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    AuthorMills, Mary Etta C. (13)Augsburger, Larry L. (10)Belcher, John R. (9)Belcher, Anne E. (7)Hollenbeck, R. Gary (7)Neal, Maggie T. (7)Aurelian, Laure (6)Ephross, Paul H. (6)Falkler, William A., Ph.D. (6)Soeken, Karen (6)View MoreSubjectBiology, Molecular (120)Health Sciences, Nursing (82)Biology, Cell (71)Social Work (65)Biology, Microbiology (49)Biology, Animal Physiology (48)Health Sciences, Pharmacology (48)Chemistry, Biochemistry (44)Health Sciences, Pharmacy (42)Biology, Neuroscience (38)View MoreDate Issued1999 (49)1998 (55)1997 (38)1996 (57)1995 (71)1994 (35)1993 (59)1992 (54)1991 (52)1990 (1)

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    Induction of cytochrome P-4501A and toxicity of carbon tetrachloride and chloroform in primary cultures of rainbow trout hepatocytes

    Rabergh, Christina Maria I. (1995)
    In vitro mammalian system for the study of mechanisms of toxicity have been widely used for several years. It is, however fairly recently that in vitro models of aquatic animals have been developed and the mechanisms of toxicity of xenobiotics on the cellular level are still poorly understood. In this study a cell system based on primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes was established. The hepatocytes were cultured on polylysine coated dishes in serum-free medium. The maintenance of differentiation was studied over time in culture, by measuring the inducibility of tyrosine aminotransferase (TAT), by dexamethasone (DEX). Cells maintained TAT inducibility up to 19 days in culture. In order to further evaluate the model system, the induction of cytochrome P4501A mRNA by various xenobiotics, {dollar}\beta{dollar}-naphthoflavone (BNF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), isosafrole (ISF) and carbon tetrachloride {dollar}\rm (CCl\sb4){dollar} was studied using a CYP1A probe and Northern blotting. TCDD was shown to be the most potent inducer of CYP1A, followed by 3-MC and BNF. ISF was the weakest inducer of CYP1A. Inducibility of CYP1A was observed up to 14 days in culture. Furthermore, this study reports the first results of differential induction of two CYP1A genes in trout by using the RNase protection assay and riboprobes specific for the two genes, CYP1A1 and CYP1A2. Both genes were induced with the model substances; BNF and ISF preferentially induced CYP1A1, while TCDD showed a higher induction of CYP1A2. 3-MC induced both genes, with a preference for CYP1A1 that was less pronounced than that seen with BNF and ISF, while {dollar}\rm CCl\sb4{dollar} induced CYP1A1 exclusively. The toxicity of two chlorinated hydrocarbons, carbon tetrachloride {dollar}\rm (CCl\sb4){dollar} and chloroform {dollar}\rm (CHCl\sb3){dollar} was studied. As observed in mammals, {dollar}\rm CCl\sb4{dollar} was five times more toxic than {dollar}\rm CHCl\sb3.{dollar} In the presence of antioxidants, cells were protected from chemical induced injury at low level toxicity. Glutathione (GSH) was depleted in the presence of both chemicals but was protected by antioxidants in cells treated with {dollar}\rm CHCl\sb3.{dollar} These results indicate that fish hepatocytes are as sensitive as mammalian hepatocytes in their response to {dollar}\rm CCl\sb4{dollar} and {dollar}\rm CHCl\sb3.{dollar}
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    Analysis of the pathways of androgen metabolism in the porcine ovary: An examination of the role of androgens during follicular development and maturation

    Garrett, Wesley MacDougall (1995)
    The follicle is the compartment of the ovary responsible for estrogen production. In the pig follicular estrogen production is accomplished by a concerted cooperation between the two cell types of the follicle, the granulosa cells, and the thecal cells, under the control of the pituitary gonadotropins Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH). Under the influence of LH stimulation, the thecal cells increase their production of the androgen, androstenedione. FSH stimulation of the enzyme aromatase, increases granulosa cell estradiol production from androstenedione provided by the thecal cells. Granulosa cells from large preovulatory follicles luteinize in vitro when placed in culture. This process is characterized by a decline in estrogen production and by increased progesterone production. To better define the steroid metabolic activity of granulosa cells during this period of in vitro luteinization, studies were performed to assess the metabolism of (A-dione) by granulosa cells in culture. The results of these studies demonstrated that aromatase activity of the granulosa cells declined substantially between 12 and 48 hours of culture resulting in the loss of estrogen production. In addition to this finding, an alternative pathway of A-dione metabolism was discovered. This pathway involves the production of the novel acidic steroid 19-oic-androstenedione (19-oic-A), and two additional novel metabolites, the C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids 5(10)estrene-3{dollar}\beta,\ 17\beta{dollar}-diol (estrenediol) and 19-nor-testosterone (19-nor-T). Evaluation of the time course of A-dione metabolism suggested that aromatase was responsible for the production of 19-oicA from A-dione. The data also suggested that changes in metabolism associated with luteinization of the granulosa cells resulted in the metabolism of 19-oic-A to estrenediol and 19-nor-T. Further investigation confirmed that aromatase was the enzyme responsible for the production of 19-oic-A from A-dione. The metabolism of 19-oic-A by granulosa cells was assessed, and the results demonstrated that this steroid was indeed the metabolic precursor of the two C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids, estrenediol and 19-nor-T. Isolation of 19-oic-A, estrenediol, and 19-nor-T in pig ovarian follicular fluid provided evidence that the metabolic pathways observed in culture were reflective of in vivo follicular steroidogenesis. The effects of metabolites of A-dione were tested for their ability to modulate parameters associated with follicular development and maturation. Granulosa cells isolated from prepubertal pigs were cultured with various combinations of FSH and steroids to assess the effects of these hormone combinations on the induction of LH receptors and aromatase activity. 19-nor-T significantly augmented the ability of FSH to induce LH receptors on granulosa cells, while estradiol and estrenediol were without effect. Likewise, the androgens A-dione, testosterone(T), 19-nor-T and all significantly enhanced the FSH stimulation of granulosa cell aromatase activity, while estradiol was without effect.;To determine if the regulation of LH receptor and aromatase induction by androgens, but not estrogens could be explained by the expression of steroid hormone receptors, immunohistochemical studies were performed to localize androgen and estrogen receptors in the pig ovary. Granulosa cells from small preantral and antral follicles expressed androgen receptors, but were devoid of estrogen receptors. As follicles increased in size as a result of follicular development and maturation, the expression of androgen receptors decreased. Weak expression of estrogen receptors was observed only in mature follicles and the developing corpus luteum. Collectively, these results were interpreted to indicate that androgens are potentially important modulators of FSH action during follicular development and maturation.
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    The relationship of pain characteristics, type of cancer, and opioid consumption to quality of life, psychological distress, and pain outcomes

    Polomano, Rosemary Carol (1995)
    The relationship of pain to quality of life (QOL) outcomes has been studied; yet, for the most part, the presence and magnitude of pain have been the major variables of interest. Little is known about the impact of pain types (somatic, visceral and neuropathic) on QOL. The primary purpose of this study was to evaluate the extent to which physiological source(s) of pain, pain duration, intensity, location(s), number of sites, relief, sensory and affective components, and opioid consumption affect perceptions of QOL and psychological distress. In addition, the association and validity of pain language in predicting physiologic pain were evaluated. A convenience sample (N = 100) of subjects with chronic pain from advanced cancer was recruited for study from three outpatient medical oncology practices. Information was collected on age, sex, tumor type, treatment information, average daily opioid requirement, physiologic source(s) of pain, pain location(s), number of painful sites, and duration of pain. Each subject completed the following measures: (a) a Numeric Pain Rating (NRS) Scale for present pain intensity (PPI) and average worst pain intensity (WPI); (b) pain relief scale (VASPR); (c) the sensory and affective Short Form-McGill Pain Questionnaire (SF-MPQ); (d) The Brief Symptom Inventory (BSI) (psychological distress); and, (e) The Quality of Life Survey (QOLS). Data analyses were conducted using The SSPS-PC Program and the SAS Software System. Multiple regression analysis determined that SF-MPQ affective component, VASPR and age accounted for a significant portion (25.3%) of the variance in the QOLS scores, while WPI, VASPR and age explained 21.3% of the model for psychological distress. Discriminant Analysis, Chi-Square analyses and linear logistic regression evaluated significant associations of pain language to physiological pain categories. No significant differences in the QOLS, BSI and average pain intensity were found for pain location and cancer diagnosis using MANOVA's. ANOVA and Student's t-tests assessed differences among pain types. Subjects with a component of neuropathic pain experienced significantly more psychological distress, (p<.05), average pain (p<.01), greater sensory and affective pain (SF-MPQ) (p<.001), and present pain (p<.01).
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    Evaluation ofopd as a reporter gene in Streptomyces lividans

    Pierson, Vicki Lynn (1995)
    The transition from primary to secondary metabolism in Streptomyces is environmentally influenced, but it is unclear how environmental factors achieve this effect. In other genera, certain nonessential gene products such as virulence factors, pigments and bacteriocins can be regulated by multiple environmental factors, many of which can also alter DNA superhelicity. It was postulated that the primary to secondary metabolic transition in Streptomyces may be regulated by genes with promoters sensitive to transient changes in superhelicity. These promoters could be isolated in their native loci via an integrative vector bearing a reporter gene to monitor in situ expression. An {dollar}att\sp-{dollar} temperate promoter-probe phage utilizing the xylE gene of Pseudomonas putida used for this purpose failed to express xylE for unknown reasons. A promoter-probe suicide vector developed for interspecific conjugation gave a frequency of integration approximately equal to the rate of spontaneous mutation to resistance. As the work progressed, the need for a dependable reporter gene in Streptomyces became apparent. A gene for organophosphate degradation (o p d), parathion hydrolase, was evaluated for its utility as a reporter gene. It can be expressed from a variety of native and heterologous promoters, either inducible or constitutive, at various stages of growth. These promoters express o p d under the same conditions as they express their native products, and activity can be screened by single colony (microtiter plate) assay, quantitative by spectrophotometric assay, and detected even at low levels by immunoblotting. As a reporter gene, opd offers several advantages. It has no known analog in Streptomyces, so background interference is negligible, as is unwanted interaction with chromosonal sequences. It is metabolically benign, even at high levels of expression, and requires no cofactors for activity. Since the protein is quite stable, it may be particularly useful in detecting transiently expressed promoters. This protein is naturally secreted by Streptomyces and may be particularly useful in studying this process, although it is not limited to this application since adding intra- and extracellular fractions gives an accurate measurement of total gene expression.
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    The relationship and differences between depression and perceived enactment of autonomy scores among post-stroke elderly

    Castellucci, Deborah Dickson (1995)
    This research investigated what the relationship was between the elderly's perceived ability to be self-determined and the level of depression following a stroke. Additionally, differences between depression and perceived enactment of autonomy scores based on gender and cerebral hemisphere infarct were studied. A convenience sample of 20 post-stroke elderly, who ranged in age from 65-84 years comprised the sample. All subjects completed Beck's Depression Inventory and Hertz's Perceived Enactment of Autonomy Scale. The first research question was: What is the relationship between perceived ability to be self-determining and the level of depression in the post-stroke elderly person? It was hypothesized that for post-stroke elderly there would be an inverse correlation found between depression and perceived enactment of autonomy scores. A correlational analysis found that there was a significant inverse relationship between depression and perceived enactment of autonomy scores (r = -.79) supporting the hypothesis. A second research question was: How does the elderly person describe feelings about rehabilitation, opportunities to make decisions and thoughts about changing one life event? A guided interview was created to elicit a descriptive response to this question by the older adult. A third and fourth research question asked: Are there gender differences on depression and perceived enactment of autonomy scores? An analysis of variance found no significant differences. A final question asked: What relationship is there between the area of infarct and the level of depression and perceived enactment of autonomy scores? A statistically, significant difference was found between hemisphere of infarct and depression scores (F (19) = 11.14, p < .05). The findings supported the hypothesis of the study and suggested that future research include the perception of being able to enact self-determined behavior among the post-stroke elderly. Research on the multi-faceted etiology of depression needs to be continued related to the economic, social, psychological and physical impact post-stroke depression can have on the progress of rehabilitation.
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    Baroreceptors buffer decreased mean arterial blood pressure during positive pressure ventilation by adjusting heart rate and total peripheral vascular resistance

    Blevins, Stephen S. (1995)
    The role of arterial baroreceptors in maintaining mean arterial blood pressure was evaluated when intermittent positive pressure ventilation with and without positive end-expiratory pressure (PEEP) was used to ventilate dogs. The decrease in mean arterial pressure (MAP) during mechanical ventilation is a result of decreased cardiac output which is a result of a restriction to venous return. Dogs had monitoring devices applied so hemodynamic variables could be measured under 4 ventilatory conditions: spontaneous, intermittent positive pressure (IPPV), IPPV with PEEP (5 & 10 cmH2O), and recovery (spontaneous) in both an open-loop (carotid sinus feedback removed) and closed-loop (feedback intact) condition. Responses were also compared before and after bilateral cervical vagotomy which eliminates afferent input from cardiopulmonary receptors. When the animals were neurally intact, the initiation of IPPV resulted in no change in MAP due to a significant increase in heart rate. With the application of PEEP, there was a significant decrease in MAP which was buffered by significant increases in heart rate and vasomotor tone as compared to denervated animals. Either carotid sinus or cardiopulmonary baroreceptors were capable of regulating and maintaining an adequate MAP but were not capable of restoring cardiac output and oxygen delivery. The effects on the cardiovascular system during the use of IPPV and PEEP dictates close monitoring of patients for decreased cardiac output.
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    Identification and characterization of a novel neurofilament-associated kinase and studies on calcium/calmodulin kinase in Alzheimer's disease

    Xiao, Jinsong (1995)
    Neurofilaments (NF), the major cytoskeletal component in neuronal cells, are one of the most highly phosphorylated proteins expressed in brain. Apart from the structural role NF play in maintaining neuronal architecture, little else is known of their function. I describe here evidence suggesting that NF may support many other proteins in the neuronal axoplasm including protein kinases. In order to isolate proteins that bind NF, I first expressed the carboxyl-terminal tail domain of the mouse heavy-molecular-weight neurofilament subunit (NF-H) as a fusion protein in bacteria and then used this portion of NF-H as a ligand in affinity chromatography. A number of different proteins were isolated, from mouse brain lysate, that specifically bound to the NF-H column and which did not bind to a control column to which BSA was bound. The proteins eluted from the NF-H column contained kinases able to efficiently phosphorylate NF proteins in vitro. I characterized these kinases further by separating proteins on denaturing polyacrylamide gels and reconstituting kinase activity in situ. Using this assay I identified a number of individual kinases including a 115 kDa polypeptide which showed a significant preference for NF proteins as substrate. Native NF was found to be the best substrate for the 115 kDa kinase, followed by a bacterially expressed NF-H non-fusion protein, and NF-H fusion protein. However, NF-L was a poor substrate. Two different NF monoclonal antibodies, SMI31 and SMI32 (Sternberger Monoclonal Inc.) were used to further demonstrate that the 115 kDa kinase is associated with NF in vivo. The kinase was co-immunoprecipitated along with NF by the two NF monoclonal antibodies but appeared to be preferentially associated with phosphorylated forms of NF. I discuss here some of the novel properties of the 115 kDa NF-associated kinase I have termed NAK115 (for NF-associated kinase with a molecular mass of 115 kDa). Other biochemical properties of NAK115 were also studied. It appears to be a peripheral membrane protein with a pI of 5.4-6.2, and it is expressed at different levels in a variety of mouse tissues. NAK115 exists as a large 570 protein complex in vivo. Purification of NAK115 was also explored. After four steps of purification, NAK115 was enriched around 20 fold.;Alzheimer's disease (AD) is characterized pathologically by two distinguishable deposits in brain, namely senile plaques and neurofibrillary tangles (NFT). Senile plaques are composed of fragments of the amyloid precursor protein, whereas NFT are composed primarily of paired-helical filaments (PHF). The latter are in turn composed principally of the microtubule-associated tau protein. Tau in PHF is highly and unusually phosphorylated. However, neither the mechanism nor the identity of the protein kinases or phosphatases that govern this unusual phosphorylation is known. Using a combination of immunoblotting and kinase assays, I demonstrate that a discreet set of kinases co-purify with PHF. One of these kinases was found by immunoblotting to be {dollar}\alpha{dollar}-calcium-calmodulin dependent kinase II {dollar}(\alpha{dollar}-CaM kinase). Immunogold labeling revealed that {dollar}\alpha{dollar}-CaM kinase was localized to a novel globular structure found at the ends of PHF. Since previous studies have shown {dollar}\alpha{dollar}-CaM kinase to be involved in memory, its association with PHF may have important implications in understanding memory loss in AD. I also discuss the possibility that the association of {dollar}\alpha{dollar}-CaM kinase with PHF may indicate sites where tau protein is converted into PHF.
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    An economic and outcome evaluation of the Pharmaceutical Care Services Program for Maryland Medicaid recipients

    Lai, Li-An Leanne (1996)
    The goal of this research was to determine if the Pharmaceutical Care Services Program (PCSP) significantly changes the utilization and cost of medical services for Medicaid recipients. The University of Maryland Center on Drug and Public Policy (CDPP) has contracted with the Maryland Department of Health and Mental Hygiene (DHMH) to provide pharmaceutical care services for Maryland Medicaid recipients. The main goal of PCSP is to improve the appropriateness and cost-effectiveness of physician prescribing decisions and patient drug use. The program accomplishes this by placing clinical pharmacists in hospital clinics to review drug therapy and provide advice to physicians and counseling to patients. A quasi-experimental pretest and post-test design with three control groups was performed in this study. The subjects who received PCSP and met the study criteria were the study group. The subjects who were Medicaid enrollees and relatively similar to the study subjects in terms of age, sex, hospital, and ACG (ambulatory care group) classification were randomly selected into the control groups by using a multiple computerized matching process. Differences in utilization and cost of medical services between study and control groups were tested for statistical significance. Cost-benefit analyses were then performed from budgetary and societal perspectives by applying a net present value method. The mortality and morbidity productivity loss estimations were specifically addressed as the indirect benefits in this study. Lastly, a sensitivity analysis was performed to test the assumptions (discount rate and wage rate) underlying the analysis. In this study, the utilization and costs comparisons between the PCSP patients and control patients revealed that: (1) PCSP "capped" the total cost of services by holding them constant while the control groups' costs rose sufficiently to create a significant difference between the PCSP and control groups; (2) specialty care physician visits remained stable for PCSP while increasing in the control groups; (3) primary care physician visits remained stable for PCSP recipients while they declined in the control groups; (4) less prescription medication was used in PCSP group than in the control group; (5) while the total cost of prescriptions increased for both groups, the cost of PCSP prescriptions was less than the control group cost; (6) PCSP showed no significant impact on the use of the emergency room and hospitalizations. The cost-benefit analysis illustrates that PCSP saved the Medicaid program $204.32 per patient for the first year intervention and $2,043.20 for the future 10 years from a budgetary perspective. From a societal perspective, PCSP saved society $4,116.01 per patient for the future 10 years period. Theoretically, if PCSP were expanded to serve all Medicaid, adult, non-institutionalized patients receiving drug therapy, the state of Maryland should be able to save as much as $27 million in the next fiscal year from a budgetary perspective. The society should be able to save as much as $259 million in the next fiscal year from both direct and indirect savings.
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    Transcriptional regulation of Proteus mirabilis urease by UreR

    Nicholson, Eric Brice (1995)
    Proteus mirabilis is the most frequent cause of infection-induced bladder and kidney stones. P. mirabilis urease catalyzes the hydrolysis of urea thereby raising the pH of urine and initiating formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Genes necessary for urease activity, ureD,A,B,C,E,F, have previously been cloned and sequenced (Jones, B. D. and H. L. T. Mobley. 1989. J. Bacteriol. 171:6414-6422). Purified whole cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences probed with a 4.2 kb Bg/I fragment from within the urease operon revealed a 4.2-fold and 5.8-fold increase in urease-specific mRNA upon induction with urea. Northern blot analysis of whole-cell RNA from wild-type P. mirabilis HI4320, probed with the same 4.2 kb DNA segment revealed significantly higher amounts of mRNA transcripts of 2.8 and 3.6 kb when induced with urea which correspond to the predicted lengths of mRNA encoding transcripts of ureABCE and ureDABCE, respectively. However, the presence of putative promoters preceding both ureD and ureA makes this interpretation difficult. A 1.2 kb EcoRV-BamHI restriction fragment upstream of the urease gene sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame 400 bp upstream of ureD consisting of 882 nucleotides, designated ureR, which is transcribed in the opposite direction of the urease structural and accessory genes, and encodes a 293 amino acid polypeptide predicted to be 33,415 Da. Autoradiographs of SDS-polyacrylamide gels of ({dollar}\sp{lcub}35{rcub}{dollar}S) -methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band of 32 kDa apparent molecular size; fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence shares amino acid similarity to a number of DNA binding proteins including E. coli regulatory proteins for acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). Subsequent analysis of this sequence revealed a potential helix-turn-helix motif and an AraC family signature (M. D. Island and H. L. T. Mobley, personal communication). These data suggest that UreR governs inducibility of P. mirabilis urease. To study the specificity of induction of urease, the ability of urea analogs and urease inhibitors to induce the enzyme was studied by constructing a fusion of ureA (urease subunit gene) and lacZ ({dollar}\beta{dollar}-galactosidase gene) within plasmid pMID1010 which encodes an inducible urease of P. mirabilis expressed in E. coli JM103 (Lac). The fusion protein, predicted to be 117 kDa, was induced by urea and detected on Western blots with anti-{dollar}\beta{dollar}-galactosidase antiserum. Induction was specific for urea as no structural analog of urea induced fusion protein activity. These data suggest that induction by UreR, the protein that governs regulation of the urease operon, is exquisitely specific for urea and does not respond to closely related structural analogs.
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    The meaning, process, and consequences of nurse caring as perceived by spinal cord injured individuals during rehabilitation

    Lucke, Kathleen T. (1995)
    The purpose of this qualitative study was to describe the meaning, process, and consequences of nurse caring from the perspective of spinal cord injured (SCI) individuals while in rehabilitation. This study addressed the following research questions: (1) What is the meaning of nurse caring to SCI individuals in rehabilitation? (2) How is the process of developing a caring relationship perceived by SCI individuals during rehabilitation? (3) What are the consequences of nurse caring for SCI individuals in rehabilitation? The theoretical foundation of this study was synthesized from philosophical, ethical, feminist, and nursing literature. A purposive sample of adults with traumatic SCI were interviewed at least once during their initial rehabilitation admission. Only individuals who could speak and understand English were included in the study. Individuals with all levels of injury, both complete and incomplete lesions, were included; individuals with a documented head injury or cognitive deficits were excluded. In-depth, tape recorded interviews with participants were conducted after receiving informed consent. The constant comparative method of Glaser and Strauss was used for collection and analysis of data; appropriate strategies were used to insure scientific credibility. Twenty interviews were conducted with fifteen SCI individuals at various times during their rehabilitation stay. Interviews were conducted over a six month period at two free-standing rehabilitation centers in southwestern Pennsylvania. The core category of "getting back together" or reintegration of self, which was the major work of rehabilitation, was accomplished with nurses and therapists who were perceived as caring. Four dimensions of caring were important to SCI participants in rehabilitation: knowledge, technical skill, interpersonal skill, and competence. The process of a developing caring relationship was conceptualized, from participants' descriptions, in three phases: learning the other, learning what I need to know, and letting me find out. Consequences of nurse caring for SCI individuals were: well-being, self-care, autonomy, independence, and hope. Nurse caring is perceived by SCI individuals in rehabilitation as central to recovery and to a positive attitude toward disability. Concepts described in this study can be used to develop an instrument to measure nurse caring and to teach nurses and nursing students important aspects of caring in rehabilitation.
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