Cu is an important trace element in living systems, including animals. It is a part of many enzymes responsible for different processes occurring in the body. However, antagonists can severely decrease the amount of Cu available to the animal, which results in numerous health issues caused by Cu deficiency. The main antagonists of the Cu are thiomolybdates whose effect is especially harsh in ruminants due to their high potential to synthesize these compounds. Thiomolybdates (the ions MoOxS4-x2- ,where x = 0-3) are formed in the rumen from molybdate and sulfide (from degradation of sulfate), both of which come from the food and water ingested by the animal.<p> The first part of the project described in this thesis was to develop reproducible preparative methods of CuTM samples in solution and solids, under conditions as biologically relevant as possible. This crucial information is missing in the literature related to Cu deficiency. The second part studied the structure of the adduct formed between Cu and two of the thiomolybdates: tetrathiomolybdate, TM4 and trithiomolybdate, TM3, as these are the most important in Cu deficiency.<p> In solution, the appropriate solution medium and the concentration of reactants were chosen to study the stoichiometry of Cu:TM in solution before precipitation occurs.<p> Conditions for preparation of solid CuTM adducts were chosen to obtain the amount of solid necessary for further studies and to eliminate the formation of unwanted side-products. In the solid phase, the stoichiometry of Cu:TM was investigated by elemental analysis. EPR spectroscopy of solid CuTM samples revealed the presence of diamagnetic species which were partially identified.<p> EXAFS studies were performed on both the Mo and Cu K edges. Spectra defined the oxidation states of both main metal atoms, Cu and Mo. Fitting of the measured EXAFS spectra of solid CuTM samples prepared at three different Cu:TM ratios determined the interatomic distances involved in these compounds. This data was used to determine the structure of the CuTM units which polymerize to form the final product. IR spectroscopy was also used to confirm the presence of some of the groups in these adducts.<p> All the results from the various spectroscopic techniques were then combined to proposed a complete reaction scheme for the two-step formation of the CuTM4 and CuTM3 adduct species.

Cu is an important trace element in living systems, including animals. It is a part of many enzymes responsible for different processes occurring in the body. However, antagonists can severely decrease the amount of Cu available to the animal, which results in numerous health issues caused by Cu deficiency. The main antagonists of the Cu are thiomolybdates whose effect is especially harsh in ruminants due to their high potential to synthesize these compounds. Thiomolybdates (the ions MoOxS4-x2- ,where x = 0-3) are formed in the rumen from molybdate and sulfide (from degradation of sulfate), both of which come from the food and water ingested by the animal.<p> The first part of the project described in this thesis was to develop reproducible preparative methods of CuTM samples in solution and solids, under conditions as biologically relevant as possible. This crucial information is missing in the literature related to Cu deficiency. The second part studied the structure of the adduct formed between Cu and two of the thiomolybdates: tetrathiomolybdate, TM4 and trithiomolybdate, TM3, as these are the most important in Cu deficiency.<p> In solution, the appropriate solution medium and the concentration of reactants were chosen to study the stoichiometry of Cu:TM in solution before precipitation occurs.<p> Conditions for preparation of solid CuTM adducts were chosen to obtain the amount of solid necessary for further studies and to eliminate the formation of unwanted side-products. In the solid phase, the stoichiometry of Cu:TM was investigated by elemental analysis. EPR spectroscopy of solid CuTM samples revealed the presence of diamagnetic species which were partially identified.<p> EXAFS studies were performed on both the Mo and Cu K edges. Spectra defined the oxidation states of both main metal atoms, Cu and Mo. Fitting of the measured EXAFS spectra of solid CuTM samples prepared at three different Cu:TM ratios determined the interatomic distances involved in these compounds. This data was used to determine the structure of the CuTM units which polymerize to form the final product. IR spectroscopy was also used to confirm the presence of some of the groups in these adducts.<p> All the results from the various spectroscopic techniques were then combined to proposed a complete reaction scheme for the two-step formation of the CuTM4 and CuTM3 adduct species.

A crucial requirement for the stepwise, continued growth of a brain tumor is the acquisition of a blood supply from the host, i.e. angiogenesis. The mechanisms of copper activity linked to neovascular and neoplastic growth are largely unknown. Copper ion was shown to be a cofactor for angiogenesis. / We tested the effect of copper depletion achieved by a low copper diet and a copper chelator D-penicillamine, on the intracerebral growth of two experimental brain tumors. We developed in in vivo brain tumor model using the VX2 carcinoma. Implantation of 5 $ times$ 10$ sp5$ VX2 carcinoma cells into the parietal lobe of normocupremic rabbits consistently yielded large hemorrhagic, necrotic, vascularized tumors. The cortical surface revealed numerous, hypertrophied, tortuous new vessels with feeding arteries and draining veins similar to the angioarchitecture of malignant human brain tumors. We report here that copper depletion prevents tumor neovascularization and restricts tumor growth of the VX2 carcinoma in the rabbit brain. Low copper diet and penicillamine are both necessary to achieve angiogenic inhibition. We also tested the effect of copper depletion on the 9L gliosarcoma. We observed that invasive growth of the tumor was blocked in rats depleted of copper. Electron microscopy revealed the absence of cytoplasmic extensions, including pseudopodia, by contrast, in normocupremic controls, cytoplasmic extensions, typical of mobile cells, invaded the surrounding neuropil. Our findings link the activity of copper in vascular and neoplastic growth. / We found an increase in the peritumoral brain water content in the copper depleted animals and that copper depletion by itself in nontumor implanted animal has no effect on brain water content. / Because of the ability to pharmacologically suppress capillary growth induced by the VX2 carcinoma, we could test the relative contribution of breakdown of the blood-brain barrier compared with that of angiogenesis in the appearance of contrast enhancement in computed tomographic examinations. We conclude from our data that tumor neovascularization, in our brain tumor model, is the key determinant for the appearance of contrast enhancement. / The same protocol used in the brain failed to prevent tumor neovascularization and growth of the VX2 carcinoma in the muscle of the rabbit thigh indicating the crucial role played by the milieu (muscle versus brain) for the growth of malignant tumor. In the same manner, lung metastases were not prevented.

The rates of plasma clearance of human native low density lipoproteins (LDL) and acetylated human low density lipoproteins (acetyl-LDL) were compared between copper-deficient (CuD) and copper-adequate (CuA) rats. Purified human LDL (d 1.02-1.063) were labeled with 125I and injected to fasted recipient rats intravenously. At different time intervals plasma clearance of 125I radioactivity was measured. The percent of clearance was calculated based on the total plasma volume, as determined by a radioisotopic dilution method. Native human 125I-LDL were cleared at a faster rate in CuD, compared with CuA rats. The half-times (t 1 2) of 125I-LDL clearance are 4.90 ± 0.20 and 5.80 ± 0.30 hours in CuD and CuA rats, respectively. The plasma trichloroacetic acid-soluble 125I-radioactivity was significantly and steadily increased in CuD rats at each interval, reflecting the faster clearance and degradation of LDL in those rats. The plasma removal of 125I-acetyl-LDL was faster compared with that of 125I-LDL. The half-times (t 1 2) of acetyl-LDL in CuD and CuA rats are 5.20 ± 0.06 and 5.16 ± 0.08 minutes, respectively, with no significant difference between the groups. The data indicates that the uptake of LDL via the "scavenger" receptor remains unaffected in copper-deficient rats. The faster removal of the unmodified (native) LDL in CuD group suggests that the apoB,E receptor is up-regulated in copper-deficient rats and that the hypercholesterolemia observed in copper deficiency is not associated with the defective uptake of LDL by the apoB,E-receptor dependent mechanism.

No description available.

Copper deficiency causes significant annual losses in grain yield due to poor grain set. Cereals such as wheat and barley are particularly susceptible to low copper soils whereas,crops such as rye and triticale are better able to grow and yield under such conditions of nutrient stress. The ability of rye and triticale, which carries a complete set of rye chromosomes, to tolerate low copper conditions has been attributed to a gene on rye chromosome 5R. Wheat-rye translocation lines have previously been produced carrying segments of the long arm of chromosome 5 of rye (5RL). Although these lines have expressed copper efficiency in University of Adelaide trials, until now they have been considered agronomically inferior and so have not been used as commercial cultivars. The physical size of rye segment of the 4BS.4BL-5RL translocation in a Chinese Spring background derived from the Cornell Wheat Selection 82a1-2-4-7 was measured using GISH (genomic in situ hybridization) and found to be 16% of the long arm. The size of this translocation was similar to GISH measurements of another 4BS.4BL-5RL translocation in Viking wheat background, although both these lines arose spontaneously and at different times. Molecular maps of both 4BS.4BL-5RL translocations in the two different wheat backgrounds were developed and used to screen for rare recombinants between wheat and rye in a background homozygous for the Sears' ph1b mutant. The maps revealed the approximate genetic location of the translocation breakpoint involved in these two 4BS.4BL-5RL translocations to be similar even though they are known to have arisen at different times and in different experimental populations. The similarity of these translocations suggests a unique property of the region at or near the translocation breakpoint that could be responsible for their similarity and spontaneous formation. After screening 703 critical seedlings for evidence of recombination between the 5RL segment and wheat homoeologues, no confirmed recombinants were identified. Lines containing the 4BS.4BL-5RL translocation were shown to yield equally as well as their recurrent parent under normal field conditions. In addition the presence of the 4BS.4BL-5RL had no adverse effects on a range of grain quality characteristics measured in these lines. A pot trial using lines derived from a cross between the CSHN translocation and the wheat cultivar Warigal (five backcrosses) revealed that they provided copper-efficiency even under the severest of deficiency conditions. While the results of this pot trial did not show the outstanding copper efficiency previously observed in these lines, the translocation did consistently out yield the recurrent parent under severe copper deficiency conditions. Finally, a reliable PCR marker was developed for the rapid identification of lines containing the distal portion of the 5RL chromosome. / Thesis (Ph.D.)--School of Agriculture and Wine, 2004.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

Copper deficiency causes significant annual losses in grain yield due to poor grain set. Cereals such as wheat and barley are particularly susceptible to low copper soils whereas,crops such as rye and triticale are better able to grow and yield under such conditions of nutrient stress. The ability of rye and triticale, which carries a complete set of rye chromosomes, to tolerate low copper conditions has been attributed to a gene on rye chromosome 5R. Wheat-rye translocation lines have previously been produced carrying segments of the long arm of chromosome 5 of rye (5RL). Although these lines have expressed copper efficiency in University of Adelaide trials, until now they have been considered agronomically inferior and so have not been used as commercial cultivars. The physical size of rye segment of the 4BS.4BL-5RL translocation in a Chinese Spring background derived from the Cornell Wheat Selection 82a1-2-4-7 was measured using GISH (genomic in situ hybridization) and found to be 16% of the long arm. The size of this translocation was similar to GISH measurements of another 4BS.4BL-5RL translocation in Viking wheat background, although both these lines arose spontaneously and at different times. Molecular maps of both 4BS.4BL-5RL translocations in the two different wheat backgrounds were developed and used to screen for rare recombinants between wheat and rye in a background homozygous for the Sears' ph1b mutant. The maps revealed the approximate genetic location of the translocation breakpoint involved in these two 4BS.4BL-5RL translocations to be similar even though they are known to have arisen at different times and in different experimental populations. The similarity of these translocations suggests a unique property of the region at or near the translocation breakpoint that could be responsible for their similarity and spontaneous formation. After screening 703 critical seedlings for evidence of recombination between the 5RL segment and wheat homoeologues, no confirmed recombinants were identified. Lines containing the 4BS.4BL-5RL translocation were shown to yield equally as well as their recurrent parent under normal field conditions. In addition the presence of the 4BS.4BL-5RL had no adverse effects on a range of grain quality characteristics measured in these lines. A pot trial using lines derived from a cross between the CSHN translocation and the wheat cultivar Warigal (five backcrosses) revealed that they provided copper-efficiency even under the severest of deficiency conditions. While the results of this pot trial did not show the outstanding copper efficiency previously observed in these lines, the translocation did consistently out yield the recurrent parent under severe copper deficiency conditions. Finally, a reliable PCR marker was developed for the rapid identification of lines containing the distal portion of the 5RL chromosome. / Thesis (Ph.D.)--School of Agriculture and Wine, 2004.

Made available in DSpace on 2016-12-08T16:24:04Z (GMT). No. of bitstreams: 1 PGCA12MA038.pdf: 2912659 bytes, checksum: a707fa201b75de04c816893aa22d474d (MD5) Previous issue date: 2012-06-15 / It has been described the epidemiological, clinical signs and lesions of a disease of cattle characterized by seasonal occurrence of outbreaks of diarrhea, chronic fever, responsive to treatment with copper sulfate. The disease occurs on properties with steeply sloping topography of the different regions of the state of Santa Catarina, with greater frequency in the west Highlands and Serra, in the months from October to January and after periods of heavy rain. Morbidity ranged from 6.16 to 66.6% and mortality from 2,1 to 20%. Besides of diarrhea, the animals showed weight loss, the bristling, sometimes with discoloration around the eye, culminating in death. In the necropsy of animals that died from disease was observed cachexia, pale mucous membranes, spleen and intestine with brown liquid content. Microscopically the only significant finding was splenic hemosiderosis. The dosage of the liver concentrations of copper and molybdenum by mass spectrometry method of inductively coupled plasma source ICP-MS, with introduction of the sample by pneumatic spraying of the animals were necropsied showed low levels of copper (4.70 to 70.35 ppm) of molybdenum and normal (from 0.07 to 1.35 ppm). In the experimental treatment was administered orally copper sulphate for two animals with clinical signs of disease during a period of 30 days, the animals had clinical improvement and body weight gain. The epidemiological, clinical and pathological, low concentrations of copper found in liver samples and the positive response to treatment with copper sulphate define the disease as deficiency disease by copper deficiency / Descrevem-se os dados epidemiológicos, sinais clínicos e lesões de uma enfermidade de bovinos de ocorrência sazonal caracterizada por surtos de diarreia crônica, afebril, responsiva ao tratamento com sulfato de cobre. A enfermidade ocorre em propriedades com topografia de declive acentuado nas diferentes regiões do estado de Santa Catarina, com maior frequência na região Oeste e Planalto Serrano, nos meses de outubro a janeiro e posterior a períodos de chuva intensa. A morbidade oscilou de 6,16 a 66,6% e mortalidade de 2,1 a 20%. Além da diarréia os animais apresentam emagrecimento progressivo, pelos arrepiados, às vezes com descoloração dos pelos ao redor dos olhos, culminando com a morte. Através da necropsia de animais que morreram pela enfermidade foi observado caquexia, palidez das mucosas, baço de coloração marrom e intestino com conteúdo líquido. Na microscopia o único achado significativo foi hemossiderose esplênica. A dosagem da concentração hepática de cobre e molibdênio através do método espectrometria de massa com fonte de plasma indutivamente acoplado ICP-MS, com introdução da amostra por nebulização pneumática dos animais que foram necropsiados revelou baixos valores de cobre (4,70 a 70,35 ppm) e normais de molibdênio (0,07 a 1,35 ppm). No tratamento experimental foi administrado sulfato de cobre via oral a dois bovinos, com os sinais clínicos da doença durante um período de 30 dias, os animais obtiveram melhora do quadro clínico e ganho de peso corporal. O quadro epidemiológico, clínico e patológico, as baixas concentrações de cobre encontradas nas amostras de fígado e a resposta positiva ao tratamento com sulfato de cobre definem a enfermidade como doença carencial por deficiência de cobre