Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). As part of this disorder the myelin sheath undergoes degeneration, leading to alterations in the conductivity of axons, and impaired function. The onset of the disease occurs in young adults and clinical pathology is characterised by varying severity. These include i) Relapsing Remitting MS (RR-MS), ii) Secondary Progressive MS (SP-MS) and iii) Primary Progressive MS (PP-MS). MS is more prevalent in women and accounts for more than two thirds of all MS sufferers. MS is considered to be a multifactorial disorder with both genetic and environmental components. The prevalence of MS is dependent on geographical localisation, with lower sunlight exposure linked to higher prevalence. Also, studies show an increased risk in close relatives, or in identical twins, indicating a significant genetic component to the disorder. There are a number of genes that may plausibly be involved in MS pathophysiology. These include myelin-related genes, such as the myelin basic protein (MBP), immune-related genes, such FC receptor and osteopontin, and heat shock proteins such as xb crystallin. These candidate genes have been implicated in a variety of ways but usually through immunological and/or genetic studies. One of the most consistent findings in recent years has been the association of disease with alterations in the specific major histocompatibility complex (MHC) localised to chromosome 6p21.3, and includes MHC I, II, III. Genome wide screens have permitted the identification of loci in the genome, which are associated with MS susceptibility. The number of genes involved in MS is unknown and several case-control association studies have been undertaken to reveal the involvement of potential candidate genes. In general terms, current research is aimed at determining allelic variation of candidate genes. Such genes have been implicated in MS because they reside within susceptible regions of the chromosome associated with MS or they have a plausible potential pathophysiological role in MS. Candidate loci investigated in this study, for association with MS susceptibility, include members of the nitric oxide synthase family of metabolic proteins (inducible NOS, iNOS/NOS2A and neuronal NOS, nNOS), methylenetetrahydrofolate reductase (MTHFR), catechol-O-methyl transferase (COMT), and vitamin D receptor (VDR). The MS population used in all studies consisted of over 100 MS cases and gender, age and ethnicity matched controls. In our study of inducible and neuronal NOS genes, PCR based assays were developed to amplify a region of both promoters that contained known microsatellite variation. Supporting phyisological data suggests that the neuroinflammatory aspects of MS are associated with aberrant NO production, which may be due to aberrant regulation of NOS activity. Specific amplified products were identified by fluorescent capillary electrophoresis and allele frequencies were statistically compared using chi-squared analysis. In the nNOS and iNOS study, no association was identified with allele frequency variation and MS susceptibility (nNOS: ?2=5.63, P=0.962; iNOS: ?2=3.4; P=0.082). Similarly, no differences in allele frequencies were observed for gender or clinical course for both markers (Pvalue greater than 0.05). In short, results from this study indicate that the NOS promoter variations studied do not play a significant role in determining susceptibility to MS in the tested population. The COMT and MTHFR genes are localised at 22q12-13 and 1p36.3 respectively, regions of the genome that have been found to be positively associated with MS susceptibility. In our research, we set out to examine the G158A change in the 4th exon of the COMT gene. This functional mutation leads to an amino acid change (valine to methionine) that is directly associated with changes in the activity of COMT. The MTHFR enzyme plays a role in folate metabolism, and can be implicated in the turnover of homocysteine. Previous investigations have shown that high levels of homocysteine are encountered in MS patients, where it is also linked to demyelination in the CNS. In our study the aim was to examine the C677T variation (alanine to valine amino acid change) in the exon 4 coding region of the MTHFR gene and the G158A variation in the COMT gene. Restriction fragment length polymorphism (RFLP) analysis and gel electrophoresis was used to identify specific alleles for both COMT and MTHFR. However, as with the NOS study, no specific association was identified between MS susceptibility and variation for either of the tested COMT or MTHFR (Pvalue greater than 0.05) variants. In a final genomic investigation of the MS population, three variations in the VDR gene were analysed for association with MS susceptibility and pathology. Using RFLP analysis, three VDR variants were investigated with genotypes detected using the Taq I, Apa I and Fok I restriction enzymes. In contrast to previous genotypic analyses, this study did show a positive association, specifically between the functional variation in exon 9 of the VDR gene and MS (Taq I, 2= 7.22, P= 0.0072). Interestingly, the Apa I variant of VDR was also found to be associated with MS ( 2=4.2, P=0.04). The Taq I and Apa I variants were also found to be in very strong and significant linkage disequilibrium (D'=0.96, Pvalue less than 0.0001) and their associations were more prominent with the progressive forms of MS (SP-MS and PP-MS). In addition to genotypic analysis of a clinical population, additional research was undertaken to identify novel targets for MS susceptibility studies. Global gene expression analysis was undertaken using comparative subtractive fluorescent microarray technology to examine differences in gene activity (expression) in age and sex matched MS plaque tissue and anatomically matched normal white matter (NWM). MS plaques were obtained post mortem from MS sufferers with no drug history in the last two months before death and matched anatomically to healthy white matter from donors with no previous neurological disorders. Target arrays consisted of 5000 cDNAs and analysis was conducted using the Affymetrix 428 scanner. In this way, 139 genes were shown to be differentially regulated in MS plaque tissue compared to NWM. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue less than 0.0001, a=0.73); while 70 transcripts were uniquely differentially expressed ( 1.5-fold) in either acute or chronic active lesions. To validate the gene expression profile results, quantitative real time reverse transcriptase (RT) PCR (Q-PCR) analysis was performed. 12 genes were selected because they were shown to be differentially expressed by array analysis in this study, or because of their involvement in MS pathology. These included transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), glutathione S-transferase pi (GSTP1), crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1), tubulin beta-5 (TBB5), inositol 1,4,5-trisphosphate 3-kinase B (ITPKB), calpain 1 (CAPNS1), osteopontin (SPP1 or OPN), as well as the signal transducer and activator of transcription 1 (STAT1) and protein inhibitor of activated STAT1 (PIAS1). Both absolute (copy number) and comparative differences in the relative levels of expression in MS lesions and NWM were determined for each gene. The results from this study revealed a significant correlation of real time PCR results with the microarray data, while a significant correlation was also found between comparative and absolute determinations of fold. As with the results of array analysis, a significant difference in gene expression patterning was identified between chronic active and acute plaque pathologies. For example, a up to 50-fold increase in SPP1 and ITPKB levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P less than 0.0.1, unpaired t-Test). Of particular note, gamma-amino butyric acid receptor ?2 (GABG2), integrin ?5 (ITGB5), complement component 4B (C4B), parathyroid hormone receptor 1 (PTHR1) were found up-regulated in MS and glial derived neurotropic factor ?2 (GDNFA2), insulin receptor (INSR), thyroid hormone receptor ZAKI4 (ZAKI4) were found down-regulated in MS. Data also revealed a decreased expression of the immune related genes STAT1 and PIAS1 in acute plaques. In conclusion, this research used both genomic analysis and technologies in gene expression to investigate both known and novel markers of MS pathology and susceptibility. The study developed tools that may be used for further investigation of clinical pathology in MS and have provided interesting initial expression data to further investigate the genes that play a role in MS development and progression.

No description available.

In the past decades, tumors have progressively been perceived as highly integrated systems in which the genetically unstable tumor cells and the genetically stable host cells cooperate to promote tumor growth. This view suggests that, beside tumor cells (that are targeted by conventional anticancer treatments such as radio- and chemotherapy), host cells within the tumor microenvironment can be targeted by antitumor therapy. Such alternative strategies are strongly supported by the need to overcome several limitations of the conventional therapies targeting tumor cells, such as collateral toxicity due to lack of tumor selectivity, limited tumor accessibility, and the selection of treatment-resistant variants. By contrast to tumor cells, the genetically stable host cells should not develop resistance to treatments. In this context, the observation that tumor growth is fundamentally dependent on the onset of a private tumor neovasculature (tumor angiogenesis) has revolutionized the field of cancer research. Several treatments have been developed aimed to prevent tumor angiogenesis (anti-angiogenic strategies) or to erase the existent tumor vasculature (anti-vascular approaches) supporting the survival and growth of thousands of tumor cells. However, although such therapies achieved cancer cure in animal models, they turned out to be rather inefficient when tested in patients. This can be attributed to differences in the angiogenic status between fast-growing animal tumors and slow-growing human tumors at the time of clinical detection. Another reading of the above-mentioned observations is that anticancer treatments could benefit from interventions aimed at increasing their efficiency. For instance, radiotherapy could benefit from tumor reoxygenation while a decrease in tumor interstitial pressure could facilitate tumor accessibility to circulating agents. In this context, the mature vasculature is an attractive target since it controls tumor blood supply and is highly accessible for therapy. Therefore, strategies aimed at exploiting its functional reactivity by inducing vasorelaxation have the potential to improve tumor perfusion/drug delivery and oxygenation/radiosensitivity. To be exploited in the clinics, such pro-vascular approaches have to fulfill essential requirements. First, they need to achieve high selectivity for tumor vessels. It should prevent systemic toxicity as well as the stealing of the blood flow towards the peripheral vasculature. Second, vasodilation has to be transient, so that the tumor should not take advantage of an increased energetic supply to grow faster. Third, the therapeutic effects have to be achieved in several tumor types and in different host strains to gain a wide therapeutic range of applicability. Finally, vasomodulation has to be achieved with interventions relevant to the clinical situation, ensuring direct therapeutic significance. However, the therapeutic exploitation of agents modulating tumor perfusion was generally hampered by confounding effects on the systemic blood pressure. In our studies, we have documented that this lack of tumor selectivity can be overcome by identifying vasomodulatory pathways that are selectively altered within the tumor microenvironment, allowing selective vasomodulatory interventions. According to the criteria detailed above, to identify a differential tumor vascular reactivity, we had to work with mice models of mature tumor vascularization. We reasoned that preexisting host arterioles in mice, if coopted, should retain architectural characteristics (such as a muscular coat) necessary for functional reactivity but also be influenced by the tumor microenvironment at both molecular and functional levels. To gain in reproducibility, this model was developed by injecting syngeneic tumor cells in the vicinity of the saphenous arteriole (i.e., a collateral branch of the femoral artery) in the rear leg of mice. With tumor growth, this arteriole was progressively included in the tumor cortex (coopted), with side branches running deeply into tumors. This model was developed using several tumors and mice strains. It provides the unique advantage to allow the easy identification and isolation of mature tumor vessels from fast-growing animal tumors. To evaluate differential vasoreactivity in those tumor-coopted vessels, we adapted pressure myography, a device initially dedicated to the study of the reactivity of coronary arterioles (see annex 1). In our hands, the unprecedented application of pressure myography to the study of small tumor vessels proved to be very efficient. Indeed, this technique not only served us to confirm that arterioles remain sensitive to vasomodulation under tumor cooption, but also allowed us to evidence two major adaptations of host vessels to the tumor microenvironment: the acquisition of an ET-1-mediated basal constrictive tone and a defect in the vasodilatory NO pathway. Furthermore, we used pressure myography to identify and characterize vasomodulatory strategies exploiting these differential reactivities. More particularly, we showed that both BQ123 (an ETA inhibitor) and ionizing radiations (that restored a functional NO pathway) promoted the vasodilation of the tumor-coopted vessels. In vivo, we verified that these strategies fulfilled the essential requirements of pro-vascular approaches: tumor selectivity, transient effects, broad range of applicability, and therapeutic significance in clinically relevant regimens. This latter study led us to further explore the effects of radiotherapy on the status of the tumor vasculature. Hence, we showed that fractionated radiotherapy induced tumor angiogenesis, thereby providing a rationale to combine radiotherapy to anti-angiogenic therapies.

No description available.

Diabetic long-term complications, despite intensive treatment, cause serious handicaps at relatively young age in diabetic patients. Diabetic nephropathy (DN) develops in up to 30% of patients with type 1 diabetes (T1D). Besides the eventual loss of kidney function, with need for dialysis treatment and transplantation, this complication also increases the risk of early death from cardiovascular disease. In addition to hyperglycaemia, the risk of developing DN is influenced by a number of life-style related factors, such as smoking and diet, but the mechanisms of action of these factors are largely unknown. The incidence of DN is not linearly related to diabetes duration. There is a peak incidence of DN at 15-20 years and this, together with results from family studies, shows that genetic factors are important contributors. Possible candidate genes are those involved in regulation of intraglomerular pressure and blood pressure, oxidative stress and inflammation. The main aims of this thesis were: ● To investigate the risk of DN associated with polymorphisms in; A) the endothelial NO-synthase gene (NOS3) and genes in the renin-angiotensin-system (RAAS) (all involved in the regulation of intraglomerular pressure). B) the manganese superoxide dismutase gene (SOD2) (involved in the regulation of oxidative stress). C) the ICAM1 gene (involved in activation and migration of lymphocytes) ● To investigate gene-smoking interactions ● To investigate the influence of normal diet on risk of microalbuminuria. The aims were addressed in different case-control settings, including 347 T1D patients from Sweden and 1163 patients from Finland, with or without DN, defined as; overt DN – having albumin excretion rate (AER) ≥200 μg/min, incipient DN – AER between 20 and 200 μg/min, non-DN controls – having AER <20 μg/min and at least 20 years of diabetes duration. In one study also non-diabetic healthy individuals were included to asses the risk of T1D associated with the ICAM1 gene. Results: The RAAS genes were investigated in the Swedish sample set and there was an association between a polymorphism in the angiotensin II type 1 receptor (AGTR1) gene and overt DN, when adjusting for age, duration of diabetes, HbA1c, sex and smoking (adjusted OR=3.04, 99% CI=1.02-9.06). Also a synergistic interaction with smoking was indicated. The ICAM1 gene was investigated in the Swedish sample set, but no association with DN was found. There were, however, associations between T1D and two polymorphisms in this gene, rs281432 (OR=1.64, 95% CI=1.14-2.38) and rs5498 (OR=2.46, 95% CI=1.59-3.80). In the combined Swedish/Finnish sample set, the Glu/Glu genotype of the Glu298Asp polymorphism in the NOS3 gene was associated with DN when age at diabetes onset, duration of diabetes, HbA1c, blood pressure, sex and smoking were taken into account (adjusted OR=1.46, 95% CI=1.12-1.91). There was also association between a polymorphism in the MnSOD gene and DN in this sample set. Homozygosity for the valine-allele of the Val16Ala polymorphism was associated with increased risk of DN in a model including age at diabetes onset, duration of diabetes, HbA1c, sex and smoking (adjusted OR=1.32, 95% CI=1.00-1.74). Smoking was associated with DN (OR=2.00, 95% CI=1.60-2.50) and in the Swedish sample set there were indications of interactions between smoking and the NOS3 and SOD2 genes, but these results could not be confirmed in the Finnish sample set. A high protein intake can enhance glomerular filtration rate and accelerate progression to DN, also other dietary components such as fat, fibres, vitamins and the ratio red/white meat have been discussed as important for DN development. In a nested case-control study including young T1D patients, the normal dietary intakes of protein and other nutrients were assessed using a semiquantitative questionnaire. The results showed that T1D patients consuming more than 6.5 g fish protein (>75th percentile) per day were at slightly lower risk to have microalbuminuria in both crude (OR=0.49, 95% CI=0.25-0.97) and adjusted analyses (OR=0.26, 95% CI=0.09-0.76, adjusted for age, duration of diabetes, sex, HbA1c, mean arterial pressure, BMI, region, smoking, energy intake and fish fat intake). Conclusions: The risk of having diabetic nephropathy is influenced by at least two genes controlling blood pressure and one gene protecting against oxidative stress. Smoking also increases the risk of DN and our findings indicate that smoking may accentuate the effect of the AGTR1, NOS3 and SOD2 genes. Normal dietary intake of protein was not associated with risk of having microalbuminuria in young T1D patients, on the other hand, an intake of fish protein above the 75th percentile decreased the risk of microalbuminuria.

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Initially upon infection of the host, intramacrophage proliferation of F. tularensis occurs but after activation of the acquired host immunity, the phagocytes become activated to kill the bacterium. In my thesis, I focused on mechanisms utilized by F. tularensis to survive intracellularly and on host mechanisms responsible for macrophage-mediated killing and control of infection. The F. tularensis-specific protein IglC has been previously shown to be essential to the intramacrophage proliferation and virulence of the bacterium in mice. By electron microscopy of macrophages infected with either the live vaccine strain of F. tularensis or an isogenic mutant, denoted ∆iglC, expression of IglC was found to be necessary for the bacterium to escape from the phagosome. IFN-g-activated macrophages significantly inhibited the escape of the live vaccine strain of F. tularensis from the phagosome. iNOS and phox generate NO and O2-, respectively. These molecules and their reaction products possess both bactericidal and immunoregulatory properties. We investigated the capability of IFN-g-activated peritoneal exudate cells from gene deficient iNOS-/- or p47phox-/- mice to control an intracellular F. tularensis LVS infection. iNOS was found to contribute significantly to the IFN-g induced killing, while phox contributed only to a minor extent. Unexpectedly, bacteria were eradicated even in the absence of both a functional phox and an active iNOS. The eradication was found to depend on ONOO-, the reaction product of NO and O2-, because addition of a decomposition catalyst of ONOO- completely inhibited the killing. Studies on iNOS-/- or p47phox-/- mice infected with F. tularensis LVS showed phox to be important during the first days of infection, a stage when iNOS seemed dispensable. Eventually, iNOS-/- mice died of the infection, suggesting a role of iNOS later in the course of infection. iNOS-/- mice exhibited elevated IFN-g serum levels and severe liver damage suggesting that the outcome of infection was at least in part related to an uncontrolled immune response. Several pathogenic bacteria express Cu,Zn-SOD, which in combination with other enzymes detoxifies reactive oxygen species produced by the host. A deletion mutant of F. tularensis LVS lacking the gene encoding Cu,Zn-SOD was attenuated at least 100-fold compared to LVS in mice. In peritoneal exudate cells from mice, Cu,Zn-SOD was found to be required for effective intramacrophage proliferation and, in mice, important for bacterial replication at the very early phase of infection. In summary, the most conspicuous findings were a capability of IFN-g activated macrophages to retain F. tularensis LVS in the phagosome, an essential role of ONOO- in intracellular killing of F. tularensis, and an importance of Cu,Zn-SOD to the virulence of F. tularensis LVS.

No description available.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 118-135).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.