Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. PLC activation causes hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and generates two second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. Each has distinct role in depending on the cell type in mammalian cells, IP3 binds to intracellular receptors, stimulating the release of sequestered Ca2+. DAG remains in the membrane, where it can activate members of the protein kinase C (PKC) family. In plant absence of PKC keeps the question open as to what is the role of DAG in plants. The role of IP3 apart form triggering calcium release is not known, although the phosphorylated product of IP3 by groups of kinases has been implicated in certain nuclear signalling pathway. Using various sequence-analysis methods on plant PLC sequences, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. we have used Saccharomyces cerevisiae as a model system to investigate physiological function of PLC in regulation of lipid metabolism. S. cerevisiae synthesizes membrane phospholipids via a pathway which appears to be similar to that of higher eukaryotes. The synthesis of glycerolipid begins with the formation of phosphatidic acid which is quantitatively a minor lipid but is responsible for the repression of UNAINO-containing phospholipid biosynthetic gene by governing localization of Opi1. When the levels of phosphatidic acid are lowered which causes translocation of Opi1 from endoplasmic reticulum membrane to nucleus, where it binds to INO2 of the INO2-INO4 activator complex thereby attenuating transcriptional activation. The expression of phospholipid biosynthetic gene is affected by many conditions which include carbon source, nutrient availability, growth stage, pH and temperature. The well studied conditions which regulate phospholipid biosynthetic genes transcription are through exogenous supplementation of inositol, which is achieved by lowering of phosphatidic acid levels by its utilization for the synthesis of phosphatidylinositol. Since inositol was able to change regulates phospholipid biosynthetic gene we proposed to investigate inositol triphosphate role in such regulation. We overexpressed a plant phospholipase C in yeast to study its effect on lipid biosynthesis. The overexpressed yeast cells were subjected to microarray analysis and the result were confirmed by Q-PCR. The result obtained indicated that there was decrease in the expression of UNAINO-containing genes. To further validate our observation we carried out an in vivo assay to determined activity of enzyme involved in phospholipid biosynthesis. These results were in accordance with our expression analysis further supporting our hypothesis. Our study indicates that phospholipase c regulates phospholipid biosynthesis at transcription level in response to various stimuli. Overall, these data suggest that the C2 domain of plant PLC plays a vital role in calcium signalling. Further it can be inferred from this study that PI-PLC regulates lipid metabolism in S. cerevisiae.

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

La membrane cellulaire est principalement une bicouche phospholipidique constituant une barrière qui régule les échanges entre la cellule et son environnement. Son intérieur hydrophobe empêche le passage d’espèces hydrophiles, chargées, de grande masse moléculaire et polaires, qui sont généralement transportées par des protéines à travers la bicouche. Dans certains cas de systèmes défectueux (e.g. les canalopathies), l’équilibre des concentrations en ions à l’intérieur et à l’extérieur des cellules est perturbé et les cellules sont compromises. C’est pourquoi le développement de transporteurs transmembranaires synthétiques est nécessaire. De nombreux travaux ont été faits dans le développement de transporteurs synthétiques d’anions (particulièrement du chlorure). Dans cette thèse, nous présentons nos travaux sur un nouveau transporteur d’anion appelé axe parapluie, capable de changer de conformation dépendamment de la polarité de son environnement. Dans un premier temps, nous avons conçu le design, puis synthétisé ces axes parapluie qui montrent une importante activité en tant que transporteur de chlorures. Ces composés réunissent deux concepts : - Le parapluie, constitué d’acides biliaires amphiphiles (une face hydrophile, une face hydrophobe). La flexibilité des articulations combinée à la grande surface des acides choliques permettent d’empêcher les interactions défavorables entre les parties hydrophiles et hydrophobes, ce qui facilite l’insertion dans la bicouche. - Un site ammonium secondaire en tant que site de reconnaissance, capable de former des ponts hydrogène avec des ions chlorure. De plus, l’axe peut complexer une roue de type éther couronne pour former un pseudo-rotaxane ou rotaxane parapluie ce qui résulte en l’inhibition partielle de leurs propriétés de transport. Ceci nous mène au second objectif de cette thèse, le développement d’un nouveau moyen de transport pour les médicaments cycliques. Certains macrocycles polaires et biologiquement actifs tels que les nactines ont besoin d’atteindre leur objectif à l’intérieur de la cellule pour jouer leur rôle. La membrane cellulaire est alors un obstacle. Nous avons imaginé tirer profit de notre axe parapluie pour transporter un médicament cyclique (en tant que roue d’un rotaxane parapluie). Les assemblages des rotaxanes parapluie furent accomplis par la méthode de clipage. Le comportement de l’axe et du rotaxane parapluie fut étudié par RMN et fluorimétrie. Le mouvement du parapluie passant d’une conformation fermée à exposée dépendamment du milieu fut observé pour le rotaxane parapluie. Il en fut de même pour les interactions entre le rotaxane parapluie et des vésicules constituées de phospholipides. Finalement, la capacité du rotaxane parapluie à franchir la bicouche lipidique pour transporter la roue à l’intérieur de la vésicule fut démontrée à l’aide de liposomes contenant de la α-chymotrypsine. Cette dernière pu cliver certains liens amide de l’axe parapluie afin de relarguer la roue. / The cell membrane is a phospholipid bilayer barrier that controls the exchanges between the cell and its environment. Its hydrophobic core prevents the entrance of hydrophilic, charged or large polar species that are transported through the bilayer by proteins. In some dysfunctional systems e.g. channelopathies), the balance of ion concentrations between the interior and exterior of the cell is no longer insured and the cell’s health is compromised. That is why the synthesis of synthetic transmembrane transporters is needed. There have been many synthetic anion carriers (especially chloride) developed in this area using different strategies. In this thesis we present our work on a new anion transporter, an umbrella thread. First, we designed and synthesized umbrella threads that showed significant chloride transport activity. These compounds combine two concepts: - the umbrella moiety, made from facial amphiphilic bile acids. The flexibility and large surface of the cholic acids can shield disfavored interactions between hydrophilic and hydrophobic elements that should ease their insertion into the bilayer. - a secondary ammonium recognition site on the thread that can form hydrogen bonds with chloride ions. Furthermore, the thread moiety is able to complex a crown-ether like wheel to form an umbrella pseudo-rotaxane or rotaxane that showed partially inhibited properties for chloride transport. This leads us to the second goal of this thesis, i.e. the development of a new vehicle for drug delivery. Some biologically active polar macrocycles (e.g. nactins) need to reach their target inside the cell to be efficient. The cell membrane also represents an obstacle here. In our work, we imagined using an umbrella thread as the vehicle for the cyclic drug as the wheel of the umbrella rotaxane). The umbrella rotaxanes were successfully assembled by the clipping method. The behavior of both the umbrella thread and umbrella rotaxane was extensively studied by NMR and fluorimetry. The umbrella motion from a shield conformation to an exposed one depending on the environment was observed for the rotaxane. Interactions between the umbrella rotaxane and phospholipid vesicles were also noticed. Finally, its ability to cross the lipid bilayer to deliver the wheel inside the vesicle was shown with α-chymotrypsin-filled liposome assays. This enzyme was able to cleave amide bonds on the umbrella thread to release the wheel.

Abstract ACCURATE METHODOLOGY FOR MONITORING BIOMEMBRANE EVENTS By Christine A. Winschel, Ph.D. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Dr. Vladimir A. Sidorov ASSOCIATE PROFESSOR, DEPARTMENT OF CHEMISTRY This study describes the synthesis and characterization of a new receptor (cyclen 1) capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive pyranine dye, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench pyranine’s fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of ion transport and nonselective leakage mechanisms within a single set of experiments. In the second part of this study a new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of a newly synthesized cascade-blue-labeled lipid through complex formation with cyclen 1. This receptor-dye complexation also has high affinity for binding at micromolar concentrations and can be reversed by either competitive displacement of the lipid probe or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. This new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments, and is less invasive to the model and cell membranes than the commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Lastly, new pH-sensitive lipids were synthesized and utilized in the formulation of liposomes suitable for controlled drug release. These liposomes contain various amounts of internal NaCl and undergo internal acidification upon the exogenous addition of an HCl co-transporter in a physiologically relevant NaCl solution. Therefore, acidification ultimately leads to the hydrolysis of the pH-sensitive lipids and subsequent contents release. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.

This work expands the knowledge on phospholipid metabolism in the kinetoplastid parasites: T. brucei, T. cruzi, Leishmania spp. that cause neglected tropical diseases and the related non-human pathogenic Crithidia fasiculata. As a close relative of parasitic kinetoplasts, specifically Leishmania, it is hypothesised that Crithidia fasiculata possesses a similar lipid biosynthetic capability and therefore represent an attractive model organism. Database mining the Crithidia genome revealed the ability to biosynthesise all of the main phospholipid species. Utilising various lipidomic techniques, a high level of an ω-6 18:3 fatty acid was observed, alongside an uncommon Δ19:0 fatty acid that was later identified to be exclusive attributed to PE species. Sphingolipid metabolism was shown to resemble that of Leishmania and T. cruzi, given the exclusive production of inositol-phosphoceramide species and no sphingomyelin species being observed. Using labelled precursors, Crithidia were seen to uptake and incorporate extracellular inositol into both phosphatidylinositol and inositol-phosphoceramide species. Crithidia were also shown to utilise both the Kennedy pathway and methylation of phosphatidylethanolamine to form phosphatidylcholine. The phospholipidome of T. cruzi revealed several phosphatidylserine species for the first time, suggesting a functional phosphatidylserine synthase. Current knowledge of T.cruzi sphingolipid biosynthesis was also confirmed as only inositol xxxi phosphoceramide species were observed. The identification and subsequent characterisation of novel phosphonolipid species are reported for the first time. Utilising lipidomic methodologies and labelled precursors, the relative contribution of the intracellular inositol pools within bloodstream and procyclic T. brucei towards PI biosynthesis was examined. This highlighted that the synthesis/turnover rates for specific phosphatidylinositol and inositol-phosphoceramide species are unequal. Efforts to optimise media conditions highlighted that under reduced levels of serum/glucose/inositol, bloodstream T. brucei unexpectedly adjusts its inositol metabolism. The procyclic parasite exemplifies this fact, as under inositol/glucose deficient media conditions they appear to have adapted to utilising glucogenesis and inositol de-novo synthesis. This work highlights that these parasites are rapidly dividing, their unique features of lipid metabolism may be exploitable for drug discovery purposes.

Objetivos: Muitas questões permanecem sobre as causas da aterosclerose acelerada nos pacientes com doenças inflamatórias sistêmicas como a artrite reumatóide (AR). Estudos na população geral sugeriram que além da inflamação existe uma participação patogênica da auto-imunidade na aterosclerose e discutem a possível associação dos anticorpos contra fosfolípides e proteínas de choque térmico (Hsp). O objetivo deste estudo foi investigar a presença de anticorpos contra fosfolípides, beta2-glicoproteína 1 (beta2-gp1), lipoproteína lipase (LPL) e Hsp em pacientes com AR e avaliar a associação entre estes anticorpos com a presença de aterosclerose subclínica de carótidas. Métodos: Anticorpos contra cardiolipina (aCL) IgG e IgM, beta2-gp1 IgG, IgM e IgA , Hsp 60 e Hsp 65 foram testados por ELISA em um grupo de 71 pacientes com AR comparado com 53 indívíduos controles não portadores de AR, de idade e sexo similar. Foram excluídos os pacientes com HAS, diabetes melitos e os fumantes em ambos os grupos. Níveis de lipoproteínas, parâmetros clínicos da AR, questionário de avaliação de saúde (HAQ), escore de atividade da doença (DAS) 28, velocidade de hemossedimentação (VHS) e proteína C reativa (PCR) foram avaliadas. A associação entre a presença dos anticorpos aCL, beta2-gp1, Hsp 60 e Hsp 65 com os parâmetros clínicos de atividade da doença, com a presença das placas de aterosclerose e com a medida da espessura íntimomedial (IMT) da carótida comum, usando ultra-som (US) modo B de alta resolução foram pesquisadas. Resultados: A idade média no grupo com AR foi 48,93 ± 12,31 vs. 45,37 ± 9,37 no grupo controle saudável (p = 0,20); 90,1% no grupo com AR eram do sexo feminino vs. 86,8% no grupo controle (p = 0,56); índice de massa corporal (IMC) foi 25,72 ± 4,57kg/m² no grupo com AR vs. 26,40 ± 4,52kg/m² no grupo controle (p = 0, 69); Os níveis de colesterol, LDL, triglicerídeos e a relação CT/HDL não foram diferentes quando comparamos os 2 grupos (p > 0,05). O nível de HDL foi maior no grupo com AR vs. grupo controle com 60,56 ± 14,40mg/dl e 54,52 ± 11,55 respectivamente (p = 0,05). A média da medida da IMT foi 0,721 ± 0,16 mm na AR e 0,667 ± 0,14mm no grupo controle, e a IMT dos pacientes com AR foi maior naqueles com idade acima dos 50 anos (P < 0,001). No grupo com AR, 14,1% dos pacientes tinham placas nas carótidas vs. 1,9% dos indivíduos saudáveis (p = 0,02) e no grupo com AR, as placas foram mais frequentes nos pacientes acima dos 50 anos (p = 0,004). No grupo AR, 5,6% tinham anticorpos aCL IgG vs. 3,8% no grupo controle (p > 0,05); 14,1% apresentavam aCL IgM vs. 7,5% (p > 0,05); 43,7% tinham anti-beta2-gp1 IgA vs. 40,8% no grupo controle (p > 0,05). A prevalência de anti-beta2-gp1 IgG e IgM e anti-LPL não foi diferente entre os pacientes com AR e o grupo controle ( p > 0,05). A presença dos anticorpos anti-Hsp 60 e 65 na AR e no grupo controle não foram diferentes (p > 0,05), mas os títulos de anticorpos contra Hsp 65 e beta2-gp1 IgM foram maiores no grupo com AR ( p = 0,007 e p = 0,03 respectivamente). Nós não encontramos associação entre a presença e os títulos dos anticorpos aCL IgG e IgM, beta2-gp1 IgG, IgM e IgA, LPL e Hsp 60 e 65 com a presença de placas nas carótidas ou com a medida da IMT (p > 0,05). Discussão: Este estudo confirma achados anteriores da maior prevalência de aterosclerose carotídea nos pacientes com AR e sua correlação com idade, colesterol e LDL. Embora tenha se encontrado uma tendência a maior presença de anticorpos nos pacientes com AR, não houve relação entre a presença da aterosclerose mais prevalente nos pacientes com AR, com a auto-imunidade dirigida contra cardiolipina, beta2-gp1 ou Hsp. / Purpose: Many questions remain unanswered about the causes of accelerated atherosclerosis in patients with inflammatory systemic diseases such as rheumatoid arthritis (RA). Some studies have suggested the role of autoimmunity besides inflammation in the pathogenesis of atherosclerosis in general population and have also discussed the possible association with antibodies directed to phospholipids and heat shock proteins (Hsp). The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase (LPL) and Hsp in RA subjects and evaluate the association between these antibodies with the presence of subclinical carotid atherosclerosis. Methods: Tests to antibodies against cardiolipin (aCL) IgG and IgM, beta2-gp1 IgG, IgM and IgA ,Hsp 60 and Hsp 65 were done by ELISA test in a group of 71 RA subjects compared with 53 age and sex-matched non-RA subjects. Smoking, diabetic and hypertensive patients were excluded in both groups. The lipoprotein levels, clinical parameters of RA, Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) 28, Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were evaluated. The association between the presence of antibodies against cardiolipin, beta2-gp1 and Hsp 60 and 65 with the clinical parameters of disease activity in RA, and with the presence of plaques and mean intimo-medial thickness (IMT) of common carotid using high-resolution B-mode ultrasound were assessed. Results: Mean age in RA group was 48.93 ± 12.31 vs. 45.37 ± 9.37 in healthy control group (p = 0.20); 90.1% were women in RA group vs. 86.8% in healthy control (p = 0.56); body mass index (BMI) were 25.72 ± 4.57 in RA group vs. 26.40 ± 4.52 in healthy control (p = 0.69). The levels of cholesterol, LDL, triglycerides, CT/HDL didn t have difference between the two groups (p > 0.05). The HDL was higher in RA group vs. control group with 60.56 ± 14.40mg/dl and 54.52 ± 11.55 respectively (p = 0.05). The mean IMT was 0.721 ± 0.16mm in RA and 0.667 ± 0.14mm in control group, and the IMT was higher in patients older than 50 years among RA subjects (p < 0.001). In RA subjects, 14.1% had carotid plaques vs. 1.9% in healthy controls (p = 0.02). In RA group, the carotid plaques were more frequent in patients older than 50 years (p = 0.004). In RA group, 5.6% had antibodies against cardiolipin IgG vs. 3.8% in control group (p > 0.05); 14.1% in RA group had anti-cardiolipin IgM vs. 7.5% (p > 0.05); 43.7% had anti-beta2-gp1 IgA vs 40.8% in control group (p > 0.05). The presence of anti-beta2-gp1 IgG and IgM, and anti-LPL didn t have significant difference between the groups (p > 0.05). The prevalence of antibodies to Hsp 60 and Hsp 65 were similar in RA and in control group (p > 0.05), but the titers of antibodies against Hsp 65 and beta2-gp1 IgM were higher in RA group (p = 0.007 and p = 0.03 respectively). We didn t find relationship between antibodies against cardiolipin IgG and IgM, or beta2-gp1 IgG, IgM and IgA, LPL, Hsp 60 and 65 with mean IMT or plaque carotid (p > 0.05). Discussion: This study confirms the great prevalence of carotid atherosclerosis in RA subjects and its correlation with age, cholesterol and LDL. Although it was found a tendency to have more autoantibodies in RA subjects, there weren t any link between atherosclerosis in RA with autoimmunity against cardiolipin, beta2-gp 1, LPL or Hsp.

Objetivos: Muitas questões permanecem sobre as causas da aterosclerose acelerada nos pacientes com doenças inflamatórias sistêmicas como a artrite reumatóide (AR). Estudos na população geral sugeriram que além da inflamação existe uma participação patogênica da auto-imunidade na aterosclerose e discutem a possível associação dos anticorpos contra fosfolípides e proteínas de choque térmico (Hsp). O objetivo deste estudo foi investigar a presença de anticorpos contra fosfolípides, beta2-glicoproteína 1 (beta2-gp1), lipoproteína lipase (LPL) e Hsp em pacientes com AR e avaliar a associação entre estes anticorpos com a presença de aterosclerose subclínica de carótidas. Métodos: Anticorpos contra cardiolipina (aCL) IgG e IgM, beta2-gp1 IgG, IgM e IgA , Hsp 60 e Hsp 65 foram testados por ELISA em um grupo de 71 pacientes com AR comparado com 53 indívíduos controles não portadores de AR, de idade e sexo similar. Foram excluídos os pacientes com HAS, diabetes melitos e os fumantes em ambos os grupos. Níveis de lipoproteínas, parâmetros clínicos da AR, questionário de avaliação de saúde (HAQ), escore de atividade da doença (DAS) 28, velocidade de hemossedimentação (VHS) e proteína C reativa (PCR) foram avaliadas. A associação entre a presença dos anticorpos aCL, beta2-gp1, Hsp 60 e Hsp 65 com os parâmetros clínicos de atividade da doença, com a presença das placas de aterosclerose e com a medida da espessura íntimomedial (IMT) da carótida comum, usando ultra-som (US) modo B de alta resolução foram pesquisadas. Resultados: A idade média no grupo com AR foi 48,93 ± 12,31 vs. 45,37 ± 9,37 no grupo controle saudável (p = 0,20); 90,1% no grupo com AR eram do sexo feminino vs. 86,8% no grupo controle (p = 0,56); índice de massa corporal (IMC) foi 25,72 ± 4,57kg/m² no grupo com AR vs. 26,40 ± 4,52kg/m² no grupo controle (p = 0, 69); Os níveis de colesterol, LDL, triglicerídeos e a relação CT/HDL não foram diferentes quando comparamos os 2 grupos (p > 0,05). O nível de HDL foi maior no grupo com AR vs. grupo controle com 60,56 ± 14,40mg/dl e 54,52 ± 11,55 respectivamente (p = 0,05). A média da medida da IMT foi 0,721 ± 0,16 mm na AR e 0,667 ± 0,14mm no grupo controle, e a IMT dos pacientes com AR foi maior naqueles com idade acima dos 50 anos (P < 0,001). No grupo com AR, 14,1% dos pacientes tinham placas nas carótidas vs. 1,9% dos indivíduos saudáveis (p = 0,02) e no grupo com AR, as placas foram mais frequentes nos pacientes acima dos 50 anos (p = 0,004). No grupo AR, 5,6% tinham anticorpos aCL IgG vs. 3,8% no grupo controle (p > 0,05); 14,1% apresentavam aCL IgM vs. 7,5% (p > 0,05); 43,7% tinham anti-beta2-gp1 IgA vs. 40,8% no grupo controle (p > 0,05). A prevalência de anti-beta2-gp1 IgG e IgM e anti-LPL não foi diferente entre os pacientes com AR e o grupo controle ( p > 0,05). A presença dos anticorpos anti-Hsp 60 e 65 na AR e no grupo controle não foram diferentes (p > 0,05), mas os títulos de anticorpos contra Hsp 65 e beta2-gp1 IgM foram maiores no grupo com AR ( p = 0,007 e p = 0,03 respectivamente). Nós não encontramos associação entre a presença e os títulos dos anticorpos aCL IgG e IgM, beta2-gp1 IgG, IgM e IgA, LPL e Hsp 60 e 65 com a presença de placas nas carótidas ou com a medida da IMT (p > 0,05). Discussão: Este estudo confirma achados anteriores da maior prevalência de aterosclerose carotídea nos pacientes com AR e sua correlação com idade, colesterol e LDL. Embora tenha se encontrado uma tendência a maior presença de anticorpos nos pacientes com AR, não houve relação entre a presença da aterosclerose mais prevalente nos pacientes com AR, com a auto-imunidade dirigida contra cardiolipina, beta2-gp1 ou Hsp. / Purpose: Many questions remain unanswered about the causes of accelerated atherosclerosis in patients with inflammatory systemic diseases such as rheumatoid arthritis (RA). Some studies have suggested the role of autoimmunity besides inflammation in the pathogenesis of atherosclerosis in general population and have also discussed the possible association with antibodies directed to phospholipids and heat shock proteins (Hsp). The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase (LPL) and Hsp in RA subjects and evaluate the association between these antibodies with the presence of subclinical carotid atherosclerosis. Methods: Tests to antibodies against cardiolipin (aCL) IgG and IgM, beta2-gp1 IgG, IgM and IgA ,Hsp 60 and Hsp 65 were done by ELISA test in a group of 71 RA subjects compared with 53 age and sex-matched non-RA subjects. Smoking, diabetic and hypertensive patients were excluded in both groups. The lipoprotein levels, clinical parameters of RA, Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) 28, Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were evaluated. The association between the presence of antibodies against cardiolipin, beta2-gp1 and Hsp 60 and 65 with the clinical parameters of disease activity in RA, and with the presence of plaques and mean intimo-medial thickness (IMT) of common carotid using high-resolution B-mode ultrasound were assessed. Results: Mean age in RA group was 48.93 ± 12.31 vs. 45.37 ± 9.37 in healthy control group (p = 0.20); 90.1% were women in RA group vs. 86.8% in healthy control (p = 0.56); body mass index (BMI) were 25.72 ± 4.57 in RA group vs. 26.40 ± 4.52 in healthy control (p = 0.69). The levels of cholesterol, LDL, triglycerides, CT/HDL didn t have difference between the two groups (p > 0.05). The HDL was higher in RA group vs. control group with 60.56 ± 14.40mg/dl and 54.52 ± 11.55 respectively (p = 0.05). The mean IMT was 0.721 ± 0.16mm in RA and 0.667 ± 0.14mm in control group, and the IMT was higher in patients older than 50 years among RA subjects (p < 0.001). In RA subjects, 14.1% had carotid plaques vs. 1.9% in healthy controls (p = 0.02). In RA group, the carotid plaques were more frequent in patients older than 50 years (p = 0.004). In RA group, 5.6% had antibodies against cardiolipin IgG vs. 3.8% in control group (p > 0.05); 14.1% in RA group had anti-cardiolipin IgM vs. 7.5% (p > 0.05); 43.7% had anti-beta2-gp1 IgA vs 40.8% in control group (p > 0.05). The presence of anti-beta2-gp1 IgG and IgM, and anti-LPL didn t have significant difference between the groups (p > 0.05). The prevalence of antibodies to Hsp 60 and Hsp 65 were similar in RA and in control group (p > 0.05), but the titers of antibodies against Hsp 65 and beta2-gp1 IgM were higher in RA group (p = 0.007 and p = 0.03 respectively). We didn t find relationship between antibodies against cardiolipin IgG and IgM, or beta2-gp1 IgG, IgM and IgA, LPL, Hsp 60 and 65 with mean IMT or plaque carotid (p > 0.05). Discussion: This study confirms the great prevalence of carotid atherosclerosis in RA subjects and its correlation with age, cholesterol and LDL. Although it was found a tendency to have more autoantibodies in RA subjects, there weren t any link between atherosclerosis in RA with autoimmunity against cardiolipin, beta2-gp 1, LPL or Hsp.

Recent declines in returns from primary forest products in New Zealand and projected increases in world food prices have led to the land-use conversion from plantation forest to pastoral farming in many lowland areas. After decades of forest cover the soils are in many cases less than adequate for pastoral farming, as they are acidic, with toxic levels of exchangeable aluminum, and contain low levels of available nitrogen (N), very high carbon (C):N ratio, and are devoid of earthworms and structural integrity. Overcoming the major site limitations of low soil pH and available N was a major priority and a field experiment was established in April 2005 to determine the impact of various rates of lime and N in relation to pasture establishment and production. Concerns about the short and long-term effects of these inputs on biological soil quality gave rise to the present study. The effects of land-use change and establishment inputs were assessed by comparison of selected treatment plots with two adjacent reference sites (long-term pasture and a 60–year Pinus radiata forest) on the same soil type. The effects of lime and N on soil biological quality were investigated under field and controlled environment conditions by determination of: microbial community structure (phospholipid fatty acids - PLFA), microbial biomass (total PLFA), and microbial activity (dehydrogenase activity). Soil physical (percentage water-stable aggregates) and chemical (pH, and total C and N) properties were also determined. Similarly, the effects of earthworm addition on soil biological properties were explored in a short-term glasshouse pot experiment. The role of earthworms as indicators of soil biological quality in the field was assumed by nematodes and these were assessed in field trial plots and the reference sites mentioned above. Land-use change and applications of lime and N contributed to changing the microbial community structure determined by principal component analysis of transformed PLFA data. However, the effect of lime was more pronounced in the field, while N contributed most to changing microbial community structure in the glasshouse. Mean microbial activity in the field increased from 4 µg dwt/hr without lime to 16 and 21 µg dwt/hr where lime was applied at 5 and 10 tons/hectare (t/ha), respectively. Mean microbial activity in the field was markedly higher (7-fold) than in the glasshouse at similar rates of lime. Lime application also increased soil moisture retention in the field, mean gravimetric soil moisture increased from 0.33 in control plots to 0.38 and 0.39 in plots treated with 5 and 10 t/ha lime, respectively. Lime application was associated with greater soil aggregate stability. Soils from test plots treated with 5 and 10 tons/ha lime had 45-50% water-stable aggregates compared to 34% in treatments without lime. After 16 weeks in pots, earthworm treatments increased mean plant dry matter (DM)/pot by at least 19% above the control. The increase was attributed primarily to greater N mineralization in the presence of earthworms. For the duration of the trial the earthworm species tested (Apporectodea caliginosa and Lumbricus rubellus, individually or combined) did not affect any of the measured soil microbial properties. However, the survival rate of A. caliginosa was 83% compared to 25% for L. rubellus. The control not receiving any lime or N and plots treated with 10t/ha lime and 200 kgN/ha had similar nematodes species composition, comprising 40% each of bacterial and fungal feeding nematodes. They differed markedly from the reference sites as the forest soil was dominated by plant associated species (38%) and the long-term pasture had 44% plant parasitic nematodes. Accordingly, the soil food web condition inferred from nematode faunal analysis characterized all test plots as basal, stressed and depleted, while the forest soil was categorized as highly structured and fungal dominated. The findings of this thesis demonstrated that land-use change from forest to pasture can have significant impacts on soil biological properties, earthworms can contribute to pasture productivity even in the short term, and nematode faunal analysis is a robust and reliable indicator of soil biological quality.

Recent declines in returns from primary forest products in New Zealand and projected increases in world food prices have led to the land-use conversion from plantation forest to pastoral farming in many lowland areas. After decades of forest cover the soils are in many cases less than adequate for pastoral farming, as they are acidic, with toxic levels of exchangeable aluminum, and contain low levels of available nitrogen (N), very high carbon (C):N ratio, and are devoid of earthworms and structural integrity. Overcoming the major site limitations of low soil pH and available N was a major priority and a field experiment was established in April 2005 to determine the impact of various rates of lime and N in relation to pasture establishment and production. Concerns about the short and long-term effects of these inputs on biological soil quality gave rise to the present study. The effects of land-use change and establishment inputs were assessed by comparison of selected treatment plots with two adjacent reference sites (long-term pasture and a 60–year Pinus radiata forest) on the same soil type. The effects of lime and N on soil biological quality were investigated under field and controlled environment conditions by determination of: microbial community structure (phospholipid fatty acids - PLFA), microbial biomass (total PLFA), and microbial activity (dehydrogenase activity). Soil physical (percentage water-stable aggregates) and chemical (pH, and total C and N) properties were also determined. Similarly, the effects of earthworm addition on soil biological properties were explored in a short-term glasshouse pot experiment. The role of earthworms as indicators of soil biological quality in the field was assumed by nematodes and these were assessed in field trial plots and the reference sites mentioned above. Land-use change and applications of lime and N contributed to changing the microbial community structure determined by principal component analysis of transformed PLFA data. However, the effect of lime was more pronounced in the field, while N contributed most to changing microbial community structure in the glasshouse. Mean microbial activity in the field increased from 4 µg dwt/hr without lime to 16 and 21 µg dwt/hr where lime was applied at 5 and 10 tons/hectare (t/ha), respectively. Mean microbial activity in the field was markedly higher (7-fold) than in the glasshouse at similar rates of lime. Lime application also increased soil moisture retention in the field, mean gravimetric soil moisture increased from 0.33 in control plots to 0.38 and 0.39 in plots treated with 5 and 10 t/ha lime, respectively. Lime application was associated with greater soil aggregate stability. Soils from test plots treated with 5 and 10 tons/ha lime had 45-50% water-stable aggregates compared to 34% in treatments without lime. After 16 weeks in pots, earthworm treatments increased mean plant dry matter (DM)/pot by at least 19% above the control. The increase was attributed primarily to greater N mineralization in the presence of earthworms. For the duration of the trial the earthworm species tested (Apporectodea caliginosa and Lumbricus rubellus, individually or combined) did not affect any of the measured soil microbial properties. However, the survival rate of A. caliginosa was 83% compared to 25% for L. rubellus. The control not receiving any lime or N and plots treated with 10t/ha lime and 200 kgN/ha had similar nematodes species composition, comprising 40% each of bacterial and fungal feeding nematodes. They differed markedly from the reference sites as the forest soil was dominated by plant associated species (38%) and the long-term pasture had 44% plant parasitic nematodes. Accordingly, the soil food web condition inferred from nematode faunal analysis characterized all test plots as basal, stressed and depleted, while the forest soil was categorized as highly structured and fungal dominated. The findings of this thesis demonstrated that land-use change from forest to pasture can have significant impacts on soil biological properties, earthworms can contribute to pasture productivity even in the short term, and nematode faunal analysis is a robust and reliable indicator of soil biological quality.