Browsing by discipline "Biochemistry"

Proteostasis is used by cells to maintain proteome health and understanding the biological mechanisms underpinning proteostasis is important. Despite many studies on small molecule-mediated inhibition of Sec61-dependent protein translocation, a knowledge gap exists in the quality control pathway(s) of pharmacologically-displaced secretory polypeptides. Genetic screens can be used to discover proteostasis regulators of pharmacologically-displaced secretory polypeptides. Near-haploid human HAP1 cells with an inducible Tet-on GFP reporter (reporter HAP1 cells) can be used as a cellular tool to screen for human host factors pertinent to proteostasis of secretory polypeptides. The isolation of haploid-enriched reporter HAP1 cells ensures that the inability to efficiently recover bi-allelic gene trap mutants is avoided. The use of haploid-enriched cells is a prerequisite to gene trap mutagenesis screens. Here, I present data on the isolation of haploid-enriched reporter HAP1 cells that could be used as a cellular tool in gene trap mutagenesis screens. A workflow for the isolation of haploid-enriched reporter HAP1 cells was optimised using a diploid reporter HAP1 cell line as a control. Both DNA content analysis and karyotyping showed that the isolated HAP1 reporter cell lines were haploid. In the haploid-enriched HAP1 reporter cells, the GFP-reporter compartmentalised in the ER, and a Sec61-translocon inhibitor CT8 could inhibit the GFP-mediated fluorescence. The haploid reporter HAP1 cell lines produced in this study are suitable for future gene-trap mutagenesis experiments.

Lymphedema is a progressive disease, resulting from abnormalities of the lymphatic system. It is characterized by swelling of one or more parts of the body, due to impaired lymph transport. Lymphedema has no definitive treatment and it can have serious effects on the patients' quality of life. However, the recent knowledge expansion of the molecular mechanisms regulating lymphangiogenesis provides new possibilities for the treatment of lymphedema. One of these mechanisms is the angiopoietin-TIE system. It has been shown recently that ANG2 is essential for the formation of lymphatic vasculature. Mice ANG2-deletion caused widespread lymphatic dysfunction, resulting in subcutaneous edema and chylous ascites. Lymphatic development is also regulated by TIE1 receptor, as its deletion in mice resulted in malformed jugular lymph sacs and severe edema. In this study, a model of autocrine TIE receptor activation has been established in endothelial cells to investigate the effect of the WT-ANG2 and four ANG2 mutants (T299M, N304K, C435S and R492Q) in TIE receptor phosphorylation and lymphedema. The role of these variants in direct cell adhesion has also been investigated in vitro using HeLa and lymphatic endothelial cells. The findings revealed that WT-ANG2 and soluble ANG2 variants induced both TIE2 and TIE1 activation in endothelial cells. We also found that N304K, C435S and R492Q mutants are secretion-deficients and retain the co-expressed WT-ANG2 inside the cells, causing a dominant negative effect in both TIE2 and TIE1 receptor activation which is likely associated with the lymphedema in the patients where the mutants were identified.

Cornea is transparent layer of cells lying in front of lens. The corneal epithelium, a squamous epithelium, covers the ocular surface and ensures proper vision by preserving the integrity of the eye. Corneal epithelium is renewed continuously throughout life from a pool of stem cells (SC). There are still conflicting theories about the localization of stem cells required for the growth, renewal and maintenance of the corneal epithelium. Previous studies demonstrated that the limbus, located in the periphery of the cornea, serves as the stem cell niche (SCN) in adults. However, contrasting evidence from clonal analysis proposes that, in early postnatal life, renewal is fuelled by SCs located in the basal layer of the central cornea. There are alternate patterns of renewal in young and adult mouse cornea and that there is an important, transitional time frame called cornea maturation, when the adult patterns of gene expression, cell dynamics and tissue renewal are established. In the cornea, solid SC markers are still missing, yet studies on human limbal cells have suggested Bmi1 and C/EBPδ as limbal SC markers. There are, indeed, long-lived SCs in the central cornea and that the gene Bmi1 plays a role in these central corneal SCs. However, the physiological importance of these Bmi1+ cells remains obscure. The main aim of this project is to understand the fate and dynamics of these Bmi1+ cells and study the chronology of maturation of the cornea. In this study, I have also tried to correlate the growth of eye size with proliferation of corneal epithelial cells This study was conducted using few different kinds of transgenic mice (Mus musculus). To study the fate of Bmi1+ cells, two different mouse lines were crossed: Bmi1-CreER and ROSA26-LacZ. Mice carrying both alleles were used for lineage tracing experiments. Moreover, Hematoxylin-eosin staining was used to follow the eye morphology. Immunohistochemistry was performed to follow the chronology of maturation of the cornea, proliferation of corneal epithelial cells and the location of Bmi1+ cells in corneal epithelium. From this study, we can propose that cornea maturation is completed by the time of eyelid opening, which take place two weeks after birth. Krt19 is perfect for studying the chronology of the corneal epithelium, immunostaining of Krt19 separates the territory of limbus from central cornea enabling to distinguish limbus distinctly. Proliferating cells reside in basal layer of cornea. Bmi1+ cells found throughout the basal layer of the cornea that locally renews the corneal epithelium concluding Bmi1+ cells as the progenitor cells.

Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.

Gene therapy trials are becoming more common place with the first approved products having arrived onto markets within the last 5 years. Gene therapy trials have focused on diseases that are monogenic and curable through the reintroduction of autologous, gene-corrected hematopoietic stem cells with promising results. A popular method for gene-correction is through the integration of the wild-type gene into the patient’s genome with a class of retroviruses called lentivirus. APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) is a rare, monogenic autoimmune disease that is caused by a recessive mutation within the AIRE gene coding region. APECED has a high prevalence within the Finnish population (1/25,000) and is theoretically curable through the transplantation of autologous gene-corrected hematopoietic stem cells. Therefore, creating and studying a lentivector carrying the endogenous AIRE promoter and coding region could provide insight and a preliminary foundation on how to begin to develop a viable gene therapy for APECED. The aim of this thesis was to generate and characterise the first lentivector containing the wildtype AIRE gene, specifically the proximal AIRE promoter and the AIRE open reading frame (ORF). The lentivector was constructed and then transfected into a human cell-line, HEK293T, from which qPCR and immunohistochemistry were used to detect for AIRE mRNA and protein, respectively. The presence of significant amounts of AIRE mRNA and protein indicated that the constructed plasmid was transcriptionally functional. It is the first plasmid to have Aire transcription regulated by its endogenous promoter and can provide future insight into gene regulation. As a lentiviral plasmid, it demonstrates the integrating of the AIRE gene into an existing lentiviral system and serves as a first step and a technical proof-of-concept in developing a gene therapeutic cure for APECED.

Eph (Erythropoietin producing hepatocellular) receptors and their membrane-bound ligands, ephrins, form the largest family of receptor tyrosine kinases in mammals. They regulate functions such as cell migration and axon guidance during development, and wound healing and tissue boundary maintenance in mature tissues for example. Due to the membrane-bound nature of the ephrin ligands, Eph-ephrin signalling can proceed in two directions: forward (Eph-expressing cell) and reverse (ephrin expressing cell). In addition to its critical physiological functions in development and tissue homeostasis, the Eph-ephrin system has also been implicated in multiple diseases, including several cancers, in which the aberrant expression of Eph receptors and ephrins are often present. The EphA2 receptor for example has been identified as an oncogene with a dual mode of action as either tumor suppressor or an oncogene through distinct phosphorylation statuses of the receptor. High-grade serous ovarian cancer is the most common subtype of ovarian cancer, and the deadliest gynaecological malignancy with a dismal five-year survival rate. High-grade serous ovarian cancer does not present symptoms at an early stage, yet it quickly progresses into forming peritoneal metastases by cell shedding from the primary tumour. Small patient cohort studies have given indications of the correlation between the Eph-ephrin system and survival in ovarian cancer. This aim of this study was to investigate the impact of the Eph-ephrin system in high-grade serous ovarian cancer using a large patient cohort mRNA expression dataset to obtain survival association data of proteins of interest used with cell-based studies and the analysis of clinical samples in the form of tumor microarrays and fresh primary samples to investigate the functions of the found proteins of interest. A 428-patient The Cancer Genome Atlas high-grade serous ovarian cancer microarray mRNA dataset was analysed using the Kaplan-Meier estimator for each Eph-ephrin family member. Cell based studies were performed with recombinant ephrin treatments and ephrin knockdowns. These data were analysed using Western blotting and immunofluorescence stainings. Clinical high-grade serous ovarian cancer samples obtained from Turku University Hospital were analysed using immunohistochemistry, immunofluorescence stainings, and mRNA sequencing. EfnA5 had a significant correlation of high expression and poor survival, which is atypical to ephrins. Low EfnA3 correlated with poor survival. High levels of known oncogenes EphA2 and EphA4 also correlated with poor survival. EfnA5 treatment resulted in increased oncogenic EphA2 signaling in comparison with canonical Eph-ephrin signalling mediated by EfnA1. Knockdown of EfnA5 increased canonical, tumour suppressive EphA2 signaling, while EfnA1 knockdown increased oncogenic EphA2 signaling. Immunohistochemical analysis of tumour microarrays with multiple ovarian cancer subtypes displayed an association between the highly malignant high-grade serous subtype and EfnA5 expression. In addition to this, EfnA5 expression was increased during high-grade serous ovarian cancer progression. The Eph-ephrin system is implicated in the survival associations of multiple cancers, but the exact functions facilitated by Eph-ephrin signalling in cancer have remained relatively unknown, with the exception of EphB4-EfnB2 driven angiogenesis. This study offers insights into oncogenic Eph-ephrin signalling in ovarian cancer, displaying that oncogenic EphA2 functions can be altered by ephrins in addition to the known kinase crosstalk pathway. The noncanonical nature of EfnA5 is highlighted by its oncogenic functions in comparison to typical Eph-ephrin signalling, and the significant increase of EfnA5 expression during high-grade serous ovarian cancer progression and association with this highly malignant subtype of ovarian cancer. Although the reverse signalling effects of EfnA5 were not studied, this study highlights the importance of ephrins in Eph-ephrin signalling in cancer, presenting that the focus should not be only on the Eph receptors when studying the oncogenic signalling facilitated by the Eph-ephrin system.

The Hodgkin and Huxley (HH) model of action potential has become a central paradigm of neuroscience. Despite its ability to predict action potentials with remarkable accuracy, it fails to explain several biophysical findings related to the initiation and propagation of the nerve impulse. The isentropic heat release and optical phenomena demonstrated by various experiments suggest that action potential is accompanied by a transient phase change in the axonal membrane. In this study a method was developed for preparing a giant axon from the crayfish abdominal cord for studying the molecular mechanisms of action potential simultaneously by electrophysiological and optical methods. Also an alternative setup using a single-cell culture of an Aplysia sensory neuron is presented. In addition to the description of the method, the preliminary results on the effect of phloretin, a dipole potential lowering compound, on the excitability of a crayfish giant axon are presented.

Yhdeksän valmennettua ratsuhevosta suorittivat kilpailurasitustestin juoksumattotestinä viidesti kahden viikon välein. Puoli tuntia kilpailurasitustestin päättymisestä hevosille letkutettiin mahalaukkuun vettä tai liuoksia, joista yksi oli pelkkä glukoosi-elektrolyyttiliuos, toinen sisälsi leusiinia ja kolmas propionihappoa. Yksi ryhmä sai peroraalisesti betaiinia kahden viikon ajan ennen rasitustestiä. Vuorokauden kuluttua kilpailurasitustestin alusta hevoset suorittivat juoksumatolla standardoidun rasitustestin, jonka avulla tutkittiin hevosten palautumista edellisen päivän kilpailurasitustestistä. Hevosista otettiin rasitustestien aikana ja niiden välillä veri- ja lihasnäytteitä, joista tutkittiin glukoosi-, rasvahappo-, triglyseridi-, insuliini-, glukagoni- ja glykogeenipitoisuudet ja kahden lihassolujen metaboliaa kuvaavan entsyymin, sitraattisyntaasin (CS) ja 3-hydroksiasyyli-koA dehydrogenaasin (HAD), aktiivisuudet. Lihasrasituksen keston ja intensiteetin mukaan lihasrasituksessa rekrytoidaan eri lihassolutyyppejä ja käytetään erilaisia energialähteitä. Toistuvalla lihasharjoittelulla saattaa lihasten solutyypeissä ja solujen koossa tapahtua muutoksia. Kestävyysharjoittelulla lihassolujen oksidatiivinen kapasiteetti kasvaa, ja lihassolujen hiilihydraatti- ja rasva-aineenvaihduntaa kuvaavien entsyymien (CS ja HAD) aktiivisuudet nousevat. Palautumisvaiheessa liikuntarasituksen jälkeen lihasten glykogeenivarastoja täydennetään. Insuliini lisää glukoosin pääsyä lihassoluun ja stimuloi glykogeenisyntaasientsyymiä. Leusiinin tiedetään lisäävän insuliinin eritystä. Tämän tutkimuksen aikana ei hevosten lihassolujen solutyypeissä, poikkileikkauspinta-aloissa eikä solutyyppien mukaisissa pinta-alojen prosentuaalisissa osuuksissa tapahtunut muutoksia. Seuratuista metaboliaa kuvaavista entsyymeistä sitraattisyntaasin aktiivisuus nousi tutkimuksen aikana. Tässä tutkimuksessa tutkittiin edellä mainittujen liuosten vaikutusta hevosen palautumiseen liikuntarasituksen jälkeen, mutta mikään käsittely ei merkittävästi nopeuttanut koehevosten lihasten glykogeenivarastojen palautumista. Annettu leusiini lisäsi insuliinieritystä palautumisvaiheessa, propionihappo vähensi sitä.

Iän ja harjoittelun vaikutus lihassyykoostumukseen suomenhevosella -tutkielman tavoitteena oli selvittää, miten lihassyykoostumus muuttuu suomenhevosvarsan kehittyessä ja miten lihassyykoostumus vaikuttaa hevosen kilpailumenestykseen. Aiemmissa tutkimuksissa on havaittu muilla roduilla nopeasti supistuvien aerobisten ( IIA ) ja hitaasti supistuvien aerobisten lihassyiden ( I ) lisääntyvän ja nopeasti supistuvien glykolyyttisten ( II B ) vähenevän iän ja harjoittelun myötä. Seurantajakso aloitettiin ennen varsojen valmennuksen aloittamista kaksivuotiaana ja lopetettiin niiden ollessa neljä - viisivuotiaita. Kilpailumenestystä seurattiin kuusi - seitsemänvuotiaaksi. Seuranta tehtiin ottamalla takaraajan suuresta gluteaali-lihaksesta biopsioita puolen vuoden - vuoden välein ja tunnistamalla saaduista biopsiosta histokemiallisen myosiiniATPaasi-värjäyksen avulla I-, IIA- ja IIB-tyypin lihassyyt. Tutkimuksessa havaittiin I-tyypin solujen määrän lisääntyminen ja IIB-tyypin solujen vähentyminen. Muutos ajoittui suurimmaksi osaksi kaksivuotis- ja kolmevuotiskevään väliseen aikaan. Lihassyytyypin yhteyttä kilpailumenestykseen ei voitu osoittaa