In this thesis we attempted to elicit patterns of gene expression that influence phenotype, and that may also have been altered by thousands of years of domestication and selection, between red junglefowl and White Leghorn chickens. Red junglefowl are the wild ancestor to all domesticated chickens, and poultry in general are highly valued as a research animal and food resource. The project was also begun in order to complement an earlier study of an intercross between White Leghorn and red junglefowl, which identified several regions that were linked with phenotypic differences between the two birds. We began by creating our own cDNA microarray via generating four cDNA libraries from red junglefowl/White Leghorn brain and testis. We generated 12,549 unique transcripts. This included 400 new putative transcripts specific to chickens, and 180 transcripts that were not found in any other database. When investigating polymorphisms between White Leghorn and red junglefowl we found a SNP rate of 1.9/kb coding region, and a synonymous and non-synonymous percentage for these SNPs of 80 and 20% respectively. In the last two studies we used the cDNA microarray to measure gene expression differences between White Leghorn and red junglefowl in both hypothalamus/thalamus and liver. We found that there appears to be a significant number of genes down-regulated in White Leghorn hypothalamus/thalamus, plus an over-representation of up-regulated genes from well-known pathways, as compared with red junglefowl. We hypothesize that domestication/selection may be connected with this characteristic. We also found that the p-arm of chicken chromosome 4, which is an ancestral microchromosome, was over represented with differentially expressed genes in hypothalamus/thalamus. A number of differentially expressed genes are shared between the two tissues, and these genes are expressed in same manner between red junglefowl and White Leghorn.

Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G>A and m.11778G>A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes. / Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.

Oestrogen is one of the most important hormonal regulators and is known to play a key role in the development and growth of breast cancer. The majority of tumours have a hormone dependent growth, and this is indicated by the presence of oestrogen receptors (ERs). About two thirds of breast cancers occur after the menopause when the ovaries have ceased to produce oestrogen and despite the low levels of circulating oestrogen’s the tumour concentrations of oestrone, oestradiol and their sulfates have been shown to be significant. Patients with hormone dependent tumours are candidates for treatment with the anti-oestrogen tamoxifen, which acts by competing with oestrogen for binding to the ER thereby, diminish the transcription of oestrogen regulated genes. The drug is mainly metabolised by cytochrome P450 enzymes in the liver and to a lesser extent locally in the breast, where upon several produced metabolites have higher affinity for the ER than the mother substance. Patients treated with tamoxifen have in general a prolonged disease-free survival. Even if most patients respond well to tamoxifen about 30-50 % either fail to respond or become resistant by incompletely understood mechanisms. Therefore, the aim of this thesis was to investigate possible mechanisms responsible for tamoxifen resistance. In paper I and II we studied genetic variants of enzymes participating in the metabolism of tamoxifen and assessed whether these variants correlated to breast cancer prognosis and/or to the benefit of tamoxifen. The results indicate an influence of CYP2D6, CYP3A5, and SULT1A1 genotypes in tamoxifen response. Further, tamoxifen has shown to compete with oestrogen for the binding to ER. In paper III we measured the expression levels of enzymes involved in the local synthesis of oestrogens in order to see if they correlated to clinical outcome. The protein expression of stromal aromatase was shown to have a prognostic significance, especially in ER-positive patients. Finally, tamoxifen and its ER-active metabolites have shown to induce both cell cycle arrest and apoptosis and one central mediator in these processes is the tumour suppressor protein p53. The proapoptotic activity of p53 is dependent on a proline rich domain containing a common Pro-to-Arg polymorphism. In paper IV we examined the value of this genetic variant as a predictive marker for anti-cancer therapy and found that patients carrying the Pro-allele might be good responders of tamoxifen therapy. The present thesis further indicates the complexity of the mechanisms underlying tamoxifen resistance. In summary, genetic variants of metabolic enzymes, genetic variants in p53, as well as expression levels of enzymes involved in local oestrogen synthesis, may have influence on breast cancer prognosis and may be useful markers in the prediction of tamoxifen response.

Rhesus macaques are a widely used animal model of human diseases and related immune responses. Fc receptors (FcRs) mediate the interaction between antibody molecules and innate killing mechanisms, consequently eliminating the pathogen. In rhesus macaques, FcRs are highly polymorphic. To evaluate the potential influence of FcgR polymorphisms on the interaction with antibody molecules, we performed in silico analysis using SIFT, Provean, nsSNPAnalyzer, I-Mutant, MuSTAB and iPTREE-STAB web servers. V20G in FcγRI, I137K in FcγRII and I233V in FcγRIII were further analyzed structurally using FOLD-X, AMMP and Chimera to calculate changes in folding and interaction energy and for structure visualization. Results from our analysis suggest that the selected variations destabilize protein structure. Additionally, Q32R increases the binding affinity of FcγRI, whereas A131T decreases the binding affinity of FcγRII towards IgG1. Together, our results indicate that these substitutions might influence effector and regulatory mechanisms resulting from antibody/FcR interactions.

No description available.

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

Abstract Primary lactose intolerance has been associated with a single nucleotide polymorphism located upstream of the lactase gene. The most common diagnostic tests for lactose intolerance are time-consuming and the patient is not allowed to eat and drink for 12 hours before the test is carried out. A method that can establish the genotype would be an easier way of diagnosing lactose intolerance compared to fenotypic lactose intolerance tests. Optimization and validation of a previously published method was performed with real-time polymerase chain reaction. We used whole blood from de-identified blood donors. During the optimization and validation we used a positive control, genotype C/T from Laboratoriemedicin Västernorrland, Sundsvall. The whole-blood was extracted using the MagNa Pure LC instrument. The reagent used was KAPA PROBE FAST qPCR Master Mix. The optimized program for real-time PCR was established to be 95°C 3min [95°C x 3sec, 55°C x 20sec, detection, 72°C x 15sec] x 50 cycles. Optimal probe concentration was found to be 0.2µM and primer concentration will be 0.5µM. This genotyping method is a good first-stage screening test for lactoseintolerance. Before it can be used as a routine method further validation will be necessary in order to ensure that the evaluation of the results can be done in an easy and secure way.

All life is organized hierarchically. Lower levels, such as cells and zooids, are nested within higher levels, such as multicellular organisms and colonial animals. The process by which a higher-level unit forms from the coalescence of lower-level units is known as “individuation”. Individuation is defined by the strength of functional interdependencies among constituent lower-level units. Interdependency results from division of labor, which is evidenced in colonial metazoans as zooid polymorphism. As lower-level units specialize for certain tasks, they become increasing dependant on the rest of the collective to perform other tasks. In this way, the evolution of division of labor drives the process of individuation. This study explores several ways in which polymorphism evolves in colonial marine invertebrates such as cnidarians, bryozoans, and urochordates. A previous study on the effect of environmental stability on polymorphism is revisted and reinterpreted. A method for quantifying colonial-level individuation by measuring the spatial arrangement of polymorphic zooids is proposed and demonstrated. Most significantly, a comparison across all colonial marine invertebrate taxa reveals that polymorphism only appears in those colonial taxa with moderately to strongly compartmentalized zooids. Weakly compartmentalized and fully compartmentalized taxa are universally monomorphic. This pattern is seen across all colonial marine invertebrate taxa and is interpreted as a “rule” governing the evolution of higher-level individuation in the major taxa of colonial marine invertebrates. The existence of one rule suggests that there may be others, including rules that transcend levels of biological hierarchy. The identification of such rules would strongly suggest that new levels in the hierarchy of life evolve by a universal pattern that is independent of the type of organism involved. / Dissertation

Kawasaki disease (KD) is the most common cause of paediatric acquired heart disease, which may be attributed to the combined effects of infection, immunological response, and genetic susceptibility. The most severe complication in KD is acute coronary artery lesions (CALs), including myocardial infarction and coronary artery aneurysms. Mounting evidence indicates that adhesion molecules and chemokines play an important role in inflammation and cardiovascular disease on basis of pathogenesis. Thus, this study aimed to investigate the association of seven single nucleotide polymorphisms (SNPs) of adhesion molecules and chemokines (P-selectin 290G>A, PSGL-1 62G>A, MCP-1 -2518A>G, SDF-1 -801G>A, PECAM-1 L125V, PECAM-1 S563N and PECAM-1 R670G) with the risk of KD, sequelae of CALs and initial intravenous immunoglobulin (IVIG) treatment failure. A total of 301 KD children (185 without acute and chronic CALs, 81 with acute but without chronic CALs, and 33 with acute and chronic CALs) and 246 sex-matched healthy controls were recruited in the case-control study. In addition, 166 cases from the above KD children and 332 parents were recruited to carry out case-parent trio study. We found that PECAM-1 3 SNPs polymorphisms were not associated with above several risks, except for CALs in chronic stage. As compared with non-Leu-Ser-Arg haplotype, Leu-Ser-Arg haplotype was associated with a significant increased risk for CALs in the chronic stage (AOR 2.50, 95% CI 1.05-6.00, P=0.039). Analyses based on the diplotypes of PECAM-1 also showed that Leu-Ser-Arg allele had a significant increased risk of CALs in chronic stage in dominant manner (AOR 2.98, 95% CI 1.15-7.72, P=0.024). In addition, carriers of Leu-Ser-Arg allele had significant increased counts of platelet (¡Ñ1000/Cumm) (672.6¡Ó207.6 versus 563.1¡Ó196.8; P=0.027) within 10 days of diagnosis of KD. Moreover, we also found a significant correlation between each SNP and polymorphonuclear neutrophil counts by genotype analysis. As for other genes, there were no markedly different outcomes regardless of the risk of KD, sequelae of CALs or initial IVIG treatment failure. In conclusion, the haplotype Leu-Ser-Arg of PECAM-1 is a genetic marker of susceptibility to sequelae of chronic CALs for KD patients. However, the role of PECAM-1 SNPs in CALs formation in the chronic stage in KD patients still needs further evaluation.

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.