| 1. |
- Afrakhte, Mozhgan
(författare)
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Growth control mechanisms in normal and neoplastic mammalian cells
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main theme of the studies presented in this thesis is, the growth control mechanisms whose loss in normal cells predispose to or cause cancer. The balance between growth inhibitory and stimulatory mechanisms is crucial for the development and maintenance of a normal animal.PDGF, a growth factor for cells of mesenchymal origin, is implicated in normal developmental processes as well as neoplasia. The alternative splicing of exon 6 in PDGF-A gene transcripts gives rise to two different proteins with different compartmentalization properties. The PDGF-A chain homodimers, PDGF-AAL, encoded PDGF A-splice variant remain associated with the cell membrane. Studies of a human fibrosarcoma cell line, U-2197, revealed a high expression level of the cell associated PDGF-AAL which upon release increased autophosphorylation of the endogenous PDGF receptors, suggesting an autocrine loop. PDGF-A gene and PDGFR-α gene found to be co-amplified in the U-2197, indicating an optimised system for growth in these cells, i.e. amplified growth factor receptor as well as a local autocrine supply of the mitogen.Members of TGFβ superfamily are potent regulators of the growth and differentiation of a wide range of cell types. Intracellular mediators of TGF-β signalling, SMADs, transduce signals from serine/threonine kinase receptors to the nucleus where they affect transcription of target genes. A new class of SMAD proteins has been identified whose members, the inhibitory SMADS, antagonise TGF-β signals by interfering with agonistic SMADs activity. Smad6 and Smad7 are two closely related TGF-β antagonists identified in mammalian cells. Overexpression of Smad7 inhibited the cellular response to TGF-β whereas expression of an anti-sense Smad7 construct showed an enhancing effect on this response. The inhibitory SMADs may act in a negative feedback loop, as their expression is induced by the same ligands whose action they antagonise.Density dependent growth inhibition is a growth control mechanism often lost in transformed and malignant cells. Cells in dense culture are refractory to the mitogen stimulation although, the mitogenic signals were shown to be processed to some extent. The expression of immediate-early genes in dense culture stimulated with mitogen was induced. The activity of cyclin dependent kinases (CDKs), the pivotal kinases in G1/S transition, showed to be density dependent and decreased by increasing cell density. pRb, a tumour suppressor and growth regulatory protein, remained unphosphorylated in mitogen treated dense culture. The cessation of CDKs kinase activity in dense cultures was shown to be accompanied with increasing expression of inhibitory proteins of these kinases, CKIs. The impaired expression of a positive regulator of CDKs, Cdc25A phosphatase, was another feature of dense cultures.
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| 2. |
- Ata, Ahmad Khaled
(författare)
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Expression of TGF- isoforms, their receptors and related SMAD proteins in brain pathology : Immunohistochemical studies focusing on infarcts, abscesses and malignant gliomas
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on the immunohistochemical expression of transforming growth factor beta(TGFβ) isoforms, their receptors and TGF-β-related SMAD proteins in brain pathology, chiefly in-farcts. One key question was whether the expressions of these compounds are altered within glial cells, endothelial cells of microvessels and other cell types in the vicinity of infarcts. Studies on human and animal brain infarcts were made. Immunoreactivities to TGF-β isoforms -β1, -β2 and -β3, and TGF-βreceptor (TβR) type I were seen in astrocytes, macrophages, neurons, endothelial and vascular smooth muscle cells of human brain infarcts. Similar observations were made in an experimental model of rat brain infarct at day 1 and 3 following occlusion of the middle cerebral artery (MCA). Increased expression of Smad2, -3, -4, -6 and -7 was seen already at 6 h after MCA occlusion in neurons, microvascular endothelial cells, astroglial cells and inflammatory cells. Later on, immunopositive macrophages were present in the infarcts. The changes persisted even at day 7 after MCA occlusion.Several alterations thus occur during the evolution of brain infarcts with regard to the immunohistochemical expression of TGF-β, its receptors and related SMAD proteins. Such changes are, however, not unique to brain infarcts. Thus, patterns of high expression for TGF-β- isoforms -β1, -β2 -β3, and TβR-I in cases of brain abscess (human), and of Smad2, -3, -4, -6 and -7 in tumor cells and neoplastic blood vessels of malignant gliomas (human) were also observed.In addition, immunohistochemical expression of vascular endothelial growth factor (VEGF) andits receptors was investigated since this growth factor is involved in angiogenesis and edemaformation, two cardinal features of brain infarcts. Increased immunoreactivities, seen particularly in the edges of infarcts, were observed already at day 1 after MCA occlusion.In conclusion, the various TGF-β isoforms, receptors and related SMAD proteins, together with other factors, seem to be involved in the very complicated and important changes taking place in the vicinity of brain infarcts.
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| 3. |
- Fedorov, Vadim B.
(författare)
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Phylogeography and mitochondrial DNA diversity in arctic lemmings
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis is to evaluate the effects of Quaternary environmental fluctuations on the present patterns of mitochondrial DNA variation in the wood lemming, Myopus schisticolor, and the two genera of Arctic lemmings: collared lemmings, Dicrostonyx, and true lemmings, Lemmus.The phylogeographic pattern and the limited mtDNA diversity in the Scandinavian populations of the wood lemming reflect recent colonization by a limited number of founders during the Holocene boreal forest expansion.The mtDNA phylogeny in the two genera of Arctic lemmings gives no support for the existence of a single Beringian refungium since the mid Pleistocene. Isolation by intermittent inundation of the Bering Strait during the interglacials was most probably important for speciation in both genera, Dicrostonyx and Lemmus.Comparative phylogeography of the two genera of Arctic lemmings, Lemmus and Dicrostonyx gives evidence for the vicariant separation by the glacial barriers in the Eurasian Arctic. There is genetic support for the importance of the Asian Beringia as a refugial area for the tundra specialist, D. torquatus, during warming periods in the interglacials.There is no evidence for the direct effect of the last glaciation on the present level of mtDNA diversity and population structure in Arctic lemmings. Intrapopulation and intraregion mtDNA diversity estimates in each genus reflect the demographic events which were likely a result of environmental changes in the Holocene. Comparisons of mtDNA diversity estimates across the two genera of Arctic lemmings suggest that historically and presently codistributed Lemmus and Dicrostonyx responded differently to environmental changes resulting from the Holocene warming events in the Eurasian Arctic.The mtDNA phylogeny supports the importance of vicariant events generated by the glacial-interglacial periods for allopatric speciation in chromosomally variable collared lemmings, Dicrostonyx, in the North America Arctic. In the Eurasian Arctic, the congruence between the phylogeographic structure in mtDNA variation and geographic distribution of chromosome races indicates that the fragmentation by the glacial barriers during the late Pleistocene and bottleneck events due to warming events in the Holocene were important for the origin of intraspecific chromosome races in the collared lemming, D. torquatus
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| 4. |
- Lindqvist, Anna-Karin B.
(författare)
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The genetics of systemic lupus erythematosus : Mapping of susceptibility loci for human SLE
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Systemic lupus erythematosus (SLE) is a systemic autoimmune disease affecting organs such as skin, jointsand kidneys. Characteristic for the disease is increased production of autoantibodies, in particular against dsDNA. Previous studies of SLE in mouse models and in humans indicate polygenic inheritance and genetic heterogeneity.For the purpose of genome scanning, we designed a set of chromosome specific panels of microsatellite markers for efficient genotyping. The 391 microsatellite markers, available from the Weber set 6.0 (CHLC), were organized according to chromosome and size of amplification products and were assigned fluorescent color for detection by ABI automated sequencers. The efficiency of the markers was evaluated regarding amplification of genomic DNA extracted from peripheral blood, formalin-fixed biopsy and pieces of hair. We conducted a genome wide search for SLE susceptibility loci in multi-case families from Iceland and Sweden using two-point linkage analysis. Prior to the analysis, the family material was stratified based on the presence of the deficiency of complement component C4A, a potential risk factor for susceptibility to SLE. Eighteen chromosomal regions with LOD scores > 1.0 were identified, some of which were shared between the two population groups. The most pronounced linkage was found on chromosome 2q37, with a LOD score of 4.24 in the combined family material. The locus was denoted hSLE1.To further evaluate the 2q37 region we analyzed additional microsatellite markers in families from Iceland; Sweden and Norway. The highest two-point lod score was obtained for the marker D2S125 and the multipoint analysis indicated the position of the locus between D2S125 and D2S2986 with a peak lod score of 5.29 at D2S125. No evidence of linkage was found in a set of families from Mexico. We also investigated the gene encoding interleukin 10 (IL-10) on chromosome 1, previously suggested to be a candidate gene for susceptibility to SLE. A microsatellite marker in the IL-10 promotor region was studied in 330 Mexican SLE patients and 368 ethnically matched controls for association to the disease. In addition, linkage to the same marker was evaluated in multi-case families from Iceland, Sweden and Mexico. No association or linkage was found.
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| 5. |
- Narain, Yolanda
(författare)
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Chromosome rearrangements do not prevent gene flow in the common shrew, Sorex araneus : Analysis of two chromosomal hybrid zones in Sweden
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Two chromosomal hybrid zones of the common shrew, Sorex araneus, have been investigated: the Hällefors-Uppsala hybrid zone in central Sweden and the Abisko-Sidensjö hybrid zone in northern Sweden. The Hällefors (Hä) and Uppsala (Up) chromosome races consisted of biarmed autosomes only (2nA=18), and differed in the Robertsonian fusions of two arms, ko, pq, and kp, oq, respectively. The Abisko (Ai) and Sidensjö (Si) races consisted of biarmed and uniarmed autosomes, and are mainly characterized by the fusions hn and hi, respectively. Robertsonian polymorphism was present in both races, affecting chromosomes g/m, h/n, i/p, j/l, kq, and j/l, k/q, n/r, respectively. The width of the Hällefors-Uppsala hybrid zone was 2.7 km and the center of the zone followed the Kolbäck river. The Abisko-Sidensjö hybrid zone had a width of 15.2 km in the area studied.In the center of the Hällefors-Uppsala hybrid zone, one type of "complex" heterozygotes occured that formed a ring of four elements during meiosis. In the Abisko-Sidensjö hybrid zone, "simple" and "complex" heterozygotes ocurred which formed trivalents and/or a chain configuration of four/six elements during meiosis, respectively. There is no evidence from fertility studies that heterozygotes are less fertile than homozygotes, and the minor differences found in certain reproductive characters might be due to different genetic background rather than to chromosomal rearrangements. Microsatellite data demonstrated that gene flow occurs across the Hällefors-Uppsala hybrid zone, which is in agreement with the results obtained from the fertility analysis.
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| 6. |
- Nilsson, Mats
(författare)
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Padlock probes : Circularizing oligonucleotides for localized detection of DNA sequence variants
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Present techniques for localized detection of DNA sequence variants have limitations due to problems with sensitivity, specificity, signal localization or ability to perform many analyses simultaneously.In this study a novel gene analytic reagent is presented - the padlock probe. Padlock probes are linear oligonucleotides with target complementary sequences at both ends, joined by a non-target complementary segment. Upon hybridization to a target DNA strand, the end-sequences are brought together and can be joined by a DNA ligase. Thereby the probe becomes circularized and threaded on the target DNA strand. Ligated probes were shown to be circular and topologically linked to circular target molecules. Thetopological link formed between circularized padlock probes and target DNA strands was shown to resist denaturing washes.A novel procedure for oligonucleotide synthesis was developed that allows for cleavage and removal of depurinated products before oligonucleotides are released from the synthesis support. In this manner molecules with intact ends can be isolated after an ordinary HPLC purification. The novel procedure is of general utility in oligonucleotide synthesis and especially suitable for syntheses of terapeutic antisense reagents and of padlock probes.Padlock probes were used to distinguish a single nucleotide difference in situ in metaphase chromosomes. Two alpha-satellite sequences present in the centromeres of human chromosomes 13 and 21 were selectively identified by two probes labelled in distinct colors. The two repeat sequences were shown to be polymorphic among different homologues of chromosomes 13 and 21, allowing identification of the parental origin of each of these chromosomes in an individual.For enhanced detection circularized padlock probes can be used to template a rolling circle replication reaction, providing a linear signal amplification suitable for quantitative analyses. The highly processive Φ 29 DNA polymerase was superior to the Kleenow DNA polymerase in initiating rolling circle replication by displacing the circular padlock probes from target DNA strands. Efficient amplification requires release of probes from target DNA strands. This is accomplished when displaced probes slide off a nearby end of the target molecule.The combined properties of the padlock probes render them promising reagents for future DNA diagnostics.
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| 7. |
- Rytting, Ann-Sofie
(författare)
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HIV-1 reverse transcriptase activity assays based on monoclonal antibodies to native enzyme : Applications on studies of antiviral substances and immunological variation of reverse transcriptase
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Specific detection of HIV-1 reverse transcriptase (RT) activity in crude materials was enhanced using a capture assay for immunopurification and subsequent activity assay. The purification step was achieved by anti-HIV-1 RT monoclonal antibodies (Mabs) immobilized onto a solid carrier. The polymerization reaction was performed in the same tube or microtitre plate well and terminated by washing away surplus substrate, while the newly synthesized DNA remained bound to the immobilized enzyme. The amount of synthesized DNA could either be measured as amount of incorporated radioactive nucleotide or calorimetrically by using alkaline phosphatase conjugated Mab to the nucleotide analogue 5-bromodeoxyuridine 5´-triphosphate(BrdUTP).The capture assay proved useful for: 1) Determination of HIV-1 RT activity in crude cell extracts. 2) Analysis of chain terminating capacity of RT from AZT resistant virus. 3) Screening for hybridoma cell lines producing RT reactive Mabs. 4) Evaluation of the isozyme specificity of RT reactive Mabs.Monoclonal antibodies to native HIV-1 RT were produced by immunization of mice with RT and the iscom matrix forming adjuvant Quil A. Twelve clones produced Mabs which inhibited at least 50% of the RT-activity, and 34 clones produced Mabs which immobilized catalytically active enzyme. This Mab panel was used to characterize RTs from HIV-1 isolates of different subtype identity.Surprisingly large immunological differences were found between RTs from different subtypes as well as from homologous subtype isolates.One of the monoclonal antibodies, reactive to an epitope in the RNase H region, was used for measuring RT protein binding to template/primer in presence of non-nucleoside RT inhibitors. This assay for RT protein is a part of an assay system to evaluate the mode of action of RT inhibitors.
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| 8. |
- Thorén, Peter A.
(författare)
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Mating structure and nestmate relatedness in primitively social hymenoptera as revealed by microsatellites
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mating structure and nestmate relatedness have been estimated in social Hymenoptera with different levels of sociality. Microsatellite markers have been developed for both communal bees (Andrena scottica) and for primetively eusocial wasps (Vespula rufa).In the communal bee, Andrena scottica, females of the same colony are not related to each other suggesting that the lack of inclusive fitness payoffs may effectively constrain their social evolution. There was a positive inbreeding coefficient for both examined populations of Andrena scottica as a result of a high degree of intranidal mating. This inbreeding did not lead to extensive production of diploid males as would be expected on the basis of the CSD (complementary sex determination) system of the honey bee, and thus Andrena scottica either avoids "matched matings", (i.e. either or both sexes refrain from mating with an individual carrying the same sex allele) or the sex determination departs from the honey bee model.The primitively eusocial wasp Dolichovespula saxonica is highly polyandrous.Queens (n=7) mated 4-11 times (mean 6.0) and the effective promiscuity, ke was 3.30-9.14 (mean 4.81). This resulted in low average relatedness among workers (gww=30-0.40; mean 0.37). On the contrary Dolichovespula media queens from three colonies mated with one male only. All males (n=80) originating from four D. saxonica colonies were produced by the queen, in agreement with the genetical interests of both the queen and the collective worker force ("worker policing"). One D. saxonica colony includedboth workers and male sexuals of two matrilines.Bombus hypnorum is a primitively eusocial bumble bee. Queens from 14 colonies had mated 1-5 times (mean 2.43) with an effective promiscuity of 1-2.8 (mean 1.40). Half of the colonies examined, inhabited alien workers. These workers could originate from an earlier nest usurpation event, and the colonies with such alien workers had on average a larger size. Alien workers together with polyandry results in high variation among colonies in sociogenetic organisation. Genetical data were consistent with the view that all males (n = 233 examined) were produced by a colony's queen, in conflictwith the collective worker interest.
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| 9. |
- Edman Ahlbom, Bodil
(författare)
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Molecular investigations on chromosome 21 in relation to Down syndrome and familial congenital hypothyroidism
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chromosomal abnormalities as well as mutations in single genes are genetic changes that can cause syndromes or inherited diseases. Down syndrome (DS), usually a consequence of trisomy 21, is a gene dosage disorder caused by an extra copy of genes on chromosome 21, but the mechanisms whereby specific genes contribute to the clinical features of DS are not known. This thesis presents molecular investigations undertaken to contribute to the identification of the chromosomal segments and genes involved in the pathogenesis of DS. In addition, candidate genes for familial autosomal recessive congenital hypothyroidism (CH) have been investigated.A woman with a phenotype of DS, but apparently normal chromosomes, was found to have no part of chromosome 2 1 in three copies. Autosomal recessive inheritance of the DS phenotype is suggested, based on the family history. If this is so, this is the first time that au autosomal recessive disorder is known to have resulted in a phenotype indistinguishable from DS.Partial trisomy of most of chromosome 21 and partial trisomy 7p was found in a boy without a DS phenotype but with mild facial dysmorphism and severe psychomotor retardation. The DS critical region on 21q22.2 was, however, not found in three copies, thereby confirming the importance of this region for the development of DS features.A deletion of chromosome 21 was found in a girl with mild mental retardation and CH. Although large, the deletion did not extend into the DS critical region on 21q22.2. These findings show that large proximal deletions of 21q when not involving the DS critical region can result in only mild mental impairment. The presence of CH in this girl indicates that genes located on proximal 21q could possibly be involved in the pathogenesis of CH.CH is a heterogeneous disorder affecting 1/3000-4000. Of these, some 10-20% are familial cases. By genetic linkage analysis in 22 families with a presumably autosomal recessive CH, chromosome 21 was excluded from containing any major disease-causing gene. Furthermore, linkage to the thyrotropin receptor was excluded by using markers identified by radiation hybrid mapping. Linkage to the thyroglobulin (TG) locus was found in 40% of the families, however, thereby confirming genetic heterogeneity and suggesting that defects in the TG gene is a major cause of familial CH.
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| 10. |
- El-Obeid, Adila Salih
(författare)
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Experimental studies on glioma cell motility and proliferation
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glioma is a malignant primary brain tumor associated with short patient survival. Various growth factors and cytoskeletal proteins are correlated to glioma progression. In this thesis the effects of transforming growth factor alpha (TGFα) and glial fibrillary acidic protein (GFAP) were studied.TGF-α is a potent mitogen that belongs to the epidermal growth factor (EGF) family. It binds to and activates the EGFR, which is a receptor tyrosine kinase. Glioma cell line U-1242 MG was transfected with human TGF-α cDNA in a tetracycline regulated system, to carry out in vitro and in vivo studies. TGF-α induced glioma cell motility via EGFR activation independent of cell density which implied a "private" autocrine TGF-α /EGFR loop. It was found that the binding of TGFα to the EGFR occurs in compartments that are not completely accessible to an extra-celularly added antibody.Nude mice were injected subcutaneously with the TGF-α inducible and control cells. TGF-α- inducible cells developed into the largest tumors, and i.p. administration of an EGFR-tyrosine kinase inhibitor significantly reduced the sizes of these tumors. Results indicate that an active TGF-α /EGFR autocrine loop leads to increased tumor growth in vivo.The intracellular effects of EGFR activation associated with glioma cell motility and ruffling have been investigated. Two major EGFR-signaling pathways, MAP kinase and phosphoinositide 3 kinase (PI3 kinase), were tested by using specific inhibitors. Results showed that the EGFR downstream pathway diverges. Inhibition of MAP kinase only affects motility, while inhibition of PI3 kinase can affect both motility and cell morphology.The GFAP studies were performed on two glioma cell lines, U-1242 MG and U-251 MG sp 3A which were transfected with a human GFAP cDNA. The effects of GFAP were studied on crude cultures as well as at single cell level. Results show that GFAP expression leads to inhibition of cell motility and proliferation and to a change in cell morphology.
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