This thesis is a comparative study of variables affecting differentiation and proliferation of progenitor cells from various embryonic and fetal tissue sites as they differentiate into mast cells. Embryonic stem cells from gestation day 4.5 (d4.5) differentiated exclusively into mast cells when cultured in semi-solid media with stem cell factor (SCF) and Interleukin-3 (IL-3). The same was true of d8-1 1 yolk sac cells and d8-1 1 cells from the embryo itself. However, culture with SCF and IL-3 of cells from dl4 fetal livers differentiated into mast cells, cells of the erythrocyte lineage and a few "macrophages" which may have been similar to the stromal cell found in the bone marrow that supplies iron to and removes the nucleus from erythrocytes. An rythrocyte-nurturing role for the fetal liver "macrophages" was substantiated by removing cells expressing Mac1, and sorting for cells expressing the early mast cell marker, Kit, resulting in cells that differentiated only into mast cells with SCF and IL-3. The variables affecting proliferation that were studied were brief incubation at very high cell density, and co-culture with fibroblasts. In addition, a few experiments were done to determine the mechanism of the effect of these variables on proliferation. High cell density incubation increased proliferation of yolk sac, fetal liver and bone marrow cells. The increased proliferation of yolk sac and bone marrow cells was attenuated by an inhibitor of the sodium-ionlhydrogen-ion exchanger, 5-(N,N-hexamethylene) amiloride (HMA). The exchanger was also found to play a role in the subsequent proliferation of mast cells from bone marrow cells co-cultured with fibroblasts since, if HMA was present during the first thirty minutes of contact, mast cell proliferation decreased by 75%. Using fluorescent imaging, activation of this exchanger was monitored by an increase in intracellular pH. Altogether these results demonstrate a growing concept in developmental biology regarding the normal stem cell in its niche (tissue site) and the effects of exogenous variables on it. They substantiate the concept that the differentiation path and proliferation of a stem cell depends both on its past history and on its future circumstances.

Objective Because the public health nutrition workforce may be in a state of transition, this study had three purposes: 1) describe the US public health nutrition workforce; 2) examine a new position class, breastfeeding peer counselor; and 3) determine if retirement intention of public health nutrition personnel can be predicted based on personal and workplace factors. Methods Secondary data analysis of the national research dataset of the 2006-07 Public Health Nutrition Workforce Survey was conducted (n=10,683, response rate 80.0% for overall survey; research dataset n=9,923). Subjects were personnel in nutrition professional/paraprofessional positions working in nutrition programs under the purview of the official health agency and who agreed to release their data for research purposes. Results Over one-quarter (28.0%) of respondents were in positions with a population/systems focus, while 67.5% were in client-focused, direct care positions. Two-thirds (67.0%) practiced primarily in the core public health function of assurance. Approximately 10% (11.3%) of personnel were breastfeeding peer counselors. The majority (52.6%) of breastfeeding peer counselor positions were part-time and 20.3% were contracted. Nearly half (42.0%) did not receive employee benefits. Close to one-quarter (23.9%) of the overall workforce intended to retire within 10 years. There were significant differences in both personal and workplace factors for intention to retire for personnel 45 years and older. Age category, years of experience in nutrition/dietetics and public health nutrition, agency of employment, vacation and retirement employee benefits, percent of work time spent in direct client services, full-time/part-time status, and US DHHS Region correctly predicted retirement intention 75.0% of the time. Conclusions The majority of respondents worked in client-focused positions which could indicate a potentially inadequate proportion of personnel available for assuring population health. Breastfeeding peer counselors constitute a noteworthy proportion of the overall workforce. That many positions are part-time or contracted and do not receive employee benefits could indicate inadequate funding for this position class. ‘Graying’ of the public health nutrition workforce appears to be an important concern. Results can be used to evaluate organizational characteristics for workforce succession planning and forecasting.

Activating mutations of ras genes are frequently found in human cancers. Since Ras proteins and their functions play an important role in tumorigenesis, it is important to develop targeted anticancer therapeutics against Ras-related human cancers. We observed that in addition to tumorigenic ability, oncogenic H-Ras possesses a novel proapoptotic ability to facilitate the induction of apoptosis by histone deacetylase inhibitors (HDACIs), such as FR901228 and trichostatin A (TSA). HDACIs make up a new class of structurally diverse anticancer agents and have been shown to exhibit antimetastatic and antiangiogenic activities toward malignantly transformed cells. We detected that expression of oncogenic H-Ras potentiated intracellular reactive oxygen species (ROS) in human and mouse cells to enhance HDACI-induced ROS, thereby contributing to the induction of selective apoptosis and caspase activation. The first part (Part I) of this dissertation focuses on the understanding of Ras proteins, their role in normal and transformed cell physiology, and current treatment options against Ras-related human cancers, as well as the role of HDACIs and ROS in anticancer therapeutics. The next three parts (Part II-IV) focus on revealing the mechanisms for the novel pro-apoptotic ability of oncogenic H-Ras that allow HDACIs to induce selective apoptosis of the oncogenic H-Ras expressing cells. Results in Part II & III verify the pro-apoptotic activity of oncogenic H-Ras in the increased susceptibility of human cancer cells to HDACIs. The caspase pathways, the B-Raf and extracellular signal regulated kinase pathway, p21Cip1 and p27Kip1, and core histone contents are regulated differently by FR901228 in oncogenic H-Ras–expressed cells than their counterparts in parental cells, contributing to the increased susceptibility to the induction of selective apoptosis. Results in Part IV describe the role of reactive oxygen species in the pro-apoptotic ability ofoncogenic H-Ras to enhance the cell susceptibility to HDACIs. Intracellular ROS was cooperatively up-regulated by oncogenc H-Ras and HDACI treatment to induce selective apoptosis of oncogenic H-Ras-expressing cells. The last section (Part V) summarizes the findings with their importance and discusses future directions.

The diagnosis of Von Willebrand's disease (VWD) may sometimes be difficult because of the variability of the results obtained over time in individuals. Moreover, blood group, age, pregnancy and inflammatory stimuli influence the level of Von Willebrand Factor (VWF). The purpose of this thesis was to screen and characterize antibodies to Von Willebrand factor and to evaluate the most promising ones in a gold- Sol assay for VWF on the CA-6000 analyzer. Seven different lots of Anti-VWF antibodies, 3 polyclonal and 4 monoclonal Ab's were screened and evaluated. Two of these antibodies (Sunol R01358 and MAVWF-AP) were selected for preparation of a Gold coated antibody solution. The preliminary testing of these gold coated antibodies on CA-6000 Analyzer showed no immunoreactivity toward VWF for both individual and pooled plasma (from normal healthy donors). Although measurement of VWF for normal plasma with this technique was not demonstrated, these data will be valuable for future work on the design of sensitive and accurate automated sol Gold Immunoassays for the diagnosis of VWD.

No description available.

<p>Overexpression and amplification of the neu proto-oncogene have been implicated in the development of aggressive human breast cancer. To investigate the effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying the unactivated neu gene under the transcriptional control of the mouse mammary tumor virus (MMTV) promoter/enhancer were established. Overexpression of neu in the mammary tumors was associated with elevated Neu intrinsic tyrosine kinase activity and the stochastic development of focal mammary tumors which frequently metastasized. These observations provide the first direct evidence that expression of the proto-oncogenic form of neu results in a heritable development of metastatic mammary tumors.</p> <p>Another potent tyrosine kinase activity that has been implicated in the genesis of murine mammary tumors is that associated with polyomavirus middle T antigen (PyV MTAg). Expression of MMTV/PyV middle T antigen in the mammary glands of transgenic mice resulted in the induction of multifocal mammary tumors which frequently metastasized to the lung. The potent transforming activity of PyV MTAg can, in part, be attributed to its ability to associate with an activate a number of e-Src family tyrosine kinases (e-Src, e-Yes, and Fyn). In order to assess the role of individual members of the e-Src family of tyrosine kinases in PyV MTAg induced mammary tumorigenesis, I have crossed the MMTV/PyV middle T fusion gene with mice bearing disrupted c-src or e-yes alleles. Mice expressing the PyV middle T transgene in the absence of functional c-Src rarely developed metastatic mammary tumors. However, transgenic mice expressing the PyV MTAg in mammary epithelium lacking functional c-Yes developed multifocal mammary tumors with kinetics comparable to MMTV/PyV middle T strains possessing a functional c-Yes. These findings suggest that c-Src tyrosine kinase activity is required for PyV MTAg induced mammary tumorigenesis and also illustrate a in vivo genetic approach to dissect mitogenic signal transduction pathways.</p> / Doctor of Philosophy (PhD)

Feline immunodeficiency virus is a retrovirus of domestic cats causing significant lifelong infection. Infection has also been detected in nondomestic species, including African lions. It is endemic in certain populations in east and southern Africa. Infection leads to immunologic dysfunction and immunosuppressive disease in domestic cats; however, little research exists about the pathogenic effects of infection in lions and its epidemiological impact on free-ranging and captive populations. Little is known about the lentivirus in these populations at the molecular and host level. Analysis of the virus from these populations is necessary for development of detection assays that are both sensitive and specific. Whole-blood and serum samples were collected opportunistically from free ranging lions in Kruger National Park, South Africa, and from Hlane Royal National Park, Swaziland. Whole-blood and serum samples were also collected from captive exotic felids in RSA andUS and domestic cats in RSA. A nested polymerase chain reaction assay was performed on all whole-blood samples, and all positive products were sequenced and analyzed phylogenetically. Serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and cross-reactive antibodies to puma lentivirus synthetic envelope peptide antigen. Serum samples were tested for feline haptoglobins and feline alpha-one acid glycoproteins by radial immunodiffusion. This research represents the first epidemiological study of the lion lentivirus among free-ranging lions of Kruger National Park and the first epidemiological study comparing genetic material to antibody-based methods of lentivirus detection on lions in RSA. The polymerase chain reaction assay was successful in amplifying the lion lentivirus from African lions. The conservation management of free-ranging lions must consider the infectious agents to which they are susceptible. No conclusions can be drawn from this investigation with respect to the potential virulence or pathogenic distinctions between viral subtypes, as little is known about the definitive consequences of lion lentivirus infection in African lions. Immunologic studies may uncover potential differences in immune expression among lentivirus-positive and -negative individuals with regard to increased resistance or increased susceptibility to infection or pathogenicity. To this end, lion lentivirus infection status could also be compared to the infection status of bovine tuberculosis.

A selective agar medium was developed for the isolation of Clostridium bifermentans. The formulation of the C. bifermentans selective agar (CBSA) was a modification of C. botulinum isolation agar (CBI) medium. The modifications included: a) change of the base medium to promote growth of C. bifermentans, b) increase of antimicrobial concentrations to improve selectivity and, c) lowering of the egg yolk concentration to lower the cost. The CBSA medium contains cycloserine (375 µg/ml), sulfamethoxazole (114 µg/ml) and, trimethoprim (6 µg /ml) as selective inhibitory agents. Quantitation of C. bifermentans strains on CBSA yielded counts comparable to those obtained on egg yolk agar control plates. The increased concentration of the selective agents in CBSA suppressed the growth of the lecithinase producing C perfringens, C. sordellii and other fecal organisms tested. Isolation of C. bifermentans from 20 seeded fecal specimens was readily achieved. The CBSA medium permits quantitative recovery of lecithinase positive colonies of C. bifermentans from human feces and can be used for large scale screening of various clinical specimens.

Roles organize occupational behavior. They influence time use, performance, manner, style, and content of interaction with others. It is assumed that knowledge of occupational role performance in the general population of Iceland will enable occupational therapists to be more effective in enabling a client's role performance. The purpose of this study was to add to the Icelandic occupational therapists' knowledge base about role performance among the Icelandic population. A random sample of 149 Icelandic subjects, with an age range of 18 to 75 years, was surveyed about the frequency, value, and change in value of role performance in ten occupational roles over time. Results indicated that the most frequently performed roles were: worker, family member, home maintainer, caregiver, and friend. The most valued roles were family member, worker, caregiver, friend, and student. Significant differences were found between various demographic variables and the frequency and value of occupational role performance.

Respiration introduces large tumor motion in the thoracic region which influences treatment outcome for lung cancer patients. Tumor motion management techniques require characterization of temporal tumor motions because tumor motion varies patient to patient, day to day and cycle to cycle. This work develops a markerless algorithm to estimate 3 dimensional (3D) lung-tumor trajectories on free breathing cone beam computed tomography (CBCT) projections, which are 2 dimensional (2D) sequential images rotating about an axis and are used to reconstruct 3D CBCT images. A gold standard tumor trajectory is required to guide the algorithm development and estimate the tumor detection accuracy for markerless tracking algorithms. However, a sufficient strategy to validate markerless tracking algorithms is lacking. A validation framework is developed based on fiducial markers. Markers are segmented and marker trajectories are xiv obtained. The displacement of the tumor to the marker is calculated and added to the segmented marker trajectory to generate reference tumor trajectory. Markerless tumor trajectory estimation (MLTM) algorithm is developed and improved to acquire tumor trajectory with clinical acceptable accuracy for locally advanced lung tumors. The development is separate into two parts. The first part considers none tumor deformation. It investigates shape and appearance of the template, moreover, a constraint method is introduced to narrow down the template matching searching region for more precise matching results. The second part is to accommodate tumor deformation near the end of the treatment. The accuracy of MLTM is calculated and compared against 4D CBCT, which is the current standard of care. In summary, a validation framework based on fiducial markers is successfully built. MLTM is successfully developed with or without the consideration of tumor deformation with promising accuracy. MLTM outperforms 4D CBCT in temporal tumor trajectory estimation.