This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

Human T lymphotropic virus type -1 (HTLV-1) infections are important causes of mortality and morbidity in endemic areas worldwide. There is neither a vaccine specific for the virus nor satisfactory treatment for the associated malignancy or inflammatory syndromes. HTLV-1 associated infective dermatitis (IDH) is a chronic dermatitis that has been observed in a variable proportion of HTLV-1 infected children. IDH may serve as an early clinical marker for HTLV-1 and an indicator of increased risk for developing other HTLV-1 associated conditions such as adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy or transient spastic paraparesis (HAM/TSP). However the mechanisms underlying IDH and the relationships with HAM/TSP and ATLL are poorly understood. We undertook skin biopsies from 14 cases with IDH, and controls which included five asymptomatic carriers (ACs) and 18 healthy uninfected individuals from South Africa. We conducted clinical assessments, proviral load, allergen-specific IgE, peripheral blood and cutaneous T cell and virological analyses. We obtained relevant clinical history and examined all cases and controls based on a pre-formed questionnaire. Despite the partial clinical similarities with atopic dermatitis, the individuals with IDH did not have an increased incidence of atopic disease including asthma or rhinitis. Furthermore house dust mite-specific IgE levels were not elevated in the cases compared to the controls, suggesting that atopy is not a predisposing factor for the development of IDH in HTLV-1 infected individuals. Circulating proviral load was significantly higher in those with IDH compared to asymptomatic carriers and skin biopsy revealed acanthosis, and lymphocytic epidermotropism associated with a superficial perivascular and periadnexal lymphocytic infiltration of CD8+, and CD4+ T cells. Furthermore IDH associated with an infiltrate of epidermal and dermal FoxP3+ T cells and lesional down-regulation of filaggrin expression compared to non-lesional skin. We did not observe an elevation of pro-inflammatory cytokines in the sera of individuals with IDH compared to the controls. We investigated integration patterns in the skin and blood of 10 cases with IDH, and two asymptomatic carrier (AC) individuals from South Africa. We first showed that the virus is present in the skin at high levels (total mean levels of 47.09 proviral copies per 1000 cells) as comparable to that which has been observed in blood (total mean levels 137 proviral copies per 1000 cells). Using a high throughput Illumina sequencing system in collaboration with Professor Bangham, we mapped and quantified the relationship between oligoclonal proliferation of HTLV-1 infected T cells in the skin and blood of IDH patients. It was found that in IDH, a selective outgrowth of certain clones is favoured, supporting the possibility of skin-specific factors exerting positive selection on proliferation. In IDH, there was not a preferential integration of the provirus in transcriptionally active regions of the gene sites, as had been observed in other HTLV-1 associated conditions. These observations imply that the selection forces that favour oligoclonal proliferation of HTLV-1+ T cells differ fundamentally between simple HTLV-1 infection and other events associated with the dermatitis. In conclusion, these data show that HTLV-1 is not associated with an atopic diathesis. Given the lack of elevated pro-inflammatory cytokines and presence of a cutaneous infiltrate of FoxP3+ T cells, the findings suggest that high levels of HTLV-1 replication promotes a regulatory environment leading to filaggrin down-regulation, cutaneous susceptibility to infection, and secondary inflammatory skin disease. Viral integration patterns would support the presence of skin-specific positive selection, perhaps eventually leading to expansion of particular clones with the potential to develop towards ATLL. It remains to be explained whether the high viral load in IDH changes over time, more specifically in the steps leading to ATLL.

Pathogenic gain-of-function mutations associated with increased paternal age, albeit harmful to embryonic development, are paradoxically enriched in the normal testis. Evidence from previous studies indicates that these so-called paternal age-effect mutations confer a proliferative advantage to the spermatogonia in which they arise, leading to clonal expansion within the normal testis over time. Recently, spermatocytic seminoma (SS; a rare testicular germ cell tumour that occurs mainly in older men) has emerged as a key link between the processes of somatic and germline mutation (Goriely et al, Nat Genet. 41:1247-52, 2009), suggesting that the proposed clonal expansion events can in some cases lead to testicular tumourigenesis. In this thesis, I have used immunohistochemistry to seek evidence for putative clones of cells in the normal adult testis. To address this, a screening approach was developed using markers chosen from analysis of normal testicular tissues and SS. The ontogeny of OCT2 and SSX expression in human testis, from embryonic development to adulthood, identified distinct subpopulations of spermatogonia at different maturation stages. Together, these data reveal the potential of OCT2 as a novel marker of A_dark spermatogonia (human reserve spermatogonial stem cells). In parallel with these observations, two distinct types of SS characterised by differential OCT2 and SSX immunoexpression were identified, providing new evidence for heterogeneity of this tumour. This work provided the backdrop to the detailed immunohistochemical study of normal adult testis by characterising in serial sections the expression of five spermatogonial markers, MAGEA4, SSX, FGFR3, OCT2 and SAGE1, and a proliferation marker, Ki67. Independent sections were screened with predetermined criteria set to identify unusual positively-stained cellular clusters within the seminiferous tubules. Several antigenic combinations previously described in SS were observed in a subset of these clones, suggesting differing genetic origins and a possible link with early events of testicular tumourigenesis. The size (minimum number of cells) of each clonal event was estimated and its correlation with the staining pattern of the molecular markers was investigated. In summary, the data presented in this thesis convincingly identify for the first time the previously hypothesised clonal events in the testis using immunohistochemical markers. My research will pave the way for future work involving genetic analysis of microdissected cells from these putative clones, aimed at identifying the underlying mutational events thought to be present.

No description available.

Resumo: As enteroparasitoses comumente afetam o equilíbrio orgânico dos animais de estimação, sendo isto particularmente preocupante em relação aos cães, devido ao intimo contato destes com o homem. As técnicas coproparasitológicas usualmente utilizadas na rotina laboratorial para detecção de parasitoses gastrintestinais são indispensáveis para o diagnóstico e por conseqüência para determinar a necessidade de tratamento dos animais infectados. Neste estudo objetivou-se avaliar a ocorrência de helmintoses e protozooses em cães domiciliados e sua associação com faixa etária, sexo e raça, bem como a eficiência dos Métodos Willis- Mollay, Faust, Sedimentação e Exame Direto quanto a recuperação de ovos e oocistos de parasitos gastrintestinais da espécie canina. Foram processadas 401 amostras fecais, sendo que em 300 foi efetuada a Técnica de Kinyoun para pesquisa de Cryptosporidium spp. Os parasitos encontrados foram Ancylostoma spp. em 53,1%, Toxocara canis em 20,7%, Cystoisospora ohioensis em 15,7%, Trichuris vulpis em 3,7%, Dipylidium caninum em 2,5%, Cryptosporidium spp. em 1,33% e Taenia spp. em 1,0% das amostras. A positividade para Ancylostoma spp. não foi influenciada pelas variáveis raça, sexo e idade. Houve diferença estatisticamente significativa para T. canis e C. ohioensis nos cães com até seis meses de idade quando comparados a animais acima de seis meses (P<0,0001). Quanto a variável sexo houve associação apenas com o ascarídeo T. canis com maior ocorrência nos machos quando comparado as fêmeas (P = 0,0243). A associação da presença das formas evolutivas dos parasitos com raça dos cães não foi estatisticamente significativa. / Abstract: The enteroparasitoses generally affect the organic equilibrium of pets, being particularly worrying referring to dogs, due to intimate contact with the human being. The techniques coproparasitologics usually used in laboratorial routines for detection of gastroenteric parasitoses are indispensable for the diagnosis and consequence to determine the necessary of infected animals treatment. In this study we evaluated the occurrence of helminthoses and protozooses in dogs and its association with age, sex and race, as well as the efficiency of Willis-Mollay, Faust, Sedimentation and exam Direct methods. It was processed 401 fecal samples, being accomplished in 300 parts, the technique of Kinyoun for directed research of Cryptosporidium spp. The parasite found were Ancylostoma spp. (53.1%), Toxocara canis (20.7%), Cystoisospora ohioensis (15.7%), Trichuris vulpis (3.7%), Dipylidium caninum (2.5%), Cryptosporidium spp. (1.33%) and Taenia spp. (1.0%). There was a statistically significant difference for T. canis and C. ohioensis in dogs up to six months old (P<0,0001). The occurrence of T. canis was statistically bigger in males when compared to females (P=0,0243). The association of parasites with race was not statistically significant. Of the four dogs (1.33%) positives for Cryptosporidium spp., only one presented a semi liquid consistence faeces and light dehydration (5 to 7%), having this animal age superior to six months old, with simultaneous infection for Ancylostoma spp. The Willis-Mollay was more efficient in the diagnosis of Ancylostoma spp. and T. canis eggs. The Direct method was least efficient. The techniques were statistically the same as the occurrence of the C. ohioensis oocysts. / Orientador: Silvia Helena Venturoli Perri / Coorientador: Kátia Denise Saraiva Bresciani / Banca: Alessandro Francisco Talamini do Amarante / Banca: Solange Maria Gennari / Mestre

Orientador: Renato Herman Sundfeld / Coorientador: Lucas Silveira Machado / Banca: Ticiane Cestari Fagundes Tozzi / Banca: Leandro Azambuja Reichert / Resumo: Este estudo prospectivo, randomizado e paralelo analisou a alteração de cor, sensibilidade dental, grau de cooperação e satisfação de voluntários quando submetidos ao clareamento dental com peróxido de carbamida a 10%, empregado durante 21 dias por 2, 4 e 8 horas/dia. O tempo preciso do uso diário das moldeiras/produto clareador foi mensurado por meio de um microssensor TheraMon (Sales Agency Gschladt, Hargelsberhg, Áustria). Sessenta e seis voluntários, de ambos os gêneros, com idades entre 18 e 22 anos foram selecionados e distribuídos aleatoriamente em três grupos de estudo (n = 22), os quais receberam as informações sobre a presença do microssensor em suas moldeiras. O fator estudado foi o tempo de uso das moldeiras em três níveis: 2 (GI), 4 (GII) e 8 (GIII) hrs/dia. Para a análise clínica, as variáveis de resposta foram: grau de cooperação diária dos voluntários de acordo com o tempo de uso diário das moldeiras; grau de satisfação dos voluntários com o clareamento dental; eficácia do clareamento dental nos incisivos e caninos superiores e inferiores pelo método visual (Vita Classical) e digital (Vita Easyshade), assim como o grau de sensibilidade dental que foi avaliado com método analógico-visual. Os voluntários foram avaliados no período inicial (baseline), aos 7, 14 e 21 dias após o início do tratamento clareador e 14 dias após apenas para as observações da cor, sensibilidade e grau de satisfação. Os dados foram analisados com aplicação do teste ANOVA e pelo post test... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This prospective, randomized, parallel study analyzed the color change, tooth sensitivity, degrees of cooperation, and satisfaction of volunteers when submitted to at home dental bleaching with 10% carbamide peroxide (Opalescence -Ultradent Products, Inc. South Jordan, USA) for 21 consecutive days for 2, 4 and 8 hours/day. The wear time of acetate trays/dental bleaching was measured through a TheraMon microsensor (TheraMon® microelectronic system; Sales AgencyGschladt, Hargelsberhg, Austria). Sixty six volunteers, both sexes, with ages ranging from 18 to 22 years were selected and randonly distributed into 3 study groups (n=22). The volunters received information about presence of the microsensor in their trays. The fator studied was the wear time of the trays in three levels: during 2 (GI), 4 (GII) and 8 (GIII) hours/day. For the clinical analysis the response variables were: the degree of daily cooperation of the volunteers as the wear time/daily of the trays, microelectrically collected by micro sensor TheraMon®; the degree of satisfaction of volunteers with dental bleaching; the effectiveness of dental bleaching in the upper and lower incisors and canines teeth, by the visual method (Vita Classical) and digital (Vita Easyshade) and dental sensitivity was evaluated by the volunteers with a scale visual analog method on a scale of 0 to 10. The volunteers were evaluated at baseline period, at one, two and three weeks after the beginning of the bleaching treatment, and again ... (Complete abstract click electronic access below) / Mestre

Darbo tikslas. Įvertinti klinikinės diagnostikos laboratorijos klaidas ir jų valdymo galimybes. Tyrimo metodika. Atlikta X ligoninės klinikinės diagnostikos laboratorijos klaidų registro 2007 – 2009 metų duomenų analizė ir susisteminti duomenys. Jais remiantis, identifikuotos klaidos, jų priežastys ir pasiskirstymas. Atlikta ligoninės skyrių, tiesiogiai susijusių su laboratoriniais tyrimais, darbuotojų (gydytojų ir slaugytojų) anketinė apklausa. Išdalinta 260, grąžinta 241 anketų (atsakas 92,7 proc.). Požymių ryšio reikšmingumui skaičiuotas χ2 kriterijus, dviejų proporcijų lyginimui - z kriterijus. Rezultatai. Iš visų klaidų (N=669) didžiausia dalis (80 proc.) identifikuota ikianaliziniame tarpsnyje. Dažniausios ikianalizinio tarpsnio klaidos - tyrimo užsakymo (22 proc.), ėminių ėmimo (23 proc.), užsakymo formos pildymo (21 proc.), įvykusios hemolizės (11 proc.). Kituose tarpsniuose klaidų pasiskirstymo dažnis ženkliai mažesnis: analiziniame. ir poanaliziniame po 10 proc. Analiziniame tarpsnyje didžiausią dalį užima klaidos, identifikuotos matavimo metu dėl ikianalizinių veiksnių poveikio (31 proc.), poanaliziniame - žmogiškojo faktoriaus ir elektroninių programų nesusidirbimo klaidos (41 proc.). Nustatyta, kad darbuotojų informuotumas apie laboratorijos klaidas ir jų valdymą yra nepakankamas. 55,9 proc. darbuotojų mano, kad apie galimas klaidas, lemiančias laboratorinių tyrimų patikimumą, bei jų išvengimo galimybes, žino tik iš dalies. Informacijos apie laboratorinio tyrimo... [toliau žr. visą tekstą] / Aim of the study: Evaluate clinical diagnostics laboratory errors and their management possibilities. Study methodology: Laboratory X error registry study performed (2007 – 2009 year). Survey among hospital staff related to laboratory test ordering conducted. Total responses: 241 (92.7% from 260 invitations). Criterions calculated: indication relation significance - χ2, couple proportions comparison - z. Results: Error distribution (total 669) by phase: 80% pre-analytical, 10% analytical, 10% post-analytical. Top errors in pre-analytical phase: specimen collection (23%), test ordering (22%), order form input (21%), hemolyzed sample (11%). Analytical phase most errors are influenced by pre-analytical variables (31%). Post-analytical phase most errors are caused by human factor and software malfunction (41%). Found, that hospital staff awareness about laboratory errors and their handling is insufficient. 55.9% staff responded they have only partial knowledge about errors influencing testing reliability and how to avoid errors. 51.3% staff has knowledge about test process organization. 33.3% staff knows about error classification by phase. 71.4% doctors and 42.6% nurses consider such information important and would like to learn more. 52% doctors and 33.8% nurses familiar with full set of available tests. More knowledge about available laboratory tests have staff of age >54y (58.1%), also, having employment history >24y (48.6%). Most of staff (expectation of 81.9% respondents... [to full text]

Primary aldosteronism (PA) is a common form of secondary hypertension with an international prevalence rates between 5 and 10 %. It is characterized by a high autonomous aldosterone production that causes cardiovascular damage, renin suppression, hypertension, sodium retention, potassium excretion and hypokalemia. The screening of PA is a simple test measuring aldosterone to renin ratio (ARR) with immunoassay method. This test is currently considered as the most reliable screening tool for PA.     The main objective of the study was to evaluate an ELISA-method, for detection of aldosterone and renin in blood plasma, to be used for routine analysis in the laboratory. The second aim was to investigate the effect of refreezing samples, considering that cryoactivation of prorenin might occur.     One hundred blood samples were analysed, in regard to aldosterone and renin, by using two commercial ELISA assays (DRG ELISA from DRG Diagnostics, Germany) on a Dynex DS2 instrument. In addition, the accuracy and precision of the methods were calculated. The effect of refreezing was investigated with a series of eight samples, which were analyzed twice on the same instrument.     Both assays performed well. The resulting data showed good precision and accuracy. The correlation between the original and refreezed samples was good, r = 0.989 and r = 1.0 for aldosterone and renin respectively. Considering that the study only included eight samples, further investigation is recommended.     Evaluation showed that both immunoassays are reliable in diagnostic use and the ELISA-method is suitable to implement in the laboratory for routine analysis.

Avsaknad av en ”gyllene standard” och en låg sensitivitet hos förstahandsmarkören total B12 har varit de stora begränsningarna i diagnostiken av vitamin B12-brist anemier. Med syftet att förbättra diagnostiken av bristanemier vid Länssjukhuset Ryhov, Jönköping utvärderades användningen av aktivt B12 vid bristanemiutredningar och en metodverifiering för analysen genomfördes på ARCHITECT i2000SR. Sambandet mellan aktivt B12 och totalt B12 undersöktes med en korrelationsanalys för totalt B12-nivåer: låg (n=10, &lt;148 pmol/L), gråzonen (n=10, 149–200 pmol/L) och normal (n=10, 201-250 pmol/L). För bestämning av träffsäkerheten hos aktivt B12 studerades 14 bristanemifall. Ett receiver operating characteristic (ROC) diagram skapades och arean under kurvan (AUC) beräknades. Variationskoefficient (CV) för mellanliggande- och inomserieprecision för aktivt B12 var 4,4 % respektive 2,4 %. Svag korrelation observerades i alla B12-nivåer, men sammantaget resulterade nivåerna i en medelstark korrelation (R=0,600). Aktivt B12 med ett gränsvärde på 40 pmol/L resulterade i en hög specificitet (75 %) och sensitivitet (80 %) med en AUC på 90 %. Analys av aktivt B12 på ARCHITECT i2000SR möjliggör en precis mättning av aktivt B12 i humant serum och därför är lämplig för rutinmässig användning. Träffsäkerheten hos aktivt B12 var hög och markören kan därmed rekommenderas för användning vid bristanemiutredningar vid Länssjukhuset Ryhov, Jönköping. / Diagnosis of vitamin B12-defiency anemia has been challenging due to lack of ”gold standard” and poor sensitivity of total B12 as a first line marker. With the aim of improving diagnostic of anemia investigations at county hospital Ryhov, Jönköping, we performed a method verification for active B12 on ARCHITECT i2000SR and evaluated the usefulness of active B12 in diagnosing anemia. Correlation analysis was performed using samples with different total B12 ranges: low (n=10, &lt;148 pmol/L), grey zone (n=10, 149-200 pmol/L) and normal (n=10, 201-250 pmol/L). Receiver operating characteristic (ROC) curve was created and area under the curve (AUC) was calculated for determining accuracy of active B12 using 14 deficiency cases. Coefficient of variation (CV) for within-run and total imprecision, for active B12 was 2,4 % and 4,4 %, respectively. Weak correlation was found between B12-ranges, however a moderate correlation (R=0,600) was found including all groups. Active B12 with the cut-off value of 40 pmol/L resulted in high specificity (75%) and sensitivity (80%) and had an AUC of 90 %. Active B12 assay on ARCHITECT i2000SR allows a precise measurement of active B12 in human serum and therefore is adequate for routine use. Active B12 had high accuracy and can be recommended in anemia investigations in county hospital Ryhov, Jönköping.