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Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the 'clearing-up' process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies.

<p>Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG^-allele followed by recombination-repair with a HEG⁺ allele.</p><p>The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the <i>Bacillus cereus</i> group, 28 IVSs were found in the <i>nrdIEF</i> operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the <i>nrdIEF</i> operon in the <i>Bacillaceae</i> family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)₈-YIG motif, expanding the family motif to GIY-(X)₈-₁₁-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival.</p><p>Most freestanding HEGs in bacteriophage T4 cleave both HEG⁺ and HEG^- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The <i>mobE</i> putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in <i>Escherichia coli.</i></p>

Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG- allele followed by recombination-repair with a HEG+ allele. The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the Bacillus cereus group, 28 IVSs were found in the nrdIEF operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the nrdIEF operon in the Bacillaceae family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)8-YIG motif, expanding the family motif to GIY-(X)8-11-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival. Most freestanding HEGs in bacteriophage T4 cleave both HEG+ and HEG- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The mobE putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in Escherichia coli.

Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the `clearing-up¿ process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies. / Covance Laboratories Ltd.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / DNA is a molecule susceptible to the attack of many toxic substances. So, studies on the toxic effects of chemical are very important. Tests that evaluate substances genomodifier (presenting action genoprotetora or genotoxic), how the Comet Assay, are often complex and laborious. Therefore an ultrasensitive and fast protocol no-cell is presented for the quantification DNA triggered by chemical compounds, called GEMO Assay (Genomodifier capacity assay). This assay includes a prooxidant standardized (H2O2, 3M) that is used to compare the effects on dsDNA damage of the compound-test that is evaluated with and without addition this prooxidant. The assay is performed in black 96-well plate and use Quant-iT PicoGreen® dsDNA Reagent and DNA from Calf Thymus. The vitamin C was used like compound-test in different concentrations (0.1, 0.3, 1, 3 and 10 μg/mL). For validation of GEMO Assay was used PBMCs (peripheral blood mononuclear cells) and HT29 (colon carcinoma cell line) exposed the same conditions that the proposed test and evaluated at different tests already well described in the literature: Alkaline Comet Assay, MTT, DCFH-DA and TBARS. The results showed high correlation with GEMO Assay, confirming the validation. Then the test developed in this work offers high sensitivity for detecting genomodifier substances without interference if biological systems. / Pelo fato do DNA ser uma molécula suscetível ao ataque muitas de substâncias, estudos sobre efeitos tóxicos ou fitoterapêuticos de compostos químicos são necessários. Muitos ensaios que analisam o efeito genomodificador de substâncias, às vezes, são relativamente complexos, como o Teste do Cometa. Entende-se por ação genomodificadora aquela em que a substância testada ou apresenta genoproteção ou genotoxicidade. Baseado na existência de ensaios in vitro que servem como triagem para avaliar a capacidade antioxidante de um dado composto, como o DPPH, o objetivo deste estudo foi desenvolver e validar um método in vitro da capacidade genomodificadora de compostos químicos e sintéticos. Assim, um ultrassensível e rápido protocolo que não utiliza sistemas biológicos foi desenvolvido para a quantificação do DNA dupla-fita (dsDNA) exposto a substâncias químicas, denominado de Teste GEMO (Teste de Capacidade Genomodificadora). Esse método foi concebido para placa preta de 96 poços, utilizando um corante altamente específico de dsDNA (PicoGreen®) e DNA purificado de timo de bezerro (dsDNA). O teste inclui um pró-oxidante de referência, peróxido de hidrogênio (H2O2 3M), que permite a análise comparativa dos dados obtidos, classificando a substância testada em vários níveis de genotoxicidade e ainda se a mesma apresenta potencial de genoproteção. Para o desenvolvimento do teste foi utilizado um antioxidante bem conhecido pelo seu papel genoprotetor e antitumoral, a vitamina C em diferentes concentrações (0.1, 0.3, 1, 3 e 10 μg/mL). Para a validação do Teste GEMO, foram utilizadas células mononucleares de sangue periférico (PBMCs) e células de adenocarcinoma colorretal (HT29), expostas as mesmas condições que o teste proposto e submetidas a diferentes testes já bem descritos na literatura: Teste do Cometa Alcalino, MTT, DCFH-DA e TBARS. Os resultados mostraram alta correlação com Teste GEMO, confirmando a validação. Portanto, o ensaio desenvolvido nesse trabalho oferece alta sensibilidade para detectar compostos genomodificadores, sem a interferência de sistemas biológicos.

The potentially oncogenic human pathogen BK Polyomavirus (BKPyV) was first identified in 1971 and has since been associated with a number of diseases primarily in immunosuppressed patients. Infection is established in early life and by adulthood up to 90% of populations show seroconversion for the major capsid protein VP1. Despite this infections are rarely cleared, maintaining a silent asymptomatic persistence punctuated with periods of viral shedding in the urine. The virus is non-enveloped and comprises a simple ~5.2 Kb dsDNA genome which expresses just seven known proteins, necessitating a heavy reliance on, and interactions with, host mechanisms in order to efficiently replicate and disseminate within a population. The poorly understood lifelong persistence and failure to clear infection highlights our lack of understanding of the viral life cycle and viral interactions with host processes and responses to infection. Indeed, non-enveloped viruses are thought to spread solely through infected cell lysis but such large-scale lysis should trigger an acute inflammatory response, which is rarely seen in healthy immunocompetent individuals. The research conducted for this thesis first investigates the egress of BKPyV in a non-lytic manner, presenting evidence for an active non-lytic method of viral egress that is dependent on cellular anion homeostasis. Moreover, data generated for this thesis suggests that virions egress via an unconventional secretion pathway which traffics directly from the endoplasmic reticulum (ER) to the plasma membrane in single-membraned vesicles. Further research undertook a whole cell quantitative temporal viromic (QTV) approach, post-experimentally tagging whole cell lysate peptides with isobaric labels (Tandem Mass Tagging, TMT) to provide a greater understanding of host cell proteomic changes throughout BKPyV infection in two primary human cell types over 72 hours of infection. Such an approach identified ~9000 cellular proteins, of which a surprisingly small number changed significantly in abundance in response to BKPyV infection. Of those that were changed in abundance a large proportion were related to cell cycle, revealing that BKPyV infection induces a pseudo-G2 arrest, similar to the G2/M checkpoint. Validation of TMT results in both cell types provided confidence in this robust data set, and further studies highlighted the importance of not only cell cycle status, but the activity of CDK1 for efficient viral infection and replication. Additionally, TMT generated data emphasised the lack of innate immune induction in response to BKPyV infection, suggesting BKPyV exhibits a sophisticated evasion of pathogen recognition.

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian cancers. Previous studies indicate that these genes can exhibit cooperative oncogenicity by cross-regulating one another and facilitating cancer development. Our earlier studies demonstrated that treatment of ovarian cancer cells with arsenic trioxide (As2O3) promotes cytoprotective autophagy regulated by induction of SnoN to antagonize the cytotoxic effects of As2O3. Since exact mechanisms underlying As2O3-induced SnoN expression and cytoprotective responses were unclear, we hypothesized that SnoN may be regulated by signaling pathways involving genes amplified at the 3q26 locus. Phospholipid scramblase 1 (PLSCR1) is located at 3q23 proximal to the amplified 3q26 region. It had been implicated in disruption of plasma membrane asymmetry by mediating phospholipid scrambling, a process critical for cellular events such as blood coagulation and apoptosis. However, recent findings have led to more investigations on the role and regulation of PLSCR1 in cancer development and immune responses. PLSCR1 expression is regulated by various stimuli including growth factors (EGF, G-CSF, and SCF), cytokines (IFN), and differentiation-inducing agents (ATRA). Despite these studies, transcriptional regulation of PLSCR1 remains incompletely understood. Numerous studies have suggested a critical role for PLSCR1 in the pathophysiology of various cancers including leukemia, ovarian cancer, colorectal cancer, and metastatic liver cancer. However, the precise contribution of PLSCR1 and its regulation in ovarian cancer development is unclear. Since PLSCR1 (at 3q23) is located in close proximity to SnoN/SkiL (at 3q26.2), we hypothesized that PLSCR1 expression in ovarian cancer cells could be regulated by SnoN. Herein, we present studies that primarily focus on understanding the role and regulation of SnoN/SkiL (a TGFβ pathway regulator) and PLSCR1 (an interferon-regulated gene), which are located at 3q26.2 and 3q23, respectively, in epithelial ovarian cancer. In Chapter 3, we determined that activation of the PI3K signaling pathway mediates SnoN expression and cytoprotective responses upon stimulation of ovarian cancer cells with As2O3. We first identified that As2O3 stimulation leads to activation of EGFR and its downstream signaling mediators as well as modulates its interaction with the adaptor proteins, ShcA and Grb2. Interestingly, while treatment with a general SFK inhibitor (PP2), reduced the As2O3-induced EGFR activation and SnoN induction, a more specific inhibitor SU6656 did not alter SnoN expression. Further, via studies utilizing specific inhibitors and siRNA targeting PI3K, we determined that inhibition of PI3K signaling pathway decreases SnoN induction and increases apoptosis in ovarian cancer cells in response to As2O3. This suggests that PI3K (PIK3CA) activity is required for the As2O3-mediated SnoN induction and the cell survival responses in ovarian cancer cells. Finally, we determined by siRNA-mediated knockdown that EGFR and MAPK1 alter As2O3-induced cell death response independently of SnoN induction. In Chapter 4, via bioinformatic analyses, we identified that PLSCR1 DNA copy number and mRNA expression is elevated in ovarian cancer patients and cell lines relative to immortalized (Tag/hTERT) normal ovarian surface epithelial (OSE) cells. Interestingly, altered PLSCR1 DNA and mRNA levels were correlated with SnoN in ovarian cancers. We next identified that SnoN knockdown leads to a significant (~35%, P2O3 transcriptionally downregulates PLSCR1 in a ROS-independent mechanism. Furthermore, PLSCR1 knockdown, similar to SnoN knockdown increases ovarian cancer cell sensitivity to As2O3. PLSCR1 knockdown increases cleaved PARP (marker of apoptosis) with a consequent reduction in LC3-II levels (marker of autophagosomes). Collectively, these studies implicate PLSCR1 in the pathophysiology of ovarian cancers and in altering the chemotherapeutic responses in ovarian cancer cells. PLSCR1 is an IFN-regulated gene and mediates antiviral/immune responses. More recent studies in plasmacytoid dendritic cells have implicated PLSCR1 in regulating TLR9 signaling upon stimulation with CpG ODN. However, whether PLSCR1 could mediate the innate immune responses upon stimulation with dsDNA remained unclear. In Chapter 5, we identified that stimulation of normal ovarian and mammary epithelial cells with dsDNA (empty plasmid) markedly induces PLSCR1 consequent with activation of IRF3, a downstream mediator of TLR signaling that transcriptionally regulates the expression of type 1 IFNs. Interestingly, IRF3 knockdown ablates the dsDNA-induced PLSCR1 expression suggesting that PLSCR1 induction in response to dsDNA could be mediated by IRF3. Additionally, we have determined that dsDNA stimulation induces nucleic acid sensing TLRs, TLR9 and TLR4 as well as IFN-α and IFN-β mRNAs. Interestingly, dsDNA stimulation did not induce PLSCR1 or IRF3 activation in ovarian cancer cells suggesting that the mechanisms of IRF3 activation and PLSCR1 induction in response to dsDNA might be dysregulated in ovarian cancers. Collectively, our studies demonstrate a possible synergistic role of SnoN and PLSCR1 in ovarian cancer pathophysiology and suggest a potentially dysregulated role of PLSCR1 in the dsDNA-induced immune responses of malignant epithelial cells relative to normal epithelial cells. These studies could potentially lead to development of a novel combinatorial therapeutic strategy that targets both these molecules for improving treatment of patients with ovarian carcinoma.

The present work presents electrochemical studies of Baylis-Hillman adducts, that show significant anti-tumoral activity. Electrochemical techniques used were Cyclic Voltammetry, Differential Pulse Voltammetry, Square Wave Voltammetry and Controlled Potential Electrolysis. The reduction behaviour of methyl 2-[p-nitrophenyl(hydroxy) methyl] acrylate (2) in aprotic medium (DMF + TBAP, 0.1 mol L-1) was typical of nitroaromatics, with three reduction waves, the first two related to the reduction of the nitro function. The third wave refers to the reduction of the acrylate group, similarly to the observed behavior of the pattern compound, the 2-[phenyl(hydroxy)methyl] acrylate (1). In protic medium (phosphate buffer, pH 6.9), compound 2 shows one reduction wave related to the generation of the derived hydroxylamine. In alkaline buffer (EtOH + phosphate, DMF + phosphate or EtOH + bicarbonate + NaOH, pH ~9), the electron transfer led to the formation of the stable nitro radical anion. Controlled potential electrolysis, in neutral protic medium, in 4 e-/4H+ process, furnished a dimer, after the nitro group reduction. Electrochemical studies performed on a dsDNA biosensor suggest that one of the targets for the biological action of 2 is the DNA. The DNA damage, verified by the presence of the oxidation peaks of the nucleobases guanine and adenine, is observed only, after the nitro group reduction (pharmacophore) to reactive intermediates, which reinforce the importance of the bioreduction for the biological action. The electrochemical and spectrophotometric studies, in the presence of GSH and GSSG, revealed that the reduction products of the nitro group interact with the endobiotics, in a different way. For phosphate + NaOH, pH 9.4, the addition of GSH to the solution of 2, led to the increase of current intensity for the first reduction wave that turns irreversible. The second reduction wave, relative to the hydroxylamine production, is no more observed in the voltammogram. Due to the acid nature of GSH, together with the inefficient buffering effect of the medium, glutathione acts as a protons donor, leading to a stable nitroso derivative. On the other hand, in bicarbonate + NaOH buffer, the pH is kept and glutathione is present in its dissociated form. Voltammetric changes are minimum, with a slight increase in the reversibility of the process concerning formation of nitro anion radical. At more negative potentials, the wave related to the production of hydroxylamine disappears, showing that GSH interacts with products of posterior reduction of the nitro group. The possible catalysis in the presence of O2 was not evidenced. These electrochemical results help in the understanding of the anticancer activity of 2 that can be considered a hypoxia targeted bioreductive agent with a glutathione depleting function / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No presente trabalho, foram realizados estudos eletroquímicos de compostos que apresentam expressiva atividade antiproliferativa, conhecidos como adutos de Baylis-Hillman. As técnicas utilizadas foram: voltametria cíclica, de pulso diferencial e de onda quadrada e eletrólise a potencial controlado. Os estudos eletroquímicos revelaram um comportamento padrão para o composto nitroaromático 2-[p-nitrofenil(hidroxi)] acrilato de metila (2). Em meio aprótico (DMF + TBAP, 0,1 mol L-1), o composto 2 apresentou três ondas de redução, sendo as duas primeiras referentes à redução do grupo nitro e, assim como no composto padrão não nitrado, 2-[fenil(hidroxi)] acrilato de metila (1), a onda adicional, em potencial mais negativo, relaciona-se à redução do grupo acrilato. Em meio prótico, tampão fosfato pH 6,9, uma única onda catódica relativa à formação da hidroxilamina é observada. Nos estudos em meio aquoso alcalino (EtOH + fosfato, DMF + fosfato ou EtOH + bicarbonato de sódio + NaOH, pH ~ 9), observou-se a formação de intermediário radicalar estável, o ânion radical nitro. Eletrólises em potencial controlado, em meio prótico neutro, levaram à formação e isolamento de um dímero, após redução do grupo nitro, em processo de 4e-/4 H+. Estudos eletroquímicos realizados em biossensor de dsDNA, sugerem que um dos alvos para ação biológica de 2 é o DNA. A lesão ao DNA, refletida pela presença de picos diagnósticos de oxidação das bases guanina e adenina, mensuráveis eletroquimicamente, é observada apenas após redução do grupo nitro (farmacóforo) a intermediários reativos, reforçando a necessidade de biorredução do grupo para posterior atividade biológica. Os estudos eletroquímicos e espectrofotométricos, em presença de GSH e GSSG, revelaram que os produtos de redução do grupo nitro interagem com os endobióticos, de maneira diferente. Para o meio fosfato + NaOH, pH 9,4, a adição de GSH promoveu o aumento na intensidade de corrente para o primeiro processo eletródico, bem como a perda de reversibilidade. Já a segunda onda de redução, relativa à formação da hidroxilamina, não foi observada no voltamograma. De acordo com as funções ácidas da GSH, aliada ao ineficiente efeito tamponante desse meio, a glutationa atua como doador de prótons, favorecendo a formação do derivado nitroso. Por outro lado, em tampão bicarbonato + NaOH, onde se tem um eficiente efeito tamponante e a glutationa se encontra na forma desprotonada, as alterações voltamétricas são mais discretas, com aumento da reversibilidade do processo referente à formação do ânion radical nitro. Em potenciais mais negativos, a onda relativa à geração da hidroxilamina desaparece, o que evidencia a interação de GSH com os produtos de redução estendida do grupo nitro. A possibilidade de catálise, em presença de oxigênio, não foi evidenciada para 2. Os resultados obtidos fornecem subsídios úteis para a compreensão do mecanismo de ação antitumoral de 2, que pode ser considerado um agente biorredutivo, com função adicional seqüestradora de glutationa

Autoantikroppar är antikroppar som attackerar en individs egen vävnad. Riktas dessa mot antigener från cellkärnan kallas de för ANA. Exempel på dessa är anti-dsDNA antikroppar som associeras med sjukdomen SLE. Med hjälp av metoderna ELISA, CLIFT eller FARR-radioimmunoassay kan anti-dsDNA antikroppar analyseras och tillsammans med kliniska symptom såsom ansiktserytem kan diagnosen SLE ställas. Syftet med examensarbetet var att undersöka analys av anti-dsDNA antikroppar med hjälp av instrumentet Phadia 250 vars princip är en automatiserad ELISA, för att se om det kan användas som screeningmetod istället för CLIFT. 20 EQUALIS-serumprover (externa kontrollprover) analyserades med Phadia 250 och de Phadia 250-positiva proverna analyserades ytterligare en gång med CLIFT. Därefter analyserades 44 patienters serumprover (21 positiva och 23 negativa, tidigare analyserade med CLIFT) med Phadia 250. Patientprovernas resultat gav 70 % sensitivitet och 86 % specificitet med Phadia 250. Phadia 250-resultat som ej överensstämde med förväntade resultat på EQUALIS- och patientproverna erhölls i flera fall. Vid en noggrannare granskning av EQUALIS-proverna noterades att liknande resultat erhållits av andra laboratorier som använt likartade metoder. EQUALIS-proverna var initialt analyserade och kategoriserade enligt CLIFT, vilket innebar att hög överensstämmelse kunde förväntas med den egna CLIFT-metoden. Slutsatsen av studien är att underlaget för att införa metoden i rutindiagnostiken är inte tillräckligt utifrån de erhållna resultaten. Det krävs ytterligare studier samt granskning av inkluderade patienters journaler för att säkrare bedöma metodernas korrelation till sjukdomen.