Development of adenoviral vectors for monitoring telomerase activity in living cells

ABSTRACT Telomerase activity is a potential molecular marker for cancer and primitive cells in regenerative tissues. The most commonly used method to measure telomerase activity in cells is the telomerase reverse amplification protocol (TRAP) assay, where the protein extracts derived from homogenized cells are analysed. In this thesis we focused on developing a method where telomerase activity could be monitored and used to separating cells with high telomerase activity without killing them. We used the promoter sequence of human telomerase reverse transcriptase (hTERT) for transcriptionally controlling a destabilised green fluorescence protein (d2EGFP) reporter gene since there is a strong correlation between hTERT transcriptional activity and telomerase activity in cells. Moreover, we chose to work with adenoviral vectors due to their high level of transient gene expression as a gene transfer system. Unfavourably, adenoviral vector transduction of tumour cells has been hampered by low expression of the coxsackie B and adenovirus receptor (CAR) on the host cells, which is the attachment receptor for the most commonly used adenoviral vector (Ad5). Thus, we used a fibre-modified Ad5 vector with the tropism of Ad35, which uses the ubiquitously expressed CD46 protein as attachment receptor. We transduced a variety of cell lines with the developed hTERT-d2EGFP vector and demonstrated a close correlation between expression levels of the d2EGFP, endogenous telomerase activity, and hTERT mRNA expression. Furthermore, the hTERT-d2EGFP reporter vector was able to monitor the cell cycle and differentiation stage-dependent hTERT transcription activity. Additionally, in order to study the self-renewal process of haematopoietic stem cells (HSCs), we utilised the hTERT-reporter vector for monitoring hTERT transcription activity in human HSCs. Interestingly, increasing hTERT expression was inversely correlated to the self-renewal capacity among primitive HSCs and the hTERT-reporter vector could be used to separate short-term from long-term re-populating human HSCs. In summary, we have developed a versatile adenoviral vector system with Ad35 tropism, which can be used to transiently transduce tumour cells as well as haematopoietic cells. This vector system can be used to study normal regulation of hTERT and has potential in development of novel therapeutic approaches for treatment of tumours and malignancies in the blood system

Combined expression of HOXA11 and CD10 identifies endometriosis versus normal tissue and tumors

The gold standard for diagnosing endometriosis is by laparoscopic visual demonstration of ectopic endometrial lesions outside the uterus, preferably verified by biopsy and microscopical examination. Molecular markers to facilitate the microscopical diagnosis of endometriosis and for distinguishing endometriosis from other benign and malignant lesions are lacking. Our aim was to test and validate an immunohistochemical antibody panel for improved diagnostic accuracy of endometriosis. Both CD10 and HOXA11 have been implicated in regulation of endometrial homeostasis. Here we have analyzed the expression pattern of these two proteins using immuno-histochemistry on human tissues in a tissue microarray format. CD10 and HOXA11 expression in endometriosis lesions were compared to expression patterns in a range of normal tissues and in primary-and metastatic lesions of endometrial-, cervical-and ovarian cancer. HOXA11 and CD10 were expressed in 98% and 91% of endo-metriosis lesions and the combined double-positive expression profile of both HOXA11 and CD10 was highly sensitive for ectopic endometrial tissue (90%). The specificity and sensitivity for this double-positive signature in endometriosis was significantly different from all investigated tissues, cancers and metastases except normal, eutopic endometrial-and cervical mucosa. The combination of HOXA11 and CD10 expression profiles provides a useful tool to identify ectopic endometrial tissue and for distinguishing endometriosis from various types of gynecological malignancies and metastases

Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses

GABA-A Channel Subunit Expression in Human Glioma Correlates with Tumor Histology and Clinical Outcome

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the CNS and is present in high concentrations in presynaptic terminals of neuronal cells. More recently, GABA has been ascribed a more widespread role in the control of cell proliferation during development where low concentrations of extrasynaptic GABA induce a tonic activation of GABA receptors. The GABA-A receptor consists of a ligand-gated chloride channel, formed by five subunits that are selected from 19 different subunit isoforms. The functional and pharmacological properties of the GABA-A channels are dictated by their subunit composition. Here we used qRT-PCR to compare mRNA levels of all 19 GABA-A channel subunits in samples of human glioma (n = 29) and peri-tumoral tissue (n = 5). All subunits except the ρ1 and ρ3 subunit were consistently detected. Lowest mRNA levels were found in glioblastoma compared to gliomas of lower malignancy, except for the θ subunit. The expression and cellular distribution of the α1, γ1, ρ2 and θ subunit proteins was investigated by immunohistochemistry on tissue microarrays containing 87 gliomas grade II. We found a strong co-expression of ρ2 and θ subunits in both astrocytomas (r = 0.86, p<0.0001) and oligodendroglial tumors (r = 0.66, p<0.0001). Kaplan-Meier analysis and Cox proportional hazards modeling to estimate the impact of GABA-A channel subunit expression on survival identified the ρ2 subunit (p = 0.043) but not the θ subunit (p = 0.64) as an independent predictor of improved survival in astrocytomas, together with established prognostic factors. Our data give support for the presence of distinct GABA-A channel subtypes in gliomas and provide the first link between specific composition of the A-channel and patient survival

Floraövervakning i Jönköpings län 2006: ett rikt odlingslandskap

Ett rikt odlingslandskapI den här sammanställningen har vi valt att beskriva utvecklingen för några kärlväxter inom fyra olika växtmiljöer i odlingslandskapet. Dessa berörs av miljömålet ”Ett rikt odlingslandskap” och dess delmål; ängs- och betesmarker, skogsbeten, gårdsmiljöer och åkrar med åkerogräs.Trenderna för de utvalda växterna visar att mångfalden av odlingslandskapets arter i huvudsak fortlöpande minskar i vårt län:För arterna inom gruppen naturbeten och ängsmarker är trenden sammantaget negativ. Totalt har 543 lokaler besökts mellan 1978 och 2006 (2 504 besök). Av dessa bedöms 170 lokaler som utgångna, ca 31%. Brudsporre är den art som gått kraftigast tillbaka därefter kommer fältgentiana, tätt följd av finnögontröst och granspira. Drakblomma och vityxne är de arter som har det svårast beroende på deras låga individantal på lokalerna.Inventeringarna av mosippa (skogsbete) 2006-2007 visade att så många lokaler som 71 av 106 (67 %) har försvunnit från länet. Det är svårt att se om arten har en positiv trend på någon lokal i dagsläget. I stort sett har alla, möjligen med något enstaka undantag, gått tillbaka.Även arterna inom gårdsmiljöer visar på en negativ trend. Totalt har 26 lokaler besökts mellan 1978 och 2006 (97 besök). I stort sett är det är endast två lokaler för kattmynta, en lokal för paddfot samt möjligen en lokal för vit kattost som minskar.Flera av lokalerna med åkerogräs har inte återbesökts. Sammantaget kan ändå utläsas att gruppen visar på en negativ utveckling.Orsakerna till tillbakagången är många. Landskapet har genom tiderna alltid förändrats. Speciellt efter andra världskriget och i takt med industrisamhällets framväxt har påverkan på vårt odlingslandskap varit mycket stor. Några särskilt viktiga faktorer var den ökade användningen av bekämpningsmedel och gödsel och att småbiotoperna och åkerrenarna försvann i takt med att maskinerna blev större och bättre. Man ville få större bättre ytor att bruka. I dag är problemet att enheter läggs ner och betesmarkerna planteras igen eller står ohävdade. Jämför vi antalet brukare idag med antalet vid tiden för andra världskriget är skillnaden stor. År 1944 fanns över 400 000 jordbruksföretag i Sverige. År 2005 var antalet jordbruksföretag endast 75 800. I Jönköpings län var två tredjedelar av jordbruken mellan 2-10 ha stora, idag är mer än hälften större än 10 ha. I snitt brukades 7,4 ha åker. Idag brukas i genomsnitt 23,9 ha åker per företag i länet.I länet, liksom i övrig skogs- och mellanbygd, har dock storleksrationaliseringen inte gått lika långt som i slättbygderna tack vare de naturgivna förutsättningarna. Därför finns här också relativt mycket natur- och kulturvärden bevarade.Regionala inventeringsrapporter import från MDP 2015-05</p

Human short-term repopulating cells have enhanced telomerase reverse transcriptase expression.

Telomerase activity has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. However, it has been unclear whether human HSCs have telomerase activity and how telomerase activity is regulated within the HSC and progenitor pool. Here, we isolated living cord-blood (CB) CD34+ cells with up-regulated human telomerase reverse transcriptase (hTERT) expression by using an hTERT-reporting adenoviral vector encoding destabilized green fluorescent protein (dGFP) driven by the hTERT promoter, and functionally characterized them in comparison with control vector–transduced CD34+ cells expressing GFP. Following a 2-day serum-free transduction protocol, cells were sorted into a dGFP+ and a GFP+ fraction. Cell-cycle analysis revealed that the dGFP+ cells had a greater proportion of cells in S/G2/M phase compared with the GFP+ cells, (56% ± 1.8% vs 35% ± 4.3%; P < .001) and fewer cells in G0 phase (8.1% ± 3.0% vs 20% ± 4.7%; P < .01) However, the colony-forming and short-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) B2m–/– mice bone marrow–repopulating capacities were similar between the dGFP+ and the GFP+ cells. Interestingly, the dGFP+ cells had a 6-fold lower repopulating capacity in NOD/SCID mice compared with the GFP+ cells and lacked secondary NOD/SCID B2m–/– mice bone marrow–repopulating capacity. Thus, up-regulation of hTERT expression within the CB HSC pool is accompanied by decreased self-renewal capacity

Nittonhundratalets bostadsarkitektur : Aspudden, Midsommarkransen, Hökarängen, Bredäng, Norra Hammarbyhöjden. Kungl Konsthögskolans Restaureringskonst 2017-2018

Denna publikation är ett resultat av läsåret 2017-2018 i kursen Restaureringskonst vid Kungl. Konsthögskolan, arkitekturavdelningen. Årets tema var bostadsarkitektur. Förvalta, förädla, förvanska? Under läsåret undersöktes bostadsarkitekturen som kulturarv, hur bostadshus och bostadsområden förvaltas, förädlas alternativt förvanskas genom restaureringsåtgärder, förtätningar och omvandlingar. I I läsårets projektstudie ingick fem bostadsområden i södra Stockholm, utvalda för att representera varsin epok i 1900-talets bostadsutveckling. En projektgrupp för varje bostadsområde gjorde studier i olika skalor, från den utvalda lägenhetens detaljer, trapphuset och bostadshuset, gården och gatan till stadsdelsnivån.Kursen restaureringskonst är en ettårig heltidskurs på avancerad nivå för yrkesverksammaarkitekter, antikvarier, ingenjörer m fl inom området byggnadsrestaurering. Kursen omfattar restaureringskonstens basämnen, dess ideologi, historia och tillämpningar. </p