Dissertations / Theses on the topic 'Biological markers'

Infertility affects 15 % of couples, which corresponds to 60 - 80 million worldwide. The microenvironments in which the oocyte, embryo and fetus mature are vital to the establishment and development of a healthy pregnancy. Different biological systems, such as angiogenesis, the immune system and apoptosis need to be adequately regulated for pregnancy to occur and progress normally. The overall aim of this thesis was to investigate the impact of Histidine-rich glycoprotein (HRG) and Src homology 2 domain-containing adapter protein B (SHB) on human female fertility. HRG is a plasma protein that regulates angiogenesis, the immune system, coagulation/fibrinolysis and apoptosis, by building complexes with various ligands. The impact of HRG on fertility is studied here for the first time. HRG is present in follicular fluid, the Fallopian tube, endometrium, myometrium and placenta. HRG distribution within embryo nuclei depends on developmental stage. Blastocysts express and secrete HRG. The HRG C633T single nucleotide polymorphism (SNP) appears to affect the chance of pregnancy and, correspondingly, parameters associated with pregnancy in IVF. Additionally, this HRG genotype may increase the risk in IVF of only developing embryos unfit for transfer. SHB is an adaptor protein involved in intracellular signaling complexes that regulate angiogenesis, the immune system and cell proliferation/apoptosis. Shb knockout mice have altered oocyte/follicle maturation and impaired embryogenesis. The impact of three SHB polymorphisms (rs2025439, rs13298451 and rs7873102) on human fertility is studied for the first time. The SNP prevalences did not differ between infertile and fertile women. BMI, gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer seems to be affected by SHB genotype. In conclusion, HRG and SHB appear to influence female fertility. They are potential biomarkers that might be used for predicting pregnancy chance in infertile women. Knowledge of these genotypes may improve patient counseling and individualization of treatment.

Abstract Hodgkin lymphoma (HL) is among a heterogeneous group of lymphomas. Over 80% of all patients can be cured with chemo- and radiotherapy. HL has become a model to study long-term effects of radio- and chemotherapy, because of the excellent prognosis. There are a significant number of patients who suffer or die because of the treatment-related long-term toxicity. The aim of this work was to discover new possible biological factors to predict poor prognosis and offer new aspects to individualize patient treatment in a convenient manner in HL. The retrospective study involved HL patients uniformly treated in 1997–2015. Immunohistochemistry was used to determine the expression of various biological markers, including oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine and the antioxidant enzymes manganese superoxide dismutase (MnSOD) as well as peroxiredoxins (Prx II, Prx III, Prx V, Prx VI) in HL patient samples. Using immunohistochemistry, we also evaluated expression of hypoxia-inducible factors (HIF-1α, HIF-2α), prolyl hydroxylase domain enzymes (PHD1, PHD2, PHD3), the epigenetic regulator lysine (K)-specific demethylase 4 (KDM4A, KDM4B, KDM4D) as well as sirtuins (SIRT1, SIRT4, SIRT6), the DNA-repair proteins Human Rap1 interacting factor 1 (Rif1) and O6-alkylguanine DNA alkyltransferase (MGMT) from representative classical Hodgkin lymphoma (cHL) patient samples. Low-level expression of 8-OHdG was associated with poorer relapse-free survival (RFS) in advanced-stage HL and a high extent of MnSOD predicted early relapse in the whole HL cohort. Strong expression of PHD1, KDM4B and KDM4D predicted dismal RFS in radiotherapy-treated cHL patients. The results also showed that strong expression of HIF-1α, SIRT6 and Rif1, and SIRT6 together with Rif1, were associated with prolonged RFS, especially in advanced-stage radiotherapy-treated cHL patients. In multivariate analysis, PHD1, MnSOD, 8-OHdG and Rif1 separately and together with SIRT6 were statistically significant predictors of RFS. The results reflect the significance of the studied biomarkers in HL, especially in radiotherapy-treated patients. This might be beneficial when individualizing treatment strategies, avoiding overtreatment and controlling long-term treatment-related toxicity. Further research, however, is needed to confirm these preliminary findings
Tiivistelmä Hodgkinin lymfooma (engl. HL) kuuluu heterogeeniseen imukudossyöpien eli lymfoomen ryhmään. Yli 80 % lymfoomapotilaista voidaan parantaa solunsalpaaja- ja sädehoidon avulla. Hyvän ennusteen takia HL- tutkimuksen tärkeä painopiste on säde- ja solunsalpaajahoidon pitkän ajan haittavaikutukset. Huomattava määrä potilaista kärsii tai jopa kuolee hoitoon liittyvistä pitkäaikaishaitoista johtuen. Tämän tutkimuksen tarkoituksena oli löytää uusia mahdollisia biologisia tekijöitä, jotka ennakoisivat taudin huonoa ennustetta ja samalla antaa uusia näkökulmia HL potilaiden hoidon yksilöllistämiseen. Tämä retrospektiivinen tutkimus käsitti vuosina 1997-2015 samanlaisesti hoidettuja Hodgkinin lymfooma -potilaita. Immunohistokemiallisilla värjäyksillä määritettiin biologisten merkkiaineiden, mukaan lukien oksidatiivisen stressin markkereiden 8- hydroksideoksiguanosiinin (8-OHdG) ja nitrotyrosiinin, sekä antioksidanttientsyymien mangaanisuperoksidi-dismutaasin (MnSOD) sekä peroksiredoksiinien (Prx II, Prx III, Prx V, Prx VI) ilmentymistä HL -potilasnäytteissä. Määrittelimme myös immunohistokemiallisilla värjäyksillä epigeneettisten säätelijöiden lysiinin spesifisen demetylaasientsyymin 4 (KDM4A, KDM4B, KDM4D) sekä sirtuiinien (SIRT1, SIRT4, SIRT6), hypoksiaa indusoivien tekijöiden (HIF-1α, HIF-2α), prolyylihydroksylaasientsyymien (PHD1, PHD2, PHD3) ja DNA:ta korjaavien proteiinien Rap1 vaikuttuvan tekijä 1 (Rif1) ja O6-metyyliguaniini-DNA metyylitransferaasin (MGMT) ilmentymistä edustavissa klassista Hodgkinin lymfoomaa sairastavien potilaiden (engl. cHL) näytteissä. Heikko 8-OHdG värjäytyminen ennusti ennenaikaista taudin uusiutumaa levinneessä HL:ssa ja korkea MnSOD ilmaantuvuus ennusti ennenaikaista taudin uusiutumaa koko HL -ryhmässä. Sädehoidetuilla cHL potilailla voimakas PHD1, KDM4B ja KDM4D värjäytyminen ennusti ennenaikaista taudin uusiutumaa. Tulokset osoittivat myös, että erityisesti sädehoidetuilla levinneen taudin cHL potilailla voimakas HIF-1α, SIRT6, Rif1 ja SIRT6 yhdessä Rif1:n kanssa oli yhteydessä pidentyneeseen uusiutumavapaaseen aikaan. Monimuuttuja-analyysissä PHD1, MnSOD, 8-OHdG ja Rif1 itsenäisenä ja yhdessä SIRT6 kanssa ennustivat tilastollisesti merkitsevästi taudin ennenaikaista uusiutumaa. Tulokset osoittavat näiden eri biomarkkereiden merkittävyyden HL:ssä, erityisesti sädehoitoa saaneilla potilailla. Tuloksista voi olla hyötyä, kun hoitokäytäntöjä yksilöidään, mikä voisi helpottaa välttämään liiallista hoitoa ja hallitsemaan pitkäaikaisiin hoitoihin liittyviä haittoja. Näiden alustavien havaintojen vahvistamiseksi tarvitaan kuitenkin lisätutkimuksia

L’étude des marqueurs biologiques dans la broncho-pneumopathie chronique obstructive (BPCO) et l'asthme, deux maladies respiratoires chroniques affectant des millions de personnes dans le monde, pourrait améliorer leur diagnostic, leur traitement et leur prévention.Cette thèse comprend deux parties. La première visait à évaluer l'association entre un marqueur spécifique des poumons, la protéine surfactant D (SP-D) sérique, et la BPCO, et à trouver un seuil de SP-D capable de discriminer les patients BPCO des témoins. Elle a été réalisée dans le cadre d’une étude cas-témoin au Liban incluant des patients BPCO (n=90), des asthmatiques (n=124) et des témoins (n=180). La deuxième partie visait à évaluer les associations chez les adultes des marqueurs de l’inflammation systémique (protéine C-réactive ultra-sensible, hs-CRP (n=252), et des cytokines (n=283)) et des marqueurs de dommages dus au stress oxydant (8-isoprostanes 8-IsoPs (n=258) du condensat de l’air exhalé) avec les phénotypes de l’asthme.Elle a été réalisée dans le cadre de l'étude épidémiologique longitudinale Française des facteurs génétiques et environnementaux de l'asthme (EGEA).Les résultats ont montré que les niveaux de SP-D sériques étaient associés positivement avec la BPCO et des seuils des niveaux de SP-D chez ces patients ont été identifiés avec d'excellentes valeurs discriminantes. Dans EGEA, aucune association n'a été trouvée entre les niveaux de hs-CRP sériques et le contrôle de l’asthme. Des profils de cytokines sériques (identifiés par analyse en composante principale) avec des niveaux élevés d’interleukine(IL)-1Ra et d’IL-10 ont été associés avec moins de crises d'asthme et un risque plus faible d'un mauvais contrôle de l'asthme sept ans plus tard. Les résultats des analyses préliminaires sur les associations entre les niveaux de 8-IsoPs et les phénotypes de l'asthme sont également présentés.Globalement, ces résultats ont montré l'utilité d'étudier les marqueurs biologiques en lien avec la BPCO et l'asthme
Studying the biological markers in chronic obstructive pulmonary disease (COPD) and asthma, two chronic respiratory diseases affecting millions of individuals around the world, could improve their diagnosis, their treatment and their prevention.This thesis includes two parts. The first aimed to assess the association between a lung-specific biomarker, serum Surfactant Protein D (SP-D), and COPD, and to find cut-off points able to discriminate COPD patients from controls using SP-D levels. It was performed in a case-control study in Lebanon including COPD (n=90) and asthma patients (n=124) and controls (n=180). The second part aimed to assess the cross-sectional and longitudinal associations in adults for systemic inflammatory biomarkers (high sensitivity C reactive protein hs-CRP (n=252) and cytokines (n=283) as well as biomarkers of damage due to oxidative stress (8-Isoprostanes 8-IsoPs (n=258) from the exhaled breath condensate) and asthma outcomes.It was performed in the French longitudinal epidemiological study on the genetics and environmental factors of asthma (EGEA).Results showed that serum SP-D levels were positively associated with COPD and thresholds for SP-D levels in these patients were identified with excellent discriminant values. In EGEA, no association was found between serum hs-CRP levels and asthma control. Serum cytokine profiles (identified by principal component analysis) with high levels of interleukin (IL)-1Ra and IL-10 were associated with less asthma attacks and lower risk of poor asthma control in adults seven years later. The results of the preliminary analyses on the associations between the levels of 8-IsoPs and asthma outcomes are also presented.Overall, these results have shown the usefulness of studying the biological markers related to COPD and asthma

Alzheimer's disease (AD) represents the most common type of dementia, accounting for 50% of the total amount of cognitive impairment, while vascular dementia (VD) accounts for approximately 20% of the cases. AD has traditionally been considered a neurodegenerative condition in which vascular dysfunction plays a marginal role. On the other hand, VD is thought to be caused by a subacute or chronic reduction in cerebral blood flow (CBF) leading to neuronal dysfunction and death. However, it is not clear if these two major causes of dementia also share pathogenetic mechanisms. Many evidences point out a vascular pathogenetic involvement in its etiology. The recent finding that Abeta has also harmful effects on vessels indicates that vascular damage could be involved in the pathogenesis of AD, thus explaining how AD and VD are not always distinct entities but overlap by varying degrees. Abeta peptide, which plays a central role in AD, not only exerts harmful effects on the vessel walls increasing the risk of silent hemorrhagic/ischemic strokes, but it also facilitates the ultrastructural degeneration of the vessels, reducing vessels’ diameters, cerebral blood flow and energetic metabolism. Conversely, vascular damage which results in hypoxia/ischemia, inflammation, microglia activation and oxidative stress, can influence APP processing, modulating the expression of enzymes responsible for Abeta production. These mechanisms have been described in animal models, while few independent observations have been performed in humans. Classical neuropathological markers of AD are: (i) deposits of amyloid β (Abeta) in brain tissue (neuritic plaques), as well as within the wall of cerebral blood vessels; (ii) microglia activation; (iii) dystrophic neuronal processes in proximity and within Abeta plaques; (iv) progressive loss of synapses and neurons; and (v) severe structural damage of cerebral blood vessels. Nonetheless, many vascular risk factors have been also associated to AD, i.e. ApoE-e4 genotype, diabetes and hyperinsulinemia, high systolic blood pressure in midlife to late life and low diastolic blood pressure in late life, smoking, stroke, traumatic brain injury, elevated serum homocysteine (Hcy) levels, hypercholesterolemia and atherosclerosis. Furthermore, there are many evidences of peripheral haemostatic abnormalities, in particular platelets alterations, Von Willebrand Factor and Activated Factor VII, and increased level of thrombomodulin and E-selectin in AD, suggesting that an endothelial dysfunction may be involved in AD pathogenesis. Based on these evidences, a possible hypothesis is that Abeta induces endothelial injury, thus promoting ischemic damage, which may in turn affect APP processing and Abeta production. This reciprocal interaction may provide an explanation to the pathogenetic link between these two conditions. In this contest, elevated plasma levels of Homocysteine (Hcy), also know as Hyperhomocysteinemia (HHcy), is one of the strongest independent risk factors for vascular and cerebrovascular disorders and it has been associated to the risk of develop AD in elderly people. Recently, our group has published evidences of elevated plasma levels of Hcy in AD, correlated with folate deficiencies. Moreover, we have demonstrated that Post Methionine Load (PML) test is able to reveal twice as many HHcy AD subjects with respect to the fasting analysis, suggesting PML as useful test in detecting AD patients who may have the chance of an early folate treatment. Since vascular lesions often coexist with Abeta deposits in AD, and aberrant Abeta deposition in the intima may be pathologically important, it is possible that this phenomena is not only the consequence of the AD-related aberrant APP processing but may represent the early trigger of Amyloid deposition, in response to the primary endothelial damage. Moreover, after Abeta or hypoxia exposure, the endothelium undergoes changes which trigger the inflammatory response, as demonstrated in cerebral small vessel disease where there is histopathological evidence of endothelial cell activation. The increased vascular permeability is one of the features of endothelial cell activation, and it is thought that entry of serum proteins, such as coagulation and/or inflammatory mediators, into the vascular wall and perivascular neural parenchyma may sustain toxic effects. Indeed the blood-brain barrier (BBB) microvasculature, plays a crucial role in the regulation of cerebral blood flow (CBF) and may also play a pivotal role in AD pathogenesis by regulating the entry into brain parenchyma of a plethora of circulating molecules and xenobiotics, also triggering inflammation and oxidative stress. Cerebral endothelium could be of clinical relevance to investigate BBB permeability, indicating early endothelial perturbation as a consequence of hypoxia or Abeta deposition, events involved in inflammatory and oxidative cerebrovascular activity. Indeed, it has been previously demonstrated that proinflammatory cytokines alter the expression and processing of Abeta precursor protein, and fibrillar Abeta itself in turn promotes the production of proinflammatory cytokines by microglial and monocytic cell lines. Microglia is the major component of the intrinsic brain immune system and its pivotal role in cerebral inflammation-like responses could trigger and sustain neurodegenerative events. However, clinical observations on the potential role of inflammation in AD have yielded inconsistent results. Whereas several community-based studies have linked antiinflammatory interventions to a lowered risk of developing AD, a randomized, placebo-controlled clinical trial failed to demonstrate a beneficial effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the progression of disease. It is noteworthy that, in the brain, perivascular macrophages and microglia that participate in intraparenchymal inflammation are derived from circulating macrophages. Previous studies have reported higher CSF levels of TNF-alpha than serum levels in AD patients, strengthening the hypothesis of a pivotal role of BBB and microglia activity in the pathogenetic mechanisms of AD. Activated microglia may also be involved in mechanisms of impaired glial glutamate uptake and reduced expression of glutamate transporters, or increased free radicals and nitric oxide synthesis in brain parenchyma. Central nervous system is particularly exposed to free radical injury, given its high metal content, which can catalyze the formation of oxygen free radicals, and the relatively low content of antioxidant defenses. Indeed, several studies show markers of oxidative damage (lipid peroxidation, protein oxidation, DNA oxidation and glycosidation markers) in brain areas affected by neurodegenerative disorders. Our group published several works demonstrating a link between oxidative stress and excitotoxicity in AD, and described peripheral markers of these mechanisms, that may be analyzed in patients as possible diagnostic and therapeutic tools. On the other hand, hypoxia and stroke could influence Abeta processing, as demonstrated by the hypoxia-inducible factor1 alpha (HIF-1alpha) regulation of BACE promoter or increased production of Abeta after stroke, which may increase caspase 3 cleavage of the GGA3 protein carrier resulting in decreased degradation of BACE. Studies of the effect of vascular risk factors on Abeta processing could help to elucidate whether vascular disease has only an additive effect on cognitive performance or it is also intrinsic to the pathogenesis of AD. We have recently analyzed some markers of vascular damage, in particular we have demonstrated that mean plasma levels of TF (Tissue Factor) and TFPI (TF Pathway Inhibitor) are both correlated with Hcy and they are significantly higher in AD and MCI patients than in healthy subjects (Piazza 2007). Moreover, the measurement of immunologically defined "circulating endothelial cells" (CECs) has been used to assess vascular integrity and the amount of microparticles (MP) has been reported elevated in a number of conditions where vascular dysfunction, thrombosis and inflammation are relevant. However, the identification of relevant biological markers for the state of brain microvessels in demented patients is still lacking. Such biomarkers, together with known risk factors common to AD and VD, can be used to better understand the involvement of cerebrovascular implications in the pathophysiology of dementia, with possible therapeutic interventions. In conclusion, it is possible that the initial endothelial damage in AD brains can trigger Abeta deposits which, in turn, may fuel monocytes infiltration through damaged BBB and microglia activation. Abeta deposits and inflammation can lead to the production of superoxide radicals, exacerbating endothelial injury. Moreover, all these processes can support previous findings of the generalized peripheral and CNS oxidative stress that typically defines AD.

Thesis (Masters Diploma (Technology)--Cape Technikon, Cape Town, 1986
Child psychiatrists have become increasingly aware of the existence. of affective disorders in prepubertal and pubertal patients. This has led to the investigation of possible biological factors contributing to the disorders. Due to the lack of availability of human brain material, different parameters have been investigated in the periphery in order to obtain information regarding the aetiology of major depressive disorder. The neurotransmitters, NA, 5-HT and DA have been implicated in depression. Levels of the metabolites of these transmitters have been measured in plasma, urine and CSF of adult depressed patients. Two other peripheral "tools" used in the study of major depressive disorder are blood platelets and lymphocytes. The former contain cr 2 -adrenoceptors and imipramine binding sites (indicative of 5-HT uptake into the platelet) and the latter S-adrenoceptors. Platelets have been widely used as a model for indirectly evaluating changes in central cr2-adrenoceptor and imipramine binding whereas lymphocytes have been used to measure changes in S-adrenoceptor binding and activity in adults with major depressive disorder.

Sickle cell disease (SCD) is remarkable for the variability of its phenotype despite its genetic simplicity, with family and population studies indicating a strong genetic influence. The best characterised genetic modifiers are genes controlling HbF levels and co-inheritance of alpha-thalassaemia. Other genetic and biological factors may also influence disease severity and the development of complications. King’s Health Partners have the largest adult cohort of SCD patients in the United Kingdom, approximately 2400 patients. Characterising this group (using laboratory variables, evidence of clinical complications and admission data) has formed the basis for my studies. Telomere length and Duffy antigen receptor for chemokines (DARC) status and two complications, sickle nephropathy and sickle hepatopathy were investigated. Relative telomere length (measured using qPCR) was significantly longer in patients with SCD than controls and positively correlates with white blood count. Shorter telomeres were found in patients on hydroxycarbamide treatment and those with Hb SC. We hypothesise that longer telomeres result from up-regulation of telomerase due to inflammation. Polymorphisms in DARC have a high prevalence in people of African ancestry and explain benign ethnic neutropenia. We found positive associations with Duffy positive phenotype and a reduced time to readmission and the development of leg ulcers. We investigated if sickle-related renal impairment was associated with the APOL1, DARC and HMOX1 genes. Duffy positive phenotype was associated with the development of macroalbuminuria and the presence of 1 or more APOL1 risk alleles was associated with the development of renal impairment (as measured by MDRD eGFR). We assessed the prevalence of liver disease in our population using Enhanced Liver Fibrosis score (a combination of serum markers associated with liver fibrosis) and transient elastography (Fibroscan®). Using this approach we found that transfusional iron overload and haemolysis appear to play a key role in the pathogenesis of sickle hepatopathy.

Gene expression profiles have identified five major molecular breast cancer subtypes (Luminal A, Luminal B, Basal-like, HER2+/estrogen receptor− , and Normal Breast-like) that show significant differences in survival. The cost and complexity of gene expression technology has impeded its clinical implementation. By comparison, immunohistochemistry is an economical technique applicable to the standard formalin-fixed, paraffin-embedded material commonly used in hospital labs, and has the advantage of simultaneously interpretation with histomorphology. In this thesis, I hypothesize that a surrogate panel of immunohistochemical biomarkers can be developed to discriminate the breast cancer biological subtypes. The main study cohort consists of over 4000 primary invasive breast tumors, assembled into tissue microarrays. These patients were referred to the British Columbia Cancer Agency between 1986-1992 and have staging, pathology, treatment and follow-up information. In summary, our results demonstrate that (1) the rabbit monoclonal antibody, SP1, is an improved standard for immunohistochemiscal estrogen receptor assessment in breast cancer; (2) the transcription factor, GATA-3, is almost exclusively expressed among estrogen receptor positive tumors but does not seem to predict for tamoxifen response among estrogen receptor positive patients; (3) the proliferation marker, Ki-67, together with HER2 can segregate Luminal A from Luminal B subtypes, which carry distinct risks for breast cancer relapse and death; and (4) the inclusion of the basal markers EGFR and ck5/6 to “triple negative” breast cancers provides a more specific definition of basal-like breast cancer that better predicts patient survival. These results consistently demonstrate that an immunopanel of six biomarkers (estrogen receptor, progesterone receptor, HER2, Ki-67, epidermal growth factor receptor and cytokeratin 5/6) can be readily applied to standard pathology specimens to subtype breast cancer samples based on their underlying molecular biology. These findings have been considered sufficient to justify application of this panel onto NCIC (MA5, MA12) and CALGB (9341 and 9741) clinical trials specimens. This followup work which is underway and will determine if the six marker immunopanel can guide decisions about which patients need aggressive systemic drug treatment, and thereby ensure patients get the most effective, individualized adjuvant systemic therapy for their breast tumor.

Benzoquinone-based tripartite prodrug delivery systems have been developed to selectively release an active drug under biological conditions. The make-up of the'tripartita delivery asystem is a trigger moiety (quinone) attached to a linker unit (propanoic acid) This is then attached to the active drug via an ester (or amide) link effectively rendering the drug inactive. The aim of this work was to design, synthesise and evaluate a select ct series of prodrug systems and to ascertain whether varying the substituents on the quinone and linker units would enhance the activation of the prodrug and the release of the active ive species following chemical or enzymatic activation.

Exposure to particulate matter (PM) and its components is associated with negative health outcomes. It is hypothesised that the channel through which these effects are expressed is via the mechanism of inflammation and oxidative stress. In order to assess the relationship between exposure and health outcomes in different personal and indoor exposure environments (Homes, Office, Commuter mode, Restaurant, Shisha lounge, Busy traffic site and Control environments), the association between exposure to PM/PM components and changes in local/systemic inflammation and oxidative stress biomarkers (EBC-nitrate, EBC-nitrite, EBC-pH and urinary 8-oxodG), alterations in autonomic and lung functions respectively were measured. The acute/elevated exposures showed significant increases in EBC nitrate and nitrite immediately after exposure but EBC-pH and urinary 8-oxodG, did not significantly change in post exposure measurements. In some of the exposure scenario, significant correlations were observed between the pollutants measured and the biomarkers. Urinary metals were also analysed and compared with biomarkers and PM/PM components. Indoor PM mass-size distribution, showed that PM prevailed in the 250 – 500 nm and the 2500 – 5000 nm size range. Na, Fe, Mn, Ca and Mg prevailed in the coarse mode (2500 – 5000 nm) and toxic metals such as Mn, Cu, V, Ni, Pb and Sn were observed. Si, S, K and Fe were the most prevalent elements observed in the particles collected analysed with TEM/EDS. The increases observed in post exposure measurements, significant correlations observed between the biomarkers and the measured PM/PM bound components in the different environments and alterations in lung function, suggests that exposure to PM/PM bound components have the ability to cause inflammation/oxidative stress thereby affecting human health. This study shows that biomarker expression after exposure can be used to measure acute responses.

Peach harvester exposure to azinphos-methyl (AZM) residues estimated by the Transfer Factor (TF) ratio of Dislodgeable Foliar Residue (DFR) to Daily Dermal Exposure (DDE) was compared to estimates based on the relationship between dermal exposure and dialkylphosphate metabolite excretion. DFR was monitored in four orchards from the time of pesticide application through harvest. Eleven male harvesters wore cotton tee-shirt dosimeters and provided area-specific skin washes and wipes for DDE monitoring during work operations. Urinary dialkylphosphate metabolites of AZM were also monitored and compared to DDE results. During a later harvest for which DDE was not determined, the dermal exposure estimated by the TF (1,310 μg) was comparable to the estimates based on dialkylphosphate excretion (1,456-1,534 μg). A repeated measures ANOVA showed that harvesters' cholinesterase levels were significantly lower than 'non-harvesters'. No significant reductions in cholinesterase levels were detected over time.

The key to understanding the heterogeneous behaviour of similar stage locally advance rectal cancer lies in the understanding of tumour biology. The aim of this project was to investigate the biological behaviour of rectal cancers and its alterations in response to neoadjuvant chemoradiotherapy, by studying the intrinsic radiosensitivity, pathophysiology and angiogenesis of rectal cancers. It was intended to provide information that may help risk-stratify patients for individualised treatments including optimal timing of surgery after chemoradiotherapy. Consecutive patients with locally advanced, non-metastatic rectal cancer, who were considered suitable for long-course neoadjuvant chemoradiotherapy, were prospectively recruited. Radiosensitivity was studied by investigating the timing of DNA repair analysis with single cell gel electrophoresis (comet assay). The tumour pathophysiology and angiogenesis was investigated in vivo by novel functional imaging techniques (multiparametric magnetic resonance imaging and dynamic contrast enhanced computed tomography). It is demonstrated that rectal cancer tissue consists of cells with heterogeneous radiosensitivities and functional microvascularity. Until six weeks after NCRT, the DNA repair remains inhibited with progressive devascularisation and increasing hypoxic blood volume resulting in loss of tumour cells. Thereafter, variable fractions of cancer cell may continue to perish or survive with corresponding changes in vascularity. Therefore, the period between the sixth and eleventh weeks after neoadjuvant therapy is a critical time when surviving cells from rectal cancers may develop aggressive traits with long-term consequences. Hence, biological assessment of locally advance rectal cancers after six weeks post-NCRT may help risk-stratify patients for individualised therapy.

The cross-sectional Biomarkers Study was undertaken in Canberra, Australia (2000-2002) to examine the role of psychosocial factors in the socioeconomic health gradient, via physiological changes consequent upon activation of the neuroendocrine stress response.¶ The study population was derived from healthy 40-44 year old men and women already participating in a longitudinal cohort study. Using data from the cohort study, four groups with similar occupational status were formed. The study sample was randomly selected within these groups, thus representing the socioeconomic spectrum.¶ A pilot study involved 60 participants with blood and saliva samples measured on two occasions. A further 302 people had blood and saliva samples taken on one occasion. Socioeconomic status was measured by occupational code and status, personal and household income, education and perceived position in the community and in Australia. Psychosocial and behavioural factors, including job strain, job security, coping style, anxiety, depression, optimism, self-esteem, sense of belonging and trust, social support, smoking, exercise and alcohol intake were assessed by selfreport. Five biological parameters: plasma fibrinogen, glycated haemoglobin, waisthip ratio, serum neopterin and salivary IgA were measured as outcome variables.Three hypotheses were tested:¶ 1. There is a socioeconomic gradient in measures of psychosocial stress, and of psychological resilience.¶ 2. There is a socioeconomic gradient in biological measures that have a plausible¶ association with future disease. 3. Psychosocial factors mediate the demonstrated association between socioeconomic status and the biological measures.¶ Data analysis confirmed a socioeconomic gradient in some psychosocial and behavioural variables: economic strain (r=-0.44, p<0.001), job demands (r=0.45, p<0.001), job control (r=0.26, p<0.001), active coping style (r=0.28, p <0.001), sense of optimism (r=0.24, p<0.001), social capital (r=0.26, p<0.001), job security (r=0.17, p=0.002), job marketability (r=-0.16, p=0.005), sense of belonging (r=0.22, p<0.001), number of adverse life events (r=-0.13, p=0.01) and positive interaction with family and friends (r=0.20, p<0.001 ), vigorous physical activity (r=-0.16, p=0.002), alcohol consumption (r=0.30, p<0.001) and smoking status (r=-0.25, p<0.001). There was no socioeconomic gradient in anxiety, depression, neuroticism, hostility, locus of control, self-esteem, perceived stress or mental health (SF-12). Four of the five biological markers varied with socioeconomic status: plasma fibrinogen (female (F): r=-0.26, p=0.002, male (M) r=-0.08, p=0.30), glycated haemoglobin (F: r=-0.23, p=0.01, M: r=-0.11, p=0.17), waist-hip ratio (F: r=-0.19, p=0.03, M: r=-0.27, p<0.001), serum neopterin (F: r=-0.21, p=0.009, M: r=-0.04, p=0.56), salivary IgA (F: r=-0.07, p=0.38, M: r=0.004, p=0.97). A more adverse biological profile was associated with lower socioeconomic status. Work characteristics, coping style, smoking and exercise were particularly important mediators of the association between the biological markers and socioeconomic status. Particular psychosocial factors were consistent mediators of the association between specific biomarkers and socioeconomic status (with little variation for different measures of socioeconomic status). However, the particular psychosocial factors providing significant mediation varied for the different markers.¶ In this sample of healthy 40-44 year olds, four out of five biological markers showed moderate socioeconomic variation with a more favourable profile associated with higher SES. The data provide limited support for the importance of psychosocial factors in the socioeconomic health gradient.

Current interventional pharmaceutical therapies targeted for depression are not adequate to achieve sufficient remission following treatment. Researchers explored inflammatory biomarkers as a way of understanding why treatment for depression is effective for some and not others. The purpose of this secondary data analysis study was to determine if there was a relationship between inflammatory biomarkers and treatment efficacy in persons diagnosed with depression who have demonstrated a previous lack of remission. Using the immune-cytokine paradigm of depression (POD) allowed depression to be viewed as multifaceted and a potential signal of chronic immune system activation. This secondary data analysis included findings from a clinical trial called, 'A Study of the Efficacy and Safety of CP-601,927 Augmentation of Antidepressant Therapy in Major Depression' ANOVA and linear regression were used to analyze 1 dependent variable: depression remission. The 5 independent variables included adiponectin C-Reactive Protein (hs-CRP), leptin, interleukin 1-β (IL1-β), interleukin 6 (IL6), and tumor necrosis factor-α (TNFα). The 3 mediating variables included age, race, and gender. According to the results of the study, IL6 significantly correlated with and predicted remission outcome, as measured by change in MADRS total score from baseline. None of the other biomarkers significantly correlated with remission outcome. Better remission outcomes for patients suffering from depression would lead to positive social change and improved quality of life.

The study of saliva for laboratory analyses is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, attention must be directed to sample collection and storage, to method development and validation and to variability evaluation. The analysis of saliva provides important information about the functioning of various organs within the body. In this respect, endocrine research certainly occupies a central role. In effect, some hormones commonly measured in plasma, such as steroids, non-steroid, peptide and protein hormones, can be detected in the oral fluid. The protein polypeptide hormones are maybe a new analytical approach of the medicine laboratory but, at present time, there are still too few investigations about protein and polypeptide hormone levels in saliva. Detection of steroid hormones is perhaps the most interesting application in salivary hormonal studies. Steroids have often been studied because salivary-free steroid hormones seem to reflect the serum-free levels. Among of these steroids, salivary cortisol measurement is today a widely accepted alternative to the determination in plasma. In the section I of the present dissertation, a new ELISA method is explained. Research was carried out to study and validate an assay to measure salivary free IGF-I (sIGF-I) in human saliva. The detection range, the detection limit, the imprecision, the recovery and the specificity were evaluated. The pre-analytical variation was also studied. After the method validation, sIGF-I levels were measured in sedentary subjects and in athletes (protocol A and protocol C); moreover two different acute physical exercises in two groups of athletes were investigated to assess a possible effect on sIGF-I (protocol B and protocol C). The section II describes an analysis method which used the chromatographic technique. A SPE-HPLC method with UV detection was developed and validated to simultaneously measure cortisol (sF) and cortisone (sE) in human saliva. The analytical performances, in terms of detection range, sensitivity, imprecision, recovery, were evaluated. The pre-analytical variation, with respect to collection strategy and storage conditions, was also examined. After validation, the sF and sE method was applied analyzing specimens collected from athletes, before and after a physical exercise (protocol C). The results suggest further investigation from the laboratory point of view, taking into account the aspects related to the various forms and the specific and unspecific binding proteins (for sIGF-I assay) and to other steroid hormones and related metabolites identified and probably present in human saliva (for sF/sE assay).
Lo studio della saliva nelle analisi di laboratorio è un’area di ricerca in forte crescita, per le sue implicazioni nella ricerca di base ma anche a fini clinici. Sebbene questo fluido biologico sia facile da manipolare e da raccogliere, bisogna porre attenzione ai processi di raccolta e stoccaggio del campione, nonché allo sviluppo e alla validazione di metodi analitici, assieme alla valutazione delle variabilità. L’analisi della saliva dà importanti informazioni sul funzionamento di vari organi del corpo. In relazione a questo, la ricerca endocrina occupa certamente un ruolo centrale. Infatti, alcuni ormoni normalmente misurati nel plasma, come ormoni steroidei, ma anche ormoni non steroidei, peptidici e proteici, possono essere identificati nel fluido orale. Un nuovo approccio analitico nella medicina di laboratorio è forse rappresentato dagli ormoni polipeptidici e proteici ma, tuttora, ci sono ancora troppi pochi studi su questi ormoni salivari. La misura degli ormoni steroidi, invece, rappresenta forse l’applicazione più interessante negli studi degli ormoni salivari. Spesso gli steroidi sono studiati perché la concentrazione salivare riflette i livelli sierici. Tra i vari steroidi, la misura del cortisolo salivare è oggi una alternativa alla sua determinazione plasmatici. Nella I sezione viene spiegato un nuovo metodo ELISA. Sono stati sviluppati test sperimentali per studiare e validare un metodo per la misura dell’IGF-I libero salivare (sIGF-I). Sono stati studiati il range di misura, la sensibilità, l’imprecisione, il recupero e la specificità. Inoltre è stata studiata anche la variabilità pre-analitica. Dopo la validazione del metodo, sono stati misurati i livelli di sIGF-I in soggetti sedentari ed in atleti (protocollo A e protocollo C); inoltre è stato studiato il possibile effetto di due differenti esercizi fisici (in acuto) sulle concentrazioni di sIGF-I (protocollo B e protocollo C). La II sezione prende in esame un metodo di analisi che usa la tecnica cromatografia. E’ stato sviluppato e valicato un metodo SPE-HPLC con rivelazione UV per la misura contemporanea del cortisolo (sF) e del cortisone (sE) nella saliva umana. Sono state calcolate le performance analitiche (range di misura, sensibilità, imprecisione, recupero). E’ stata considerata anche la variabilità pre-analitica con particolare attenzione alle condizioni di raccolta e conservazione del campione. Dopo la validazione, questo metodo è stato applicato a campioni raccolti da un gruppo di atleti, prima e dopo un esercizio fisico (protocollo C). I risultati ottenuti suggeriscono ulteriori approfondimenti soprattutto da un punto di vista laboratoristico, tenendo presente la possibile presenza di varie forme e di specifiche ed aspecifiche proteine di legame (per sIGF-I) e altri ormoni steroidei e loro metaboliti identificati e probabilmente presenti nella saliva umana (per sF/sE).

This thesis aimed to examine the impact of environmental tobacco smoke on biological markers of ageing in pet dogs. In order to achieve this, community-based dogs and owners were recruited, approximately half of whom lived in smoking homes and half non-smoking homes. Owners were asked to attend 2 appointments, 12 months apart. Questionnaire assessments of dog environmental tobacco smoke exposure were compared to biomarkers in hair of the dogs (Chapter 3). This gave an objective measure of exposure and hair nicotine concentrations reliably reflected information provided by owners. A qPCR technique was optimised to measure telomere lengths (Chapter 4). This was utilised to measure telomere lengths in leukocytes, buccal cells, cremaster muscle, vas deferens and epididymis samples from the study dogs. Owners were offered free-of-charge neutering for their pet, and the spare tissues used for these analyses. A negative relationship in leukocyte telomere length with hair nicotine was observed, among other changes (Chapter 5). mRNA levels of further biomarkers of ageing were measured in testes, as well as leukocyte global DNA methylation percentage (Chapter 6). Again, several significant relationships were found between tobacco smoke exposure markers and the biomarkers of ageing, including a significant negative relationship with CDKN2A expression with increased tobacco smoke exposure. Plasma testosterone and hair cortisol concentrations were measured. In addition, factors which related to weight gain after neutering were examined (Chapter 7). Increased cotinine concentrations in fur were significantly related to increased percentage weight gain. Several avenues for future research were generated, and many areas warrant further investigation.

The immune system has been suggested as crucial in brain and psychological functioning. More precisely, immune markers reflecting immune system activity are important for psychological and mental health, as evident by their role in the physiopathology of depression and in the impairment of executive functions. Frontal alpha asymmetry (FAA), an electroencephalographic marker of brain function, has also been linked to such psychopathology and is thought to reflect psychological processes underlying approach- versus withdrawal-related motivation and higher-order inhibitory control. Only a few studies have linked FAA to immune markers but notably found a negative association between IL-6, a pleiotropic pro-inflammatory cytokine, and FAA. The aim of the present work is thus to study the relationship between various immune markers (including pro-inflammatory cytokines and IL-6) and FAA. 35 healthy young male participants underwent a resting EEG recording and blood sampling from which immune markers were measured. The results did not suggest an association between IL-6 and FAA. No other immune markers were either suggested to be associated to FAA. The complexity of the immune system (e.g., effect of cytokines) is underlined and may explain the results. Despite such results, the implication of true negative correlations between FAA and circulating immune markers, as suggested in previous studies, is discussed in the light of the theoretical models of FAA.

Thesis (M.S.)--West Virginia University, 1999.
Title from document title page. Document formatted into pages; contains xi, 77 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

The work presented in the present thesis investigated the neural, behavioural and genetic markers that may be associated with the manifestation of behavioural problems during the early years of life. Across four different empirical studies, and by incorporating, behavioural, neurophysiological and genetic investigations, it was demonstrated that: (1) there are neurophysiological signatures that may be associated with the manifestation of behavioural problems early in life; (2) common genetic variations that determine serotonin variability are strongly associated with affectivity-related patterns of frontal brain activation; and that (3) normal genetic variations that modulate serotonin availability and neuroplasticity are each associated with affectivity-related patterns of visual scanning behaviours in response to faces and aversive scenes. Taken together, the results illustrate the existence of robust neural, genetic and behavioural markers that may be associated with the manifestation of behavioural problems in early childhood and prompt further investigation of the area by generating novel hypotheses. Together, the empirical findings of the thesis provide a first stage contribution to the complex mechanisms that may yield risk and resilience for behavioural problems during the early years of life by generating a more comprehensive insight on the field of affectivity.

The aim of this thesis is to examine the hypothesis that breast cancer exhibits ethnic differences in incidence, age of presentation and aggressiveness. Two approaches were used:;Firstly, International data was examined to find if differences in the age of presentation of breast cancer existed between low and high breast cancer incidence populations. Secondly, selected pathobiological parameters and genetic markers, chosen to act as surrogate markers of tumour behaviour, were examined in breast cancers from Western Region of Saudi Arabia, Leicester Asians and Leicester Europeans. Analysis of International data showed countries with the lowest incidence of breast cancer to have a significantly lower mean age of onset than countries with the highest incidence. The mean age of onset in the Western Region was less than Leicester Asians and Leicester Europeans. Statistically significant differences in ASIR were found between the study groups that varied with age. Breast cancers from Western Region and Leicester Asian women, when compared to the Leicester Europeans, were found to have a more aggressive profile of clinicopathological markers, but a less variable and possibly less aggressive nuclear morphometry. Molecular alterations at the markers studied appeared to be related to age, 16q exhibiting more Loss of Heterozygosity in older groups and p53 in the younger groups. 6q also showed differences in Loss of Heterozygosity with respect of age, but these differences varied between the populations studied. The results are consistent with a "two disease" model of breast cancer. It is proposed that the low age of onset seen in the Western Region is primarily due to a low incidence of "older type" of breast cancer rather than an absolute excess of "younger type".

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound.
AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme, ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees belowende middel, asook die geskikte formulasie vir die spesifieke roete van toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy word. Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek. Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort, finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker. In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe, insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en klein intestinale mukosa. Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer. Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste vlak verminder. Die verwydering van die vaginale epiteel het die diffusie vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK) van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met ‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes ondersteun dus die alombekende diffusie teorie. Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel. Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219 μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die drie kommersiële formulasies blootgestel was. Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC. Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n 24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine, propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes. Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en 1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van hierdie studie stem ooreen met ander in vivo studies. Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal het om sommige van die probleme en tekortkominge van ander in vitro modelle te oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde geneesmiddel/chemiese stof bevat, te bepaal.

This dissertation research is a bioarchaeological investigation of Late Neolithic through Early Bronze Age (3600-1800 BC) burial populations from the Portuguese Estremadura. In this project macroscopic and isotopic analyses of skeletal and dental materials are used to gather information pertaining to diet, health status, and inter-lifetime mobility patterns for individuals interred at different burials within a small geographic area with the goal of evaluating the level of social differentiation in the region. The archaeological record for the transition between the Late Neolithic and the Early Bronze Age in southwestern Portugal demonstrates clear evidence of the rise of a socially-complex, non-state society. During the Early Bronze Age, however, this region underwent a period of social `devolution' which cumulated in widespread settlement abandonment. To date, it is unclear to what extent sociopolitical or environmental factors contributed to this social collapse. This study seeks to expand our knowledge of social differentiation in the Late Neolithic to Early Bronze Age of the Estremadura region of Portugal and provide insight into social structure during the emergence and collapse of early complex societies in Iberia. The results of this study found that there were statistically significant differences in dietary, mobility and demographic patterns between burials that suggest socially distinct populations were interred at different sites. In particular, one burial site, Cova da Moura, diverged significantly from the other sampled burial populations. However, based upon the data presented here, it was not possible to tie these biological markers of differentiation to particular aspects of Late Neolithic to Early Bronze Age social organization. Therefore, while this study successfully identified differences between burial populations, at this time, it is not possibly to relate these to particular hierarchical structures. It is suggested that aspects of burial practices in the region confound biologically-based investigations of social organization in a similar way that they have impeded researchers' abilities to identify elite versus non-elite individuals through grave goods alone. Nonetheless, despite these obstacles, this work provides strong evidence of population heterogeneity in the region, and has implications for our understanding of the evolution of complex societies in the Iberian Peninsula and elsewhere.

Mother-to-child transmission of HIV is the leading cause of pediatric AIDS; however, mechanisms by which HIV compromises pregnancy are not understood. CD4+CD25+ T-regulatory (Treg) cells play a role in pregnancy maintenance. RNA from early and late gestation placentas and fetuses from FIV-infected and control cats were probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. High rates of vertical transmission and reproductive failure were detected in early and late pregnancy. In control animals, both FOXP3 and CTLA4 expression decreased with gestational stage, indicating a natural decline in Tregs. Expression of FOXP3 and CTLA4 was decreased at early gestation in FIV-infected queens and a trend toward decreased expression of CD25, FOXP3, and CTLA4 in placentas from FIV-infected queens producing non-viable pregnancies was observed as well. Our results suggest that FIV infection may alter placental Treg function and adversely affect pregnancy outcome.