Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

Testicular germ cell tumors (TGCTs) are thought to arise during early embryogenesis due to the arrest of germ cell differentiation at primordial germ cells (PGCs) or gonocytes. Oxidative stress (OS) is implicated in cancer development as a factor leading to DNA damage. Reactive oxygen species (ROS) -induced instability occurs as a series of progressive steps. The cell has several defense mechanisms against the deleterious effect of ROS (e.g. antioxidants and DNA repair). When the defense mechanisms are exhausted by increasing OS, DNA damage leads to genomic instability with subsequent mutations that can be transmitted during cell division. On the other hand, male infertility is a representation of testicular dysgenesis syndrome, which carries a risk for TGCTs development. The mechanisms underlying both TGCTs and male infertility are thought to be overlapping to some extent. The central hypothesis of this work is that OS induces germ line genomic instability leading to testicular germ cell tumors. To test this hypothesis, mouse germ cell lines were established and subjected to different doses of OS in the form of H2O2. The mutation frequency was associated with the treatment dose 2 uM at days 3, 6, and 9 (p<0.001, p<0.001, and p<0.0003, respectively). The mBAT27 marker showed a mutation frequency fitting quadratic response surface regression. The mutation frequencies pointed to the possible role of OS leading to accumulation of DNA damage and initiating events that lead to TGCTs development that may occur early in life, possibly during the prenatal period. In addition, different panels of microsatellite markers from across the genome were analyzed to test for differential instability in both somatic cells and germline cells. Blood and semen samples from 18 infertile patients and 7 ethnically matched controls were used. Microsatellite markers were selected; 26 on the Y chromosome, 16 on the X chromosome, and 20 on different autosomes. Microsatellite instability was detected in markers located near genes responsible for testis development, spermatogenesis, cell differentiation, and proteins involved in mismatch repair mechanisms. This supports the hypothesis that testicular germ cell tumors may arise during early embryogenesis through acquiring multiple mutations that accumulate over time.

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Master of Science / Department of Animal Sciences and Industry / Christopher D. Reinhardt / Three experiments were conducted to examine nutritional and management strategies for different segments of the beef industry. The first experiment examined the effects of feeding traditional step-up diets (STEP) vs. limit-feeding (LIMIT) the finishing diet to adapt cattle to high-concentrate diets. When all cattle reached ad libitum intake of the finishing diet there was a trend (P = 0.09) for DMI to be different between treatments. During week 1, STEP cattle had higher total VFA concentrations (P = 0.02), while LIMIT cattle had higher valerate absorption (P = 0.02) and disappearance (P = 0.08). During week 4, LIMIT cattle had higher total VFA concentrations (P = 0.03) and lower valerate disappearance and absorption (P = 0.05) than STEP cattle. These results indicate that limit-feeding the finishing diet may inhibit nutrient absorption from the rumen or this method may cause increased production of valerate by lactate utilizing bacteria due to a more acidotic rumen environment. The second experiment examined the effects of feeding 5% (DM basis) dried, full-fat corn germ (GERM) on feedlot performance and carcass characteristics of naturally raised yearling steers and heifers. Carcass-adjusted ADG was higher for GERM cattle (P = 0.04). There were no other differences in performance or carcass characteristics. Total incidence of liver abscesses and the incidence of severe liver abscesses were decreased by 12 and 8.2% (P = 0.01 and 0.02, respectively) when GERM was added to the diet. Corn germ can be added to finishing diets at 5% without affecting performance and carcass characteristics. Producers raising natural cattle may also be able to benefit from the reduced incidence of liver abscesses. The third experiment examined concurrent metaphylactic treatment of high-risk calves with tulathromycin and chlortetracycline. Calves were placed on 1 of 3 treatments: 1) no top-dress pellets; 2) diet top-dressed with pellets containing chlortetracycline; or 3) diet top-dressed with pellets containing no chlortetracycline. There were no differences in the performance or health of these calves (P > 0.25). There are no additive benefits of concurrent metaphylaxis using both tulathromycin and chlortetracycline. This information could assist producers when designing receiving health protocols for high-risk calves.

Master of Science / Food Science Institute - Animal Science & Industry / Fadi M. Aramouni, Bean, Scott R. / The biochemical, physical and baking properties of caroubin, the main protein in the carob bean, were characterized. The biochemical properties of caroubin were analyzed using reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and micro-fluidics analysis. The physical and baking properties of caroubin were characterized via SE-HPLC, laser scanning confocal microscopy, farinograph mixing, and texture profile analyzer analysis. Using a modified Osborne fractionation method, carob germ flour proteins were found to contain ~32% albumin and globulin and ~68% glutelin with no prolamins detected. When divided into soluble and insoluble protein fractions under non reducing conditions it was found that caroubin contained (~95%) soluble proteins and only (~5%) insoluble proteins. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater Mw than the soluble proteins and ranged up to 8x107 Da. These polymeric proteins appeared to play a critical role in protein network formation. Analysis of the physical properties of carob germ protein-maize starch dough showed that the dough’s functionality was dependent on disulfide bonded protein networks, similar to what is found in wheat gluten. When baked into a bread these proteins were shown to have a possible improving affect by decreasing staling in gluten-free breads. This was evident when compared to a gluten-free batter bread, and a wheat bread over a five day period.

The commonest reproductive disorders of young men (namely low sperm counts, testicular germ cell cancer) may originate in fetal life similar to established disorders (cryptorchidism, hypospadias) that manifest at birth. These disorders are interlinked and may comprise a testicular dysgenesis syndrome (TDS), a concept supported by animal model studies. The latter have identified the likely time-frame within which TDS disorders may be induced, namely within the so-called masculinisation programming window (MPW). During this critical period, sufficient testosterone (androgen) must be produced by the fetal testis to program the male reproductive tract so that it will differentiate and grow normally after the MPW. Impaired androgen production or action within the MPW can result in smaller reproductive organs and their abnormal formation and function (e.g. cryptorchidism, hypospadias). The MPW is thus of fundamental importance in determining normal, or abnormal, male reproductive development and function for later life. There are two big unanswered questions about the MPW. First, what determines its timing? Second, what mechanisms are controlled by androgens specifically within this time-window and not at later time points? Three approaches were undertaken to address the first question experimentally in rats. First, investigation of whether the availability of androgens and or androgen receptors (AR) plays a role in determining the onset or ‘opening’ of the MPW. Second, investigation of whether the expression of AR co-regulators was a factor in determining androgen sensitivity during the MPW. Third, investigation of whether prostaglandins played a role in mediating androgen action in the MPW, as studies in the 1980s had suggested this possibility. To address what mechanisms are controlled by androgens specifically within the MPW, the expression of selected genes in the genital tubercle was investigated before, during and after the MPW in fetuses that had been exposed to treatments that modulated androgen action. Selection of genes was based on microarray studies and data reported in the literature (ie candidate genes). The studies reported in this thesis show that neither availability of androgens nor the AR are important in determining onset of the MPW, and providing exogenous androgens either prior to or during the MPW does not advance or enhance masculinisation. These studies also showed that females may have a slightly different window of susceptibility to androgen action than do males. Key AR co-regulators have been characterized in the male reproductive tract for the first time, two of which (BRG1, CBP) show changes in expression through development of the testis consistent with a role in Sertoli cells. Another AR co-regulator, RWDD1, was found to switch off in the absence of androgen action in the genital tubercle, pointing to a potential role during and/or after the MPW. Studies involving gestational exposure to indomethacin (a compound which inhibits prostaglandin synthesis) during the MPW showed no detectable effect on masculinisation. Finally, evaluation of candidate genes for mediating androgen action in the genital tubercle during the MPW, failed to identify their key involvement, thus they are unlikely to be involved in penis development and disorders such as hypospadias.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy