Multiple Genes Encoding the Conserved CCAAT-Box Transcription Factor Complex Are Expressed in Arabidopsis

The CCAAT motif is found in the promoters of many eukaryotic genes. In yeast a single complex of three proteins, termed HAP2, HAP3, and HAP5, binds to this sequence, and in mammals the three components of the equivalent complex (called variously NF-Y, CBF, or CP1) are also represented by single genes. Here we report the presence of multiple genes for each of the components of the CCAAT-binding complex, HAP2,3,5, from Arabidopsis. Three independent Arabidopsis HAP subunit 2 (AtHAP2) cDNAs were cloned by functional complementation of a yeast hap2 mutant, and two independent forms each of AtHAP3 and AtHAP5 cDNAs were detected in the expressed sequence tag database. Additional homologs (two of AtHAP3 and one of AtHAP5) have been identified from available Arabidopsis genomic sequences. Northern-blot analysis indicated ubiquitous expression for each AtHAP2 and AtHAP5 cDNA in a range of tissues, whereas expression of each AtHAP3 cDNA was under developmental and/or environmental regulation. The unexpected presence of multiple forms of each HAP homolog in Arabidopsis, compared with the single genes in yeast and vertebrates, suggests that the HAP2,3,5 complex may play diverse roles in gene transcription in higher plants.

Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: Potential as EphA3-specific tumor-targeting reagents

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the low-affinity ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.

Use of the DACUM process in the identification and verification of competencies for the medical administrative assistant

Medicine has changed in recent years. Medicare will all of its rules and regulations, worker's compensation laws, managed care and the trend toward more and larger group practices all contributed to the creation of an extremely structured regulatory environment which in turn demanded highly trained medical administrative assistants.^ The researcher noted three primary problems in the identification of competencies for the medical administrative assistant position: A lack of curricula, diverse roles, and a complex environment which has undergone radical change in recent years and will continue to evolve. Therefore, the purposes of the study were to use the DACUM process to develop a relevant list of competencies required by the medical administrative assistant practicing in physicians' offices in South Florida; determine the rank order of importance of each competency using a scale of one to five; cross-validate the DACUM group scores with a second population who did not participate in the DACUM process; and establish a basis for a curriculum framework for an occupational program.^ The DACUM process of curriculum development was selected because it seemed best suited to the need to develop a list of competencies for an occupation for which no programs existed. A panel of expert medical office administrative staff was selected to attend a 2-day workshop to describe their jobs in great detail. The panel, led by a trained facilitator, listed major duties and the respective tasks of their job. Brainstorming techniques were used to develop a consensus.^ Based upon the DACUM workshop, a survey was developed listing the 8 major duties and 71 tasks identified by the panel. The survey was mailed to the DACUM group and a second, larger population who did not participate in the DACUM. The survey results from the two groups were then compared. The non-DACUM group validated all but 3 of the 71 tasks listed by the DACUM panel. Because the three tasks were rated by the second group as at least "somewhat important" and rated "very important" by the DACUM group, the researcher recommended the inclusion of all 71 tasks in program development for this occupation. ^

Literacy coaching roles in diverse contexts of teaching and learning : New ways of working

As the demands placed on the literacy coach have evolved, so too have the roles of these educational providers who are often responsible for working with school teams to turn around student performance on standardized literacy tests. One literacy coach based in a Queensland primary school recounts her experiences via open-ended interview over a two year period. We offer a theorisation of the new ways of working as a literacy coach in a context of teaching and learning marked by diversity.

VERSATILITY IN THE MODERN PERFORMER: THREE DIVERSE OPERA ROLES

This dissertation project consisted of performances of three diverse operatic roles. The goal was to examine the challenges and benefits of performing diverse repertoire (baroque, classical, and romantic opera, in this case) and to observe how vocal and acting techniques might adjust to different styles and voice classifications. On December 3, 5, 11, and 13,2003, I performed a composite role in Fatal Song, directed by Leon Major, in the Robert & Arlene Kogod Theatre. This opera pastiche, premiered in 1996, contains spoken dialogue, and the music originates from famous eighteenth and nineteenth-century operas. I explored the challenges and benefits of playing three different, well-known opera characters within the same opera, performing four different languages within the same opera, and performing spoken dialogue in an opera. My roles in this pastiche of famous opera included Lucia from Lucia di Lammermoor by Gaetano Donizetti, Manon from Manon by Jules Massenet, and Pamina from Die ZauberJlote by W.A. Mozart. On January 28 and 29, 2005, I performed the role of Nice in Eurilla e Alcindo, a serenata by Antonio Vivaldi, in Homer Ulrich Recital Hall; musical direction by Joseph Gascho and stage direction by Kate Vetter Cain. I explored the challenges and benefits of preparing baroque ornamentation, singing without a conductor, performing an unknown work, staging a work that is not traditionally staged, singing with minimal sets and costumes, and singing with period instruments. On April 16,20, and 24,2005, I performed the role of Musetta in Giacomo Puccini's La BohBme, directed by Pat Diamond and conducted by Jeffrey Rink, in the Ina & Jack Kay Theatre. I examined the challenges and benefits of singing with a large orchestra of modem instruments, singing Puccini rubato and coordinating this with the conductor, and singing a role in one of the most well-known operas in today's repertoire. As a result of this investigation, I concluded that singing different styles, and singing outside one's voice classification, within reason, is beneficial. The knowledge, skill, confidence and insight I acquired by becoming more versatile benefited me as a performer and as a teacher.

Intergroup processes in intragroup contexts: The roles of group status, group identification and communication in diverse work teams

The present research examines employee identification and communication in organisations. In Study 1, 2229 soldiers from a military organisation completed measures of perceived status and strength of identification with their unit, employment category and their brigade. As predicted, the status of a key organisational group influenced reactions to different organisational groups: full-time soldiers evaluated their work unit and the organisation as being lower in status and identified less strongly with both of these groups than part-time soldiers. The second study extended these findings to a different research context: a large psychiatric hospital undergoing downsizing and restructuring. Surprisingly, there were no differences in survivors' and victims' levels of identification with organisational groups. Instead, and consistent with Study 1, there was evidence to suggest that employees adjusted their patterns of identification and perceptions of group status through a compensatory mechanism that maximised opportunities for selfenhancement and positive distinctiveness. In the third study, employees from a public hospital (N = 142) rated communication from double ingroup members (same work unit/same occupational group) more favourably than communication from partial group members (same work unit/different occupational group). These results are considered in terms of their practical implications for identity management in organisations.

Beyond water homeostasis:diverse functional roles of mammalian aquaporins

Background - Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review - AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions - As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance - Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.

Role demands and work-family balance experience in Malaysia : the different moderating effects of collectivism and gender role identity among diverse ethnic groups

The purpose of this thesis is to examine the influence of ethnic cultural values on the relationship of role demands and the work-family balance (WFB) experience. Past studies have found that the demands from work and family roles have a different impact on the work-family experience in people of different ethnicity. Researchers attribute these results to the cultural differences across the groups. However, there has been no empirical support for these assumptions because most past studies did not explicitly measure the cultural dimension in their design. Therefore, although studies have found ethnic differences in work-family experience, as cultural variables were not measured, it cannot be determined whether these differences were due to the differing ethnic groups’ cultural styles. The present thesis is set up to address this limitation in the literature, employing the Malay and Chinese ethnic groups in Malaysia as the study samples. The investigation consisted of pilot interviews and two survey studies. The interviews were carried out to establish the perception of WFB by target participants of a non-western nation. The first survey served to identify whether the Malay and Chinese ethnic groups residing under the same economic and social systems vary in their perceptions of work and family roles. The second survey tests the research model empirically, that is, whether cultural values moderate the relationship between role demands and WFB and if these moderation effects vary across ethnic groups. From the interviews, the results indicated that work-family experience is not a universal experience, but is partly culture-specific. Specifically, in the case of Malaysia, WFB is very much observed from the role obligation perspective. In particular, balance is perceived when work duties and household affairs are both adequately fulfilled. On the other hand, the conceptualisation of WFB in terms of role satisfaction and role interference also emerged in the interviews, suggesting the universality of these constructs across cultures. The findings from Survey One indicated that participants of different ethnicities in this study do not differ greatly in their perceptions regarding their participation in work and family roles. Generally, these participants revealed the less traditional attitudes towards women’s participation in work and family roles. However, variations were observed between the two groups in terms of reasons for working, spouses’ preferences towards their employment, and the extent to which their work role is perceived to impede their normative role performance in the household. Despite these differences, the Malay and Chinese ethnic groups showed more similarities than differences in their perceptions of work and family. The findings from Survey Two, which tested the research model, produced mixed results. On the whole, the results showed that the cultural dimensions examined in this study (i.e. collectivism, work identity and family identity) did influence the relationship between role demands and WFB experience, thus providing empirical evidence for the assumption in the literature that the relationship between role demand and work-family experience is moderated by cultural values. Most importantly, support was found for the proposition that these moderation effects vary between the Malay and Chinese ethnic groups. Moreover, this study also found evidence that Malays and Chinese differ significantly on collectivism and work identity cultural dimensions where Malays are found to be more collectivist than the Chinese, while work identity is stronger in the Chinese than in the Malays. There is no difference in the levels of family identity between the two groups. Of all the three moderators, work identity was deemed the most important because many of the supported hypotheses pertained to the work identity moderating effects. In contrast, family identity does not seem to have much moderating influence on role demand-WFB relationships, while the results for the collectivism moderator are mixed. As such, although not conclusive, it can be deduced that variations in the effects of role demand on work-family experience across ethnicity are a result of the groups’ cultural differences, thereby supporting the assumption in the literature.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

Roles, responsibilities and scope of practice: describing the ‘state of play’ for infection control professionals in Australia and New Zealand

Background In the past decade the policy and practice context for infection control in Australia and New Zealand has changed, with infection control professionals (ICPs) now involved in the implementation of a large number of national strategies. Little is known about the current ICP workforce and what they do in their day-to-day positions. The aim of this study was to describe the ICP workforce in Australia and New Zealand with a focus on roles, responsibilities, and scope of practice. Methods A cross-sectional design using snowball recruitment was employed. ICPs completed an anonymous web-based survey with questions on demographics; qualifications held; level of experience; workplace characteristics; and roles and responsibilities. Chi-squared tests were used to determine if any factors were associated with how often activities were undertaken. Results A total of 300 ICPs from all Australian states and territories and New Zealand participated. Most ICPs were female (94%); 53% were aged over 50, and 93% were employed in registered nursing roles. Scope of practice was diverse: all ICPs indicated they undertook a large number and variety of activities as part of their roles. Some activities were undertaken on a less frequent basis by sole practitioners and ICPs in small teams. Conclusion This survey provides useful information on the current education, experience levels and scope of practice of ICPs in Australia and New Zealand. Work is now required to establish the best mechanisms to support and potentially streamline scope of practice, so that infection-control practice is optimised.

From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.