnologija koja omogućava vizualizaciju morfologije u nanometarskoj rezoluciji. Najčešći supstrat koji se koristi za vizualizaciju je tinjac međutim potrebna je njegova funkcionalizacija s ciljem jačeg vezivanje IV-a, pogotovo u zračnom modu AFM-a. 3-aminopropiltrietoksisilan (APTES) je amino-silanski linker koji se često koristi u funkcionalizaciji površina, a čijom adsorpcijom na površinu se povećava broj pozitivno nabijenih mjesta za vezanje IV-a. Time se poboljšava elektrostatska interakcija između negativno nabijene stanične membrane IV-a i pozitivno nabijene površine tinjca. Cilj rada je uspostaviti protokol funkcionalizacije tinjca s APTES-om i istražiti učinak vezivanja na vizualizaciju u zračnom modu AFM-a primjenjujući različite metode sušenja uzorka. Prije vizualizacije IV-e su fiksirane otopinom paraformaldehida (PFA, 3 %) i glutaraldehida (GA, 1.5 %), dehidrirane etanolom ili 2,2-dimetoksipropanom (2,2-DMP) i osušene pri kritičnoj točki CO2 ili heksametildisilazanom (HMDS). Optimalni protokol pripreme uzorka za vizualizaciju uključuje: fukcionalizaciju tinjca isparavanjem APTES-a, dehidraciju sa 2,2-DMP-om i sušenje HMDS-om. Ovaj protokol je pokazao najbolje preklapanje promjera i visine po ukupnom broju IV-a, najveći medijan promjera i srednju vrijednost te najmanji broj čestica koje odstupaju u usporedbi s ostalim protokolima. Detektirane čestice bile su okruglog, višerežnjastog i konkavnog oblika. Budući da su ove metode usredotočene na otkrivanje IV-a prema njihovom obliku, promjeru i visini, naredna istraživanja trebala bi se usredotočiti na metode koje mogu razlikovati IV-e od drugih čestica istog ili sličnog vanjskog izgleda, primjerice imunointerakcija antigena IV-a s protutijelima
The role of ATF3 in neuronal differentiation and development of neuronal networks in opossum postnatal cortical cultures
No full textActivating transcription factor 3 (ATF3), a member of the ATF/cAMP response element binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury
Personalised nutrition
No full textPrehrana je prepoznata po korisnim učincima u prevenciji bolesti i održavanja zdravlja ljudi, ali i prepoznatljiv čimbenik koji pridonosi čestim bolestima, uključujući bolesti krvožilnog sustava i srca, neurodegenerativne bolesti i bolesti mozga, dijabetes tipa 2 i raka.
Personalizirana prehrana koristi specifične informacije za pojedinca, utemeljene na znanstvenim dokazima, u promicanju promjene prehrambenog ponašanja koja može rezultirati mjerljivim zdravstvenim prednostima. Povezana je s individualnim genetskim, fenotipskim, medicinskim, prehrambenim i drugim važnim informacijama koje su namijenjene specifičnim smjernicama za zdravu prehranu i prehranu prema potrebi.
Iako pristup personalizirane prehrane u sustavu javnog zdravstva u budućnosti obećava, potrebna su daljnja istraživanja. Ona obuhvaćaju široko dostupne podatke (demografske, životne navike) i manje dostupne podatke (genetske informacije i informacije dobivene omics metodama - analize proteomike, metabolomike, transkriptomike, mikrobioma, ksenometaboloma).Diet is known for its beneficial effects in preventing disease and maintaining human health, but it is also recognized as a contributing factor to common diseases, including cardiovascular and heart disease, neurodegenerative and brain diseases, type 2 diabetes, and cancer.
Personalised nutrition uses individualised information based on scientific evidence to promote dietary behavior change that can lead to measurable health benefits. It is linked to individual genetic, phenotypic, medical, nutritional, and other important information to provide specific healthy eating guidelines as needed.
Although the public health approach to personalised nutrition is promising, more research is needed in the future. This includes commonly available data (demographics, lifestyle habits) and less accessible data (genetic information and information obtained through omics methods - analysis of proteomics, metabolomics, transcriptomics, microbiome, xenometabolome)
Analysis of PI3P-binding proteins with FYVE domain in platelet transcriptome and proteome
No full textStanje uravnoteženog proteoma – proteostaza, ključno je za sve fiziološke procese u organizmu, te je razina pojedinih proteina određena brzinom njihove sinteze, kao i razgradnje te dostupnosti i razine transkripata. Jedna od važnih obitelji fosfolipida u staničnim membranama koji stupaju u interakciju s proteinima je obitelj fosfoinozitida. Svaki odjeljak membrane sadrži neke od karakterističnih fosfoinozitida (ukupno 7 različitih): fosfatidilinozitol-3-monofosfat (PI3P), fosfatidilinozitol-4-monofosfat (PI4P), fosfatidilinozitol-5-monofosfat (PI5P), fosfatidilinozitol-3,4-difosfat [PI(3,4)P2], fosfatidilinozitol-3,5-difosfat [PI(3,5)P2], fosfatidilinozitol-4,5-difosfat [PI(4,5)P2] i fosfatidilinozitol-3,4,5-trifosfat [PI(3,4,5)P3]. PI3P čini otprilike 15% ukupnih fosfoinozitida u ljudskim stanicama, shodno tome, ovdje se daje detaljna analiza funkcije PI3P, specifične FYVE domene koju veže te proteine koji sadrže FYVE domenu. Osim FYVE domene, PI3P veže i Phox te plekstrin homolognu domenu.
U ovom radu analizirani su dostupni podaci transkriptoma i proteoma ljudskih i mišjih trombocita na prisustvo proteina s FYVE domenom te je napravljena usporedba između trombocita ovih dviju vrsta. Korištenjem dostupnih baza DepMap i Međunarodnog konzorcija za fenotipizaciju miševa analizirana je uspješnost stvaranja staničnih linija s delecijom za proteine s FYVE domenom koji su visoko eksprimirani u ljudskom i mišjem proteomu kao i posljedice nedostatka odabranih gena u miševa na njihovu letalnost, i/ili hematološki fenotip. Nadalje, dan je pregled mehanizma kojim PI3P regulira nastanak i funkciju trombocita, ali i općenito kako disfunkcija stvaranja PI3P utječe na različite procese u stanici ili u organizmu.The state of a balanced proteome - proteostasis, is crucial for all physiological processes in the organism, the level of individual proteins is determined by the speed of their synthesis, as well as their degradation and availability and transcript levels. One of the important families of phospholipids in cell membranes that interact with proteins is the phosphoinositide family. Each compartment of the membrane contains some of the more characteristic types of phosphoinositides (total of 7 different ones), of which there are 7: phosphatidylinositol-3-monophosphate (PI3P), phosphatidylinositol-4-monophosphate (PI4P), phosphatidylinositol-5-monophosphate (PI5P), phosphatidylinositol-3,4-diphosphate [PI(3, 4)P2], phosphatidylinositol-3,5-diphosphate [PI(3,5)P2], phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] and phosphatidylinositol-3,4,5-triphosphate [PI (3,4,5)P3]. PI3P accounts for approximately 15% of the total phosphoinositides in human cells, accordingly, a detailed analysis of PI3P function as well as the specific FYVE domain that it binds and FYVE domain-containing proteins is provided here. In addition to the FYVE domain, PI3P was found to bind Phox and pleckstrin homologous domains.
In this paper, the available transcriptome and proteome data of human and mouse platelets were analyzed for the presence of proteins with the FYVE domain, and a comparison was made between the platelets of these two species. Using the available databases of DepMap and the International Mouse Phenotyping Consortium, the success of creating cell lines with deletions for proteins with the FYVE domain that are highly expressed in the human and mouse proteome was analyzed, as well as the consequences of the lack of selected genes in mice on their lethality and/or hematological phenotype. Furthermore, an overview is given of the mechanism by which PI3P regulates the formation and function of platelets, but also in general how the dysfunction of PI3P formation affects various processes in the cell or in the organism
Validation of premises and equipment cleaning from the perspective of good manufacturing practice
No full textInspekcija dobre proizvođačke prakse (DPP) kod proizvođača lijekova je složen postupak za koji je potrebno planiranje i sagledavanje svih kritičnih postupaka u proizvodnji koji mogu utjecati na proizvodnju lijekova te posljedično imati utjecaj na pacijenta. U kritične procese prilikom proizvodnje se svakako ubraja čišćenje prostorija i opreme koje treba biti dobro definirano te validirano. Validacija čišćenja prostorija i opreme je dokumentirani postupak kojim se dokazuje da postupci čišćenja uklanjaju ostatke djelatnih ili pomoćnih tvari, sredstava za čišćenje ili drugih onečišćenja do unaprijed definirane granice određene kriterijima prihvatljivosti. Provedba provjere valjanosti čišćenja mora biti opisana u protokolu validacije i zabilježena u izvješću o validaciji. Izmjene u postupku čišćenja potrebno je procijeniti kroz proces kontrole izmjena, a u skladu s procjenom rizika potrebno je provesti revalidaciju. U ovome su radu razmatrana dva pristupa validacije čišćenja na temelju primjera iz prakse. Pristup validaciji čišćenja specifičan za proizvod razmatran je na primjeru proizvodnje sterilnog proizvoda koji sadrži djelatnu tvar busulfan te je izračunato MACO ograničenje za prijenos djelatne tvari uspoređeno s vrijednostima koje u protokolu validacije čišćenja navodi proizvođač. Pristup validaciji čišćenja specifičan za opremu razmatran je na drugom primjeru proizvodnje šest sterilnih proizvoda na istoj proizvodnoj liniji, a za validaciju čišćenja odabran je sterilni proizvod koji sadrži djelatnu tvar desmopresin kao najkritičniji na temelju ADE vrijednosti. Procjenom rizika utvrđeno je da validacija čišćenja ne podržava proizvodnju injekcije eptifibatida nakon proizvodnje injekcije desmopresina. Validacija čišćenja podržava proizvodnju ostalih injekcija u proizvodnoj liniji nakon proizvodnje injekcije desmopresina.Good manufacturing practice (GMP) inspection at medicines manufacturing sites is a complex procedure which includes meticulous planning. Within the inspection an overview of all processes must be performed, especially ones that are considered to be critical for the product quality and consequently for the patient. Cleaning of premises and equipment is considered to be a critical process hence it should be well defined and validated. Cleaning validation is a documented procedure that proves that removal of the of the traces of active substances and excipients, detergents or any other defined impurities is conducted successfully up to the set limit. Validation protocol has to document the validity cleaning procedures checks and later documented in the report. Any changes in the validated cleaning procedure should risk assessed and the result could be a re-validation. In this thesis two different approaches found in the practice were explained and assessed using a real life examples. Approach specific for the single sterile medicine production was assessed based on example of manufacturing of the sterile product containing active pharmaceutical ingredient busulfan. MACO limit for busulfan is calculated and compared with the values given by manufacturer in validation protocol. Equipment-specific approach was assessed based on another example of the production of six sterile medicine products on the same production line, and for cleaning validation the sterile product containing the active pharmaceutical ingredient desmopresin was selected as the most critical based on the ADE value. The risk assessment determined that cleaning validation does not support production of eptifibatide injection after production of desmopresin injection. Cleaning validation supports the production of other injections in the production line after the production of desmopressin injection
Mogućnosti liječenja sezonske depresije
No full textSeasonal affective disorder (SAD) is a subtype of major depressive disorder. It is a multifactorial disorder characterised by the seasonal appearance of its symptoms such as depressed mood, hypersomnia or insomnia, increased or reduced appetite... There are two types of seasonal affective disorder, based on the season when the symptoms appear – summer and winter type. The cause of the disorder has not yet been found, but there are a couple of hypotheses that suggest the possible origin of the disorder. They are called the photoperiodic and phase shift hypotheses, also known as the chronobiological hypotheses. Aside from them, the serotonin and melatonin disbalance may also contribute to the disorder. The goal of this thesis was to discover various treatment options for seasonal affective disorder. Well-known and used treatments are light therapy, cognitive behavioural therapy and pharmacotherapy. No treatment is significantly superior to the others, at least not in all aspects. Light therapy gives the fastest results, but cognitive behavioural therapy shows greater potential to lower the chances of relapse in the next season. Pharmacotherapy is mostly used as a combination treatment with either of the other two. Right now only two drugs have been found to work almost as effectively as light therapy – fluoxetine and bupropion, but they also have side effects. Two other treatments are dawn simulation and negative air ion therapy. Their effect is a little weaker but are still used in patients with milder symptoms. Because all mental illnesses are very hard to treat universally, medical doctors prescribe a combinatorial treatment consisting of more therapies to treat the symptoms in the best possible way for each patient individually.Sezonska depresija je podvrsta kliničke depresije. To je multifaktorijalni poremećaj kojeg karakterizira sezonska pojava simptoma kao što su depresivno raspoloženje, hipersomnija ili insomnija, povećan ili smanjen apetit... S obzirom na godišnje doba u kojem se simptomi pojavljuju, razlikujemo zimski i ljetni tip sezonske depresije. Iako je uzrok poremećaja i dalje nepoznat, postoji nekoliko teorija koje predlažu mogući uzrok. Radi se o teorijama fotoperioda i faznih pomaka, zajedno nazvanim i kronobiološke teorije. Neuravnoteženost serotonina i melatonina bi također bi također mogle pridonijeti poremećaju. Cilj ovog rada je proučiti različite metode liječenja ovog poremećaja. Dobro poznati i najčešće korištene metode su terapija svijetlom, kognitivno-bihevioralna terapija i farmakoterapija. Ni jedna metoda se ne smatra najboljom, barem ne u svim aspektima. Terapija svijetlom pokazuje najbrže rezultate, ali kognitivno-bihevioralna terapija ipak pokazuje najveći potencijal u sprečavanju ponovne pojave simptoma u sljedećoj sezoni. Farmakoterapija se uglavnom koristi kao kombinacija sa jednom od dvije prethodno navedene metode. Trenutno su samo dva lijeka uspješna kao i terapija svijetlom, fluoxetin i bupropion, ali oni imaju i svoje nuspojave. Druge dvije metode koje se malo rijeđe koriste za liječenje su simulacija zore i terapija negativnim ionima iz zraka. One imaju malo blaži efekt, ali se zato koriste kod pacijenata sa blažim simptomima. Sve mentalne poremećaje je teško liječiti na jedan univerzalan način, tako i za sezonsku depresiju liječnici pripisuju kombinaciju dostupnih metoda kako bi najbolje tretirali svakog pojedinog pacijenta
Utišavanje ATF3 u primarnim kulturama korteksa neonatalnog oposuma Monodelphis domestica
No full textThe mammalian central nervous system (CNS) develops primarily during the embryonal period, but final structuring and tuning occurs postnatally. Before or soon after birth, the mammalian CNS loses its ability to fully regenerate after injury. Therefore, neuroregeneration studies are mostly conducted on rodent embryos (mice or rats) or derived in vitro preparations, such as primary cell or tissue cultures. However, an intrauterine surgical procedure is necessary to access rodent embryos, which usually means sacrificing the mother - an ethically questionable issue. Therefore, to study regenerative mechanisms of the CNS, we have used an alternative experimental animal: the grey South American short-tailed opossum (Monodelphis domestica), a marsupial without a pouch. Opossum neonates are very immature, and their CNS develops mostly postnatally, with the cortical plate not observed until postnatal day (P)3-5. Therefore, harming the mother is not necessary in order to investigate CNS development in opossums. Furthermore, opossums are unique among mammals in their ability to successfully regenerate the spinal cord after injury during the first two weeks of their life. After that period, the regenerative capability of their spinal tissue is lost. Thus, the first aim of this work was to characterize primary cortical cell cultures obtained from opossums at two developmentally different ages, P3-5 and P16-18, using immunofluorescent microscopy. The results showed a high percentage of neurons in primary cultures derived from both P3-5 and P16-18 opossums (>96% and >80% at DIV7, respectively). The second aim was to genetically silence activating transcription factor 3 (ATF3) in opossum primary cortical cultures, via transfection with specific siRNA molecules, to further investigate its role in neuroregeneration. The cell transfection protocol was optimized to reach the optimal transfection efficiency, and the ATF3 gene and protein levels were assessed with quantitative real-time PCR and Western blot, respectively. Altogether, the results indicate that primary cortical cultures obtained from opossum M. domestica are favorable preparation that can be used to investigate mechanisms underlying CNS regeneration, particularly the involvement of regeneration-related transcription factors, such as ATF3.Središnji živčani sustav (SŽS) sisavaca se razvija primarno tijekom embrionalnog razvoja, a funkcionalno sazrijevanje živčanih mreža i procesa događa se postnatalno. Prije ili nedugo nakon rođenja, sisavci gube sposobnost potpune regeneracije SŽS-a nakon ozljede. Većina istraživanja regeneracije SŽS-a provodi se na embrijima glodavaca (miševa ili štakora) ili in vitro preparatima, poput primarnih staničnih ili tkivnih kultura. Embrijima se ne može pristupiti bez intrauterine operacije, a to obično podrazumijeva žrtvovanje majke što je etički upitno. Iz tog razloga, za istraživanje regenerativnih mehanizama SŽS-a odlučili smo se za alternativni organizam: sivi kratkorepi oposum (Monodelphis domestica), tobolčar bez posteljice. Mladunci oposuma se rađaju vrlo nezreli, te se njihov SŽS većinom razvija postnatalno, kortikalnu ploču, primjerice, nije moguće uočiti do postnatalnog dana (P)3-5. Dakle, razvoj SŽS-a se kod oposuma može istraživati bez potrebe da se pritom šteti majci. Nadalje, oposumi su posebni među sisavcima jer u prva dva tjedna života mogu uspješno regenerirati leđnu moždinu nakon ozljede. Nakon tog razdoblja, mogućnost regeneracije moždanog tkiva se gubi. Stoga, prvi cilj ovog rada je bio pomoću imunofluorescentne mikroskopije opisati primarne stanične kulture korteksa pripremljene od dvije razvojno različite dobi oposuma, starih P3-5 i P16-18. Rezultati su pokazali visok postotak neurona u staničnim kulturama pripremljenih od P3-5 (>96%) i P16-18 (>80%) oposuma nakon 7 dana u kulturi. Drugi cilj bio je genetski utišati aktivirajući transkripcijski faktor 3 (ATF3) u primarnim staničnim kulturama korteksa oposuma koristeći metodu transfekcije sa specifičnim siRNA molekulama, kako bi se pobliže istražila njegova uloga u neuroregeneraciji. Protokol je bio optimiziran kako bi se postigla optimalna učinkovitost transfekcije, a razine ATF3 mRNA i proteina, bile su provjerene kvantitativnim PCR-om u realnom vremenu, odnosno Western blot-om. Zaključno, rezultati ovog rada ukazuju na to da su primarne stanične kulture korteksa oposuma M. domestica poželjan preparat za istraživanje mehanizama regeneracije SŽS-a, osobito u istraživanju uloge transkripcijskih faktora povezanih s regeneracijom kao što je ATF3