Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes. The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems. Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis. A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner. Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression. <b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling

<p>Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes.</p><p>The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems.</p><p>Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis.</p><p>A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner.</p><p>Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression.</p><p><b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling</p>

Tumour-associated macrophages (TAM) are a major component of the inflammatory infiltrate that typifies most malignancies. Among them, Burkitt’s lymphoma (BL), a high-grade non Hodgkin lymphoma (NHL) of B-cell origin, represents a characteristic example. Studies from our group have shown that TAM in BL exert pivotal roles that are mainly supportive of tumourigenesis such as maintaining an immunosuppressive microenvironment. In order to unravel the molecular mechanisms underlying TAM functions in BL, solid tumours from a mouse xenograft model of BL have been used to obtain TAM and assess their activation status in vivo. Laser-capture microdissection has been successfully used to procure intact macrophage sections from the tumour site, allowing the production of a pure, in situ gene expression signature of TAM in BL. Tingible-body Mφ from lymph node germinal-centres and resident tissue Mφ from resting lymph nodes of non-tumour bearing mice were chosen for direct comparison with TAM. Whole-genome microarray technology has revealed a distinct TAM gene expression profile, with 454 genes being significantly up-regulated (fc ≥ 2, p<0.05) and 1293 genes being significantly down-regulated (fc ≤ -2, p<0.05) between TAM and either of the two normal Mφ populations. Further bioinformatics analysis of gene functions has highlighted matrix remodeling, phagocytosis, and immune response among the processes most highly enriched in TAM. Importantly, mRNA and tissue expression of selected differentially expressed genes relevant to these processes was validated by real-time qPCR and immunofluorescence labeling respectively. Following the generation of the TAM profile in situ, in vitro experimental approaches were undertaken in order to investigate how specific elements of the BL microenvironment drive the observed TAM signatures. Specifically, the direct role of apoptotic tumour cells, a key component of the BL microenvironment, versus that of viable tumour cells in driving TAM matrix remodelling gene expression was assessed in short-term mouse and human Mφ-NHL cell co-cultures. From the aforementioned cluster, emphasis was given to MMP12 and MMP2 transcripts: mRNA and protein expression of these MMPs was found to be up-regulated in Mφ following viable tumour cell co-cultures and this effect was further enhanced following apoptotic tumour cell co-cultures, implying that apoptotic NHL cells could directly shape TAM matrix remodeling phenotype in BL in vivo. Whereas the mRNA of both MMPs was solely Mφ-derived in this system, MMP12 and MMP2 protein was surprisingly found also to be increased in NHL cells in the apparent absence of increased mRNA. Detailed examination of MMP12 production by NHL cells revealed that it is most likely an apoptosis-dependent process, since apoptotic NHL cells generated through different apoptosis stimuli, as well as apoptotic cell-derived microparticles, showed markedly increased MMP12 protein levels. In conclusion, the data presented in this thesis, provide the first insight into the in vivo activation status of TAM in high-grade NHL, through generation of the TAM gene signature in situ. Upon further in vitro studies, apoptotic NHL cells were shown to directly modulate the matrix remodelling component of the TAM signature as well as to actively produce matrix remodelling mediators themselves, suggesting distinct roles for tumour cell apoptosis within the NHL microenvironment that can profoundly influence the disease outcome.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Licentiatavhandling (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

Thesis (Ph.D. in Biostatistics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 237-239). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.112-113

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.215-229

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 60-77).