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A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins

Journal of Virological Methods 173 (2011) 175–181

Contents lists available at ScienceDirect

Journal of Virological

Methods

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j v i r o m e

A dendritic cell-based assay for measuring memory T cells speci?c to dengue envelope proteins in human peripheral blood

Peifang Sun a ,?,Charmagne Beckett b ,Janine Danko b ,Timothy Burgess b ,Zhaodong Liang a ,Tadeusz Kochel b ,Kevin Porter b

a Henry Jackson Foundation for the Military Service,Rockville,MD,USA

Department of Viral and Rickettsial Diseases,Naval Medical Research Center,Silver Spring,MD,USA

Article history:

Received 15October 2010

Received in revised form 20January 2011Accepted 31January 2011

Available online 18 February 2011Keywords:Dengue

Human T cell

Human dendritic cell Interferon ELISPOT Dengue E protein

a b s t r a c t

Dengue envelope (E)protein is a dominant immune inducer and E protein-based vaccines elicited par-tial to complete protection in non-human primates.To study the immunogenicity of these vaccines in humans,an enzyme linked immunospot (ELISPOT)assay for measuring interferon gamma (IFN-?)pro-duction was developed.Cells from two subject groups,based on dengue-exposure,were selected for assay development.The unique feature of the IFN-?ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells.IFN-?production,ranging from 53–513spot forming units per million peripheral blood mononuclear cells (PBMCs),was observed in dengue-exposed subjects as compared to 0–45IFN-?spot forming units in dengue-unexposed subjects.Further,both CD4+and CD8+T cells,and cells bearing CD45RO memory marker,were the major sources of IFN-?production.The assay allowed quanti?cation of E-speci?c IFN-?-secreting memory T cells in subjects 9years after exposure to a live-attenuated virus vaccine and live-virus challenge.Results suggested that the dendritic cell-based IFN-?assay is a useful tool for assessing immunological memory for clinical research.

1.Introduction

Dengue viruses belong to genus Flavivirus.They are positive-sensed single-stranded RNA viruses consisted of 4serologically distinct serotypes,Dengue-1,-2,-3and -4.The viruses are trans-mitted to humans through mosquito vectors,Aedes aegypti and Aedes albopictus ,and are endemic in more than 100countries in tropical and sub-tropical regions of the world.It is estimated that over three billion people (1/3of the world’s population)live in risk areas of dengue exposure,and 50million clinical infections and over 500,000deaths are reported each year (Mukhopadhyay et al.,2005;Thomas et al.,2003).Despite countless efforts,there are no licensed vaccines or drugs.

The major virion surface protein,the E protein,is a 55–60kDa glycoprotein of 494–501amino acids long.The protein is respon-sible for virus attachment to target cells,such as human dendritic cells and macrophages.It mediates virus-speci?c membrane fusion in the acidic endocytic vesicles of the host cell,leading to the release of viral genome into the cytoplasm to initiate its intracel-lular replication cycle (Mukhopadhyay et al.,2005).This protein

?Corresponding author at:Naval Medical Research Center,503Robert Grant Ave.,Silver Spring,MD 20910,USA.Tel.:+13013197446;fax:+13013197451.

E-mail address:Peifang.sun@https://www.wendangku.net/doc/2a17967269.html, (P.Sun).is also a strong immunogen capable of eliciting long-lasting neu-tralizing antibodies and cellular immunity (Mukhopadhyay et al.,2005).Due to its unique property,the E protein is considered to be an important vaccine component.DNA vaccines expressing dengue serotype-speci?c Pre-membrane (PreM)and E genes elicited partial to full protection in non-human primates (Blair et al.,2006;Kochel et al.,2000;Raviprakash et al.,2000).Currently,antibody neutral-ization is recommended by the World Health Organization (WHO)as the most relevant mechanism of immune protection,so that in vitro evaluation of vaccine potency in humans relies heavily on the plaque reduction neutralization test (PRNT)(Hombach,2009).However,results from animal studies highlighted the urgency to discover novel protective mechanisms aside from the current stan-dard PRNT,since signi?cant numbers of the non-human primates were protected without detectable serum neutralization antibod-ies (Raviprakash et al.,2000).The focus of this study was to develop an assay that allows exploration of dengue-speci?c cell-mediated immunity.

Dengue-speci?c T cell responses have been characterized in a number of studies.Long-term memory responses of dengue-speci?c T cells were detected 20years after only one exposure (Sierra et al.,2002).Dengue-speci?c cytotoxic T cells (CTLs)are found in endemic donors during or after convalescent period as well as in dengue vaccinated donors (Kurane et al.,1989;Mongkolsapaya et al.,2003).On the other hand,IFN-?-production

176P.Sun et al./Journal of Virological Methods173 (2011) 175–181

Table1

Volunteer information.

Study subject#Previous vaccine/challenge Additional information Vaccine Challenge Vaccine date Challenge date

837-089a–Dengue-3–5/24/2002Na?ve challenged with dengue-3 1337-039a

837-090–Dengue-1–5/24/2002Na?ve challenged with dengue-1 1337-004TLAV b–11/30/2001–Serum positive pre-vaccination 1337-024TLAV–6/20/2006–

1337-034TLAV–6/20/2006–

1337-028TLAV–6/20/2006–

–:None or not applicable.

a Same person.

b Tetravalent live-attenuated virus vaccine.

by peptide-speci?c CD8+T cells are more frequently detected among hospitalized dengue hemorrhagic fever patients than among dengue patients(Sierra et al.,2002).In lymphocytic chori-omeningitis virus and in?uenza infections,T cells clearly contribute to immunopathology,but depletion of T cells delays the clear-ance of viral infection.Currently,low cost small animal models are not yet available to model human diseases from dengue infection. Therefore,?eld prospective cohort studies and vaccine models are precious alternative ways to investigate the types,the magnitude and other characteristics of T cell immune response with respect to its anti-viral or immuno-pathological roles.

The ELISPOT method reported in this study uses human den-dritic cells as antigen presenting cells and E proteins as antigens. The assay is primarily designed for clinical vaccine trials,but can be used for basic clinical https://www.wendangku.net/doc/2a17967269.html,ing this assay,IFN-?responses were detected in subjects9years after exposure to dengue viruses or vaccines.

2.Materials and methods

2.1.Human samples

De-identi?ed blood samples were collected from healthy volun-teers enrolled in two approved consecutive human use protocols: the former project WRAIR#837subproject#018and a current WRAIR#1337subproject4BR.The corresponding institutional approvals were NMRC.2005.0014and NMRC.2007.0009.A ques-tionnaire was provided to each blood donor to document his/her previous exposure to dengue,Yellow Fever,Japanese encephali-tis,West Nile,and Tick-borne encephalitis,at enrollment.Serum samples were collected and screened for serological responses to a panel of antigens including dengue(all4serotypes),Yellow Fever,West Nile,and Japanese encephalitis using an enzyme-linked immunoassay(ELISA).Subjects who reported previous exposure to dengue were traced back to previous trials using their study identi?cation number(ID).Table1shows a list of volunteers who previously participated in clinical vaccine/challenge trials of tetravalent live-attenuated vaccines.These donors were all sero-logically positive to dengue viruses,and thus their blood samples were used as positive controls for assay development.Blood sam-ples from subjects who reported no previous exposure to any of the above viruses or vaccines,and who showed negative serologi-cal responses to the above viral antigens,were selected as negative controls.As negative control donors have no other speci?cation, they were not listed in Table1;instead,they were referred collec-tively to as dengue-unexposed(or non-immune)donors.

2.2.PBMC isolation

PBMCs were isolated from peripheral blood on Ficoll-Paque TM (GE Healthcare Bio-Sciences AB,SE-75184Uppsala,Sweden)den-sity gradient centrifugation and stored in liquid nitrogen until use.

2.3.Cell culture medium

All cell cultures were carried out in medium referred to as“Complete Medium”.The Complete Medium was the RPMI 1640(w/o l-Glutamine)medium(Mediatech,Inc.,Manassas,VA) supplemented with1%penicillin(stock solution10,000IU/ml), 1%streptomycin(stock solution10,000?g/ml)(Invitrogen Corp., Frederick,MD),1%l-glutamine(stock200mM in0.85%NaCl) (Mediatech,Inc.),1%non-essential amino Acid(Mediatech,Inc.), and10%heat-inactivated fetal bovine serum(Hyclone Laboratories, Inc.,Logan,Utah).

2.4.Preparation of dendritic cells

PBMCs were thawed and separated into adherent and non-adherent cells by placing whole PBMC into surface-modi?ed polystyrene non-pyrogenic6-well plate(Primaria TM Tissue culture plates,BD Bioscience,San Jose,CA)for2h in a37?C5%CO2incu-bator.After washing the plates several times with plain RPMI1640 (not supplemented with additives such as fetal bovine serum and antibiotics),adherent cells and non-adherent cells were separated. The adherent cells,consisted of mainly CD14+monocytes,were cul-tured in the presence of10ng/ml of recombinant human IL-4(rIL-4) and rGM-CSF(R&D Systems,Minneapolis,MN)in a37?C5%CO2 incubator.After a period of5or6days,cells were differentiated into dendritic cells as described previously(Palmer et al.,2005).This method of dendritic cell preparation is a standard method which ensures a>95%pure dendritic cell population with<5%contamina-tion of T cells(CD3+),B cells(CD20+)and monocytes(CD14+).The cell surface expression of the dendritic cell lineage marker CD1a, HLA class I and II,and co-stimulatory markers CD80,CD40,CD86 were shown in Supplementary Fig.1.At the day of ELISPOT assay (day6),dendritic cells were collected,washed,counted on a Guava cell counter(Guava Technologies,Inc.,Hayward,CA)and adjusted to106/ml,and then pulsed with E proteins(80%of whole E protein sequence,Hawaii Biotech,Inc.,Aiea,HI)at20ug/ml for3h.After antigen pulsing,dendritic cells were ready to be used as antigen presenting cells in the T cell ELISPOT assay.

2.5.Dendritic cell-based IFN- ELISPOT assay

A dendritic cell-based IFN-?ELISPOT assay was developed.This assay used E protein-pulsed dendritic cells as antigen present-ing cells(described in Section2.4)and non-adherent cells freshly separated from whole PBMCs as the source of T cells.Brie?y,non-adherent cells were co-cultured with E protein-pulsed dendritic cells in an ELISPOT plate pre-coated with IFN-?capture anti-body(Mabtec,Inc.,Cincinnati,OH).Each culture should contain

P.Sun et al./Journal of Virological Methods173 (2011) 175–181177 2–4×106of non-adherent cells and2–4×105E protein-pulsed

dendritic cells(the ratio of dendritic cells:PBMCs=1:10).After a

20-h culturing period,plates were emptied by washing6times

with PBS–Tween20(0.05%).Washing was done manually by adding

200?l of washing buffer and then decanting the washing buffer.

IFN-?detection monoclonal antibody(Mabtec,Inc.)at1:1000dilu-

tion was added to the plates for a period of3h at room temperature.

After removing the detection mAb by washing,horse-radish per-

oxide(Mabtec,Inc.)was placed to the plates for2h at room

temperature.Following additional washing,AEC substrate(Vec-

tor Lab,Burlingame,CA)was added.When pinkish spots emerged

and stabilized in the plate,the assay was stopped and spots were

counted using an automated AID ELISPOT Reader(Autoimmun

Diagnostika GmbH,D-72479Strassberg,Germany).E proteins of

all4serotypes were used for most of the ELISPOT assays.Dendritic

cells pulsed with phosphate buffered saline(PBS)or Staphylococ-

cus aureus Cowan(SAC)(Sigma–Aldrich,St.Louise,MO),served as

negative and positive controls,respectively.

2.6.Conventional ELISPOT assay

The conventional ELISPOT assay was carried out by adding

directly E proteins to whole PBMCs(containing both adherent and

non-adherent cells)in an ELISPOT plate pre-coated with anti-IFN-?

monoclonal antibody(Mabtec,Inc.).The cell cultures were main-

tained for about44h in37?C5%CO2incubator.The plates were

then washed to remove cells and IFN-?spots were developed as

described in Section2.5.

2.7.Depletion of CD45RO+,CD4+or CD8+cells

Miltenyi microbeads(Miltenyi Biotec,Auburn,CA)were used

to deplete CD45RO+(kit130-046-001),CD4+(kit130-045-101)

and CD8+(kit130-045-201)cells.Brie?y,cells for depletion were

labeled with a speci?c monoclonal Ab which was later conjugated

to microbeads.Microbeads-attached cells were removed by a mag-

netic Miltenyi LS column(Miltenyi Biotec)and discarded,whereas

unlabeled cells were eluted from the column and collected for

use.To determine the ef?ciency of cell depletion,we stained the

eluted cells with appropriate?uorescent monoclonal antibodies:

CD4,CD8,CD45RO(all from BD Bioscience),and analysis of cells

was carried out on a?ow cytometry.

2.8.Data analysis

All cultures were set in triplicates.An antigen-speci?c response

was determined by subtracting the average number of spot form-

ing units in PBS control cultures from the average number of spot

forming units in antigen-stimulated cultures;this number was nor-

malized further to spot forming units/106cells based on input cell

number of each culture.The formula was:

spots in antigen-stimulated culture?spots in PBS culture

number of cells per culture

×106

Final data were presented as number of antigen-speci?c spot forming units/million PBMCs.

3.Results

3.1.Dendritic cell-based ELISPOT assay results in

dengue-immune and non-immune subjects

Using the dendritic cell-based ELISPOT assay,IFN-?responses in dengue-exposed and unexposed subjects were evaluated.Fig.1a shows an image of IFN-?spots forming units from two donors rep-resenting dengue-exposed and-unexposed groups,respectively.The IFN-?production was evaluated against all4serotypes of E proteins.Among the responses elicited by4serotypes of antigens, we used the highest response of each donor to calculate the group average.As shown in Fig.1b and c,the group of dengue-exposed subjects showed a higher IFN-?response which ranged from53to 513spot forming units/106cells with the group average of202spot forming units/106cells;whereas the group of dengue-unexposed subjects showed a much lower response which ranged from0to 45spot forming units/106cells with the group average of11.6spot forming units/106cells.Dendritic cells pulsed with E proteins with-out the addition of T cells produced0spots in most cases;only occasionally,1–2spots were observed(data not shown).Note that the089and the039were the same person who enrolled twice into two consecutive studies,the studies1337and837.This particular case was not known because subjects’vaccine identi?cations were not revealed until all the data in this study were collected.There-fore,results for the089and the039were reported independently as if they were two different subjects.

3.2.Assay reproducibility and sensitivity

Samples from3donors were selected to test the reproducibility of the ELISPOT assay for IFN-?production.The assays were per-formed on several different dates.The dendritic cell-based assay was reproducible,as results from a same sample showed a con-siderable level of consistency in several repetitive tests performed at different days(Fig.2a).Further,4donor PBMCs were used in the side-by-side comparison of the conventional and dendritic cell-based ELISPOT assay(Fig.2b).The dendritic cell-based assay appeared to be more sensitive as it produced higher IFN-?spot forming units than that of the conventional method.A paired Stu-dent’s t-test was used to statistically analyze the difference of the IFN-?responses generated by the conventional and the dendritic cell-based IFN-?ELISPOT assays.The probability(p)values were 0.007,0.009,0.045,and0.054for the D1E,D2E,D3E,and D4E, respectively;suggesting that the dendritic cell-based ELISPOT assay is signi?cantly better than the conventional ELISPOT assay.

3.3.Characterization of IFN- response

Since dendritic cells are potent antigen presenting cells with strong priming capability,it is unclear whether the IFN-?responses detected with the dendritic cell-based ELISPOT assay were artifacts of in vitro priming or a true ex vivo recall of memory responses. To investigate the contribution of memory cells for IFN-?produc-tion,the dendritic cell-based ELISPOT assays were performed in the absence of CD45RO+cells.As shown in Fig.3a and b,a greater than98%of CD45RO+cells was depleted from whole PBMCs,which resulted in a>80%loss of IFN-?responses,suggesting that the majority of the responses are not from in vitro priming,but from antigen stimulation of memory cells.Dengue E is a soluble protein, an exogenous antigen classically undergoes the MHC class II path-way for CD4+T cell stimulation(Watts,2004).To examine if the dendritic cells could present dengue antigens to both subsets of CD4+and CD8+T cells,the dendritic cell-based ELISPOT assay was performed in the absence of either subset of CD4+or CD8+T cells.As shown in Fig.3c,a greater than99%of either CD4+or CD8+subset was eliminated from whole PBMCs by magnetic bead-depletion.As shown in Fig.3d,in2out of3immune donors tested,depletion of either CD4+or CD8+T cell subset did not eliminate IFN-?response; suggesting that both CD4+and CD8+T cells are the source of IFN-?production.

178P.Sun et al./Journal of Virological Methods

173 (2011) 175–181

Fig.1.IFN-?responses in dengue-exposed and -unexposed subjects measured by the dendritic cell-based IFN-?ELISPOT assay.(a)An image of ELISPOT plate showing IFN-?spot forming units from a representative dengue-exposed and a dengue-unexposed donor.(b)IFN-?response from a group of dengue-exposed subjects;D1E,D2E,D3E and D4E are E proteins from each of the 4serotypes.Empty data space represents “not done”.(c)IFN-?response from a group of dengue-unexposed subjects.

3.4.The persistence of memory T cells in live-attenuated virus vaccine/challenge recipients

Using the ELISPOT assay,the quantity and the persistence of memory T cells were examined.Based on blood samples available during the study,2donors who received dengue live-attenuated virus vaccines and one donor who received live virus challenge (Table 1)were selected,and tested on 3time points.As shown in Fig.4,the memory T cells producing IFN-?in donor 1337-004,837-089,and 1337-024persisted for as long as 9,7and 4

years.

Fig.2.Assay reproducibility and sensitivity.(a)Repetitive assays were performed to assess the reproducibility of IFN-?ELISPOT assay.Number of assay repetition was indicated by “n ”.Responses to all 4serotypes were shown.Error bars are standard deviation.(b)Comparing the dendritic cell-based ELISPOT assay with the conventional ELISPOT assay.Data are from 4PBMC samples as indicated.Data for sample1337-039are the average of two experiments.The error bars are the standard deviation.

P.Sun et al./Journal of Virological Methods173 (2011) 175–181

179

Fig.3.Characterization of IFN-?responses.(a)Depletion of CD45RO cells shown by?ow-cytometry dot-plot images.The?gures shows a3-color staining scheme.Two separate gates were drawn on CD4and CD8subsets,and a third color showed the expression of CD45RO on each subset.(b)IFN-?responses from whole PBMCs(whole)and CD45RO-depleted PBMCs(CD45RO-)from3indicated immune subjects.All data are averages of2experiments.The error bars are standard deviation.(c)Depletion of CD4+ or CD8+cells shown by?ow-cytometry dot-plot images.The?gures shows a two-color-staining scheme,with the x-axis for CD4and y-axis for CD8.(d)IFN-?responses from whole PBMCs(whole),CD4-depleted(CD4-)or CD8-depleted(CD8-)PBMCs from3indicated immune subjects.All data are averages of2experiments except that of 837-089.The error bars are standard deviation.

Although decline of T cell response was observed in donor837-089 from year2006–2009,there seems to be no signi?cant decrease of IFN-?producing cells in the other two donors throughout the testing period.

4.Discussion

One of the major differences of the ELISPOT method compared to the published methods is the form of antigen.A number of vaccine and clinical?eld studies use live or inactivated viruses or infected Vero cell lysates as antigens for in vitro stimulation of memory T cell responses(de la et al.,2006;Edelman et al., 2003;Kurane et al.,1989).These forms of antigens are impure,ill-de?ned and likely stimulatory to innate immune cells.Potential problems of using these forms of antigens are low assay sensitivity,low speci?city,and low reproducibility.As reported previously,cell mediated immunity was evaluated for a Phase I clinical trial of35 volunteers receiving either2doses of monovalent or tetravalent live-attenuated virus vaccines(Gwinn et al.,2003).In this study, live viruses were used as antigens to stimulate human PBMCs and supernatants were collected for Th1/Th2cytokine characterization. Among30subjects who received monovalent live-attenuated vac-cine,16vaccine recipients showed IFN-?responses,and only CD4+ T cell cytokine production was detected.An alternative approach is the use of well-characterized peptide epitopes or over-lapping pep-tide pools.However,since the peptides are in large quantity,large number of cells could be required to test peptide pools and peptide variants(Vaughan et al.,2010).Furthermore,epitopes of dengue E protein are less well characterized compared to non-structural proteins.Whole protein antigens in the new assay system.

One

Fig.4.Quantity and longevity of E-speci?c IFN-?response.IFN-?responses from3immune subjects were measured at3times points during the indicated years.Responses were measured to D3E(837-089)and D4E(1337-004and1337-024).Data are averages of3experiments except those of837-089(year2006)and1337-024(year2007). The error bars are standard deviation.

180P.Sun et al./Journal of Virological Methods173 (2011) 175–181

possible problem of using E protein is the antigen processing and presentation,because soluble protein antigens may not be inter-nalized and processed as ef?ciently as particulate antigens and may not be presented as readily as small peptides(Corradin,1990; Watts,2004).Additionally,soluble proteins classically undergo the exogenous pathway for antigen processing and presentation that predominantly leads to CD4+T cell activation(Brodsky and Guagliardi,1991).Dendritic cells are characteristically potent anti-gen presenting cells since they express high levels of MHC and co-stimulatory molecules;they hold MHC–antigen complex on cell surface for a longer period of time(>2days);they are capable of pre-senting apoptotic cells or other cell-associated antigens on MHC class I molecules(cross-presentation)(Steinman,1991).Therefore, dendritic cells were used as antigen presenting cells to present protein antigens to stimulate T cells.The dendritic cell-based IFN-?ELISPOT assay detected dengue E-speci?c IFN-?responses in dengue-exposed individuals and from cells bearing the memory marker CD45RO,and from both subsets of CD4+and CD8+T cells. The dendritic cell-based assay showed good reproducibility and higher sensitivity compared to the conventional method.Note that this assay requires preparation of dendritic cells,which is an extra step comparing to the conventional method.However,techniques for preparation of dendritic cells are well established.In addition, while the adherent cells from PBMCs can be used to make dendritic cells,the non-adherent cells can be used for other purposes,such as studying of memory B cells and NK cells.

The major challenge for developing a more meaningful assay stems from the lack of understanding of the characteristics of pro-tective immunity.Dengue causes a self-limited acute febrile illness in primary infections.In endemic population,memory T cells and neutralization antibodies have been observed years after a single exposure,which confer life-long protection to infections from the homologous viruses.Severe forms of disease,the dengue hemor-rhagic fever(DHF)and the dengue shock syndrome(DSS),occur mostly in patients who are experiencing a secondary infection with an infecting serotype different from that of the previous expo-sure(Halstead and O’Rourke,1977).Cross-reactive immunity is postulated to enhance infection and cause“original antigenic sin”(Mongkolsapaya et al.,2003).It is not yet known how the T cell response contributes to immunopathology.Studies revealed that CD8+T cell clones obtained from persons with natural infection demonstrated cross-reactivity to peptide variants(Mongkolsapaya et al.,2003).In this study,although donor1337-089or donor 837-090was exposed to only one serotype,dengue-3or dengue-1,respectively;signi?cant amount of serotype cross-reactivity of memory T cells in these two donors was observed.For example, in donor1337-089,although serotype-speci?c IFN-?production to dengue-3was dominant,the cross-reactive IFN-?production to dengue-1and dengue-2were also high.Nevertheless,the study was not particularly designed to study serotype cross-reactivity; additional evaluation involving more monovalent immune sub-jects is needed.Further,T cell IFN-?responses to4seroytpes of E varied quantitatively in4donors vaccinated with the tetravalent live-attenuated virus vaccine.Again,more studies are needed as the current sample size is too small.

The dendritic cell-based ELISPOT assay was used to evaluate the duration of the memory T cells in persons who received the tetravalent live-attenuated virus vaccine and live virus challenge. Signi?cant amount of memory T cells circulating in peripheral blood were found nearly9years after the vaccination and chal-lenge.This observation is in accord with a previous report stating that anti-dengue long-term T cell proliferation was found in people 20years after an epidemic outbreak of dengue in Cuba(Sierra et al., 2002).

In summary,a dendritic cell-based IFN-?ELISPOT assay was developed for detection of memory T cell activities speci?c to dengue E proteins.This assay can be extended further to measure

immune responses to other viral structural or non-structural pro-

teins.The use of pure protein antigens in this assay systems allows

for a more standardized and accurate comparison of immune

responses among4serotypes,which can be very useful when devel-

oping tetravalent vaccines.

Con?icts of interests

The views expressed in this article are those of the authors

and do not necessarily re?ect the of?cial policy or position of the

Department of the Navy,Department of Defense,nor the https://www.wendangku.net/doc/2a17967269.html,-

ernment.

I am a military service member(or employee of the https://www.wendangku.net/doc/2a17967269.html,ern-

ment).This work was prepared as part of my of?cial duties.Title17

U.S.C.§105provides that“Copyright protection under this title is not available for any work of the United States Government.”Title

17U.S.C.§101de?nes a https://www.wendangku.net/doc/2a17967269.html,ernment work as a work prepared by a military service member or employee of the https://www.wendangku.net/doc/2a17967269.html,ernment as part of that person’s of?cial duties.

This study protocol was approved by the Naval Medical Research

Center Review Board in compliance with all applicable Federal reg-

ulations governing the protection of human subjects. Acknowledgement

This work was supported by U.S.Navy work unit number

6000.RAD1.S.A0312.

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in

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a rose for Emily 分析

A Rose for Emily 的评析(2010-06-21 23:49:34)转载▼ 标签：文化威廉.福克纳和他的《献给爱米丽的玫瑰》摘要：福克纳把南方的历史和现实社会作为自己创作源泉而成为美国南方文学的代表。《献给爱米丽的玫瑰》通过爱米丽的爱情悲剧揭示了新旧秩序的斗争及没落贵族阶级的守旧心态，福克纳运用神秘、暗语、象征、时序颠倒等写作手法来揭示这一主题。关键词：威廉·福克纳；献给爱米丽的玫瑰；南方小说一、威廉·福克纳的南方情结威廉·福克纳(William Faulkner，1897-1962)是美国文学史上久负盛名的作家之一，生于密西西比州一个在内战中失去财富和地位的没落的南方种植园家庭。福克纳的大多数作品都以美国南方为背景，强调南方主题和南方意识。在他19部长篇小说和75篇短篇小说中，绝大多数小说的故事都发生在他虚构的美国的约克纳帕塔法县(Yoknapatawpha county）和杰弗生镇。这些作品所展示的生活画卷和人物形象构成了福克纳笔下的“约克纳帕塔法世系”。 “约克纳帕塔法世系”是以该县家族的兴衰、变迁为主题，故事所跨越的时间上起自印地安人与早期殖民者交往的岁月，止于第二次世界大战后，长约二百年。他的世系小说依南方家系人物的生活而展开，以南方浓郁的泥土气息伴随着因工业文明而带来的焦虑、惶惑、无奈，把一百多年来即从1800年到第二次世界大战之后社会发展过程中，南方人所独有的情感和心态通过独特的艺术方式展示出来，可谓一部“南方生活的史诗”。在这部史诗的字里行间，留下了作家的血与泪之痕：割不断爱恋南方古老精神的一片深情，可又抵御不了现代文明进程的必然性。正如福克纳所说：“我爱南方，也憎恨它。这里有些东西我本不喜欢。但是我生在这里，这是我的家。因此，我愿意继续维护它，即便是怀着憎恨。”这种矛盾恰好构成了福克纳情感意识及其小说世界的无穷魅力。结果，约克纳帕塔法县成了旧南方的象征，而福克纳也借此成功地表现了整个南方社会的历史和意识。二、《献给爱米丽的玫瑰》暗示南方的腐朽没落福克纳素以长篇小说著称于世，但其短篇小说，无论艺术构思、意境创造、人物塑造抑或语言风格、结构艺术等，均可与其长篇小说互论短长，而其最著名的短篇之一《献给爱米丽的玫瑰》 A rose for Emily则对理解、研究福克纳的主要作品，即“约克纳帕塔法世系”具有十分重要的意义。在这篇小说中，作者以凝练的笔触、独特的结构成功地塑造了爱米丽·格里尔森Emily Grieson这个艺术典型。《献给爱米丽的玫瑰》是福克纳1930年4月发表的被誉为最负盛名的短篇小说。故事发生在约克纳帕塔法县的杰弗生镇，小说分为五小部分，在这五个写作空间里，运用回忆来描写爱米丽·格里尔森（Emily Grieson)神秘的一生，讲述了一位被剥夺了与他人建立正常人际关系的妇女如何逃避现实以至于精神失常的故事，表现了新旧南方价值观念之间的冲突。爱米丽的个性冲突、所做所为源于她南方古老而辉煌的家庭背景，福克纳用神秘、暗语、象征等写作方法来揭示爱米丽是南方腐朽传统的象征。故事的叙述采用了时序颠倒的手法，这一手法不仅使小说结构奇突，情节跳跃，更为重要的是其具有深化主题思想的艺术功效。作者用这种手法打破时空界限，把过去和现在直接放在一起，在它们之间形成鲜明的对照，从而使读者深深地感到时代的变迁和传统价值观念的沦丧。小说一开始就告诉读者爱米丽死了，全镇的人都去送丧。她的死象征着南方古老传统、价值观念、生活方式的彻底灭亡和消失。为展示新旧双方的矛盾和以爱米丽为代表的贵族阶级虽大势已去却拒不接受社会变革的心态，作者选取了一个极为典型的事件——纳税事件。

英语选修六课文翻译Unit5 The power of nature An exciting job的课文原文和翻译

AN EXCITING JOB I have the greatest job in the world. I travel to unusual places and work alongside people from all over the world. Sometimes working outdoors, sometimes in an office, sometimes using scientific equipment and sometimes meeting local people and tourists, I am never bored. Although my job is occasionally dangerous, I don't mind because danger excites me and makes me feel alive. However, the most important thing about my job is that I help protect ordinary people from one of the most powerful forces on earth - the volcano. I was appointed as a volcanologist working for the Hawaiian V olcano Observatory (HVO) twenty years ago. My job is collecting information for a database about Mount Kilauea, which is one of the most active volcanoes in Hawaii. Having collected and evaluated the information, I help other scientists to predict where lava from the volcano will flow next and how fast. Our work has saved many lives because people in the path of the lava can be warned to leave their houses. Unfortunately, we cannot move their homes out of the way, and many houses have been covered with lava or burned to the ground. When boiling rock erupts from a volcano and crashes back to earth, it causes less damage than you might imagine. This is because no one lives near the top of Mount Kilauea, where the rocks fall. The lava that flows slowly like a wave down the mountain causes far more damage because it

管理学原理与方法周三多第五版重点.docx

管理学原理与方法(周三多第五版) 总论人类活动的特点：目的性，依存性，知识性管理的概念：管理是管理者为了有效地实现组织目标（目的性有效性协调性过程性）１：管理是人类有意识有目的的活动。２：管理应当是有效的。３：管理的本质是协调。４：协调是运用各种管理职能的过程。管理的职能：决策、组织、领导、控制、创新，是一切管理活动最基本的职能。 1：决策：所有管理者必须制定符合并支持组织的总体战略目标。（制定目标、行动） 2：组织：设计岗位，授权分工，使整个组织协调地运转。（设计、授权） 3：领导：指导人们的行为，通过沟通增强互相理解，统一思想和行动，激励成员自觉地为实现组织目标共同努力。（指导、沟通、激励） 4：控制：使实践活动符合于计划，计划是控制的标准。（衡量、纠偏） 5：创新：与其他职能结合中表现。管理二重性：1、管理的自然属性--反映人与自然的关系不以人的意志为转移，也不因社会制度形态的不同而有所改变，这完全是一种客观存在。 2、管理的社会属性--反映社会关系管理者的角色：明茨伯格这十种角色可归入三类。人际角色：代表人角色、领导人角色、联络者角色信息角色：监督者、发言人、传播人决策角色：企业家、干扰对付者、资源分配者、谈判者管理者三种技能：卡次 1：技术技能，运用管理者所监督的专业领域中的过程、惯例、技术和工具的能力。 2：人际技能，成功地与人打交道并与人沟通的能力。 3：概念技能，把观点设想出来并加以处理以及将关系抽象化的精神能力。管理学的研究方法：归纳法、试验法、演绎法中国传统管理思想的要点： 1：宏观管理的治国学--（财政赋税、人口管理、货币管理、等） 2：微观管理的治生学--（农副业、手工业、运输、建筑工程等）顺道、重人、人和、守信、利器、求实、对策、节俭、法治西方早期思想产生的三个人物：亚当斯密巴贝奇罗伯特欧文泰罗创立的科学管理理论主要观点：1：科学管理的根本目的--谋求最高工作效率 2：达到最高效率的重要手段--用科学的管理方法代替旧的经验方法 3：实施科学管理的核心问题-要求管理人员和工人双方在精神上和思想上来一个彻底的改变提出的以下管理制度：1：对工人提出科学的操作方法，以便合理利用工时，提高效率 2：在工资制度上实行差别计件制3：对工人进行科学的选择，培训和提高 4：制定科学的工艺规程5：使管理和劳动分离评价：1：它冲破了传统地落后地经验管理办法，将科学引进了管理领域，创立了一套具体地科学管理方法2：科学地管理方法和科学地操作程序使生产效率提高了二三倍，推动了生产地发展，适应了资本主义地发展。 3：由于管理职能于执行职能地分离，企业中开始有一些人专门从事管理工作 4：泰罗把人看成会说话的机器，只能按照管理人员的决定、指示、命令执行劳动，在体力技能上受很大的压榨缺陷：适应历史发展的需要而产生的，同时也受到历史条件和个人经历的限制，他的科学管理所涉及的问

ARoseforEmily英文分析及简评

“A Rose for Emily” is divided into five sections. The first section opens with a description of the Grierson house in Jefferson. The narrator mentions that over the past 100 years, Miss Emily Grierson’s home has fall into disrepair and become “an eyesore among eyesores.” The first sentence of the story sets the tone of how the citizens of Jefferson felt about Emily: “When Miss Emily Grierson died, our whole town went to the funeral: the men through a sort of respectful affection for a fallen monument, the women mostly out of curiosity to see the inside of her house, which no one save an old manservant – a combined gardener and cook – had seen in at least ten years.” It is known around town that Emily Grierson has not had guests in her home for the past decade, except her black servant who runs errands for her to and from the market. When a new city council takes over, however, they begin to tax her once again. She refuses to pay the taxes and appear before the sheriff, so the city authorities invite themselves into her house. When confronted on her tax evasion, Emily reminds them that she doesn't have to pay taxes in Jefferson and to speak to Colonel Sartoris, although he had died 10 years before. In section two, the narrator explains that the Griersons had always been a very proud Southern family. Mr. Grierson, Emily’s father, believes no man is suitable for his daughter and doesn't allow her to date. Emily is largely dependent upon her father, and is left foundering when he dies. After Mr. Grierson's death, Emily does not allow the authorities to remove his body for three days, claiming he is still alive. She breaks down and allows authorities to take the body away for a quick burial. Section three introduces Emily’s beau, Homer Barron, a foreman from the north. Homer comes to Jefferson with a crew of men to build sidewalks outside the Grierson home. After Emily and Homer are seen driving through town several times, Emily visits a druggist. There, she asks to purchase arsenic. The druggist asks what the arsenic is for since it was required of him to ask by law. Emily does not respond and coldly stares him down until he looks away and gives her the arsenic. When Emily opens the package, underneath the skull and bones sign is written, "For Rats." Citizens of Jefferson believe that Miss Emily is going to commit suicide since Homer has not yet proposed in the beginning of section four. The townspeople contact and invite Emily's two cousins to comfort her. Shortly after their arrival, Homer leaves and then returns after the cousins leave Jefferson. After staying in Jefferson for one night, Homer is never seen again. After Homer’s disappearance, Emily begins to age, gain weight, and is rarely seen outside of her home. Soon, Miss Emily passes away.

管理学原理与方法周三多第六版

第一篇第一章管理与管理学第一节人类的管理活动一：人类活动的特点(目的性、依存性、知识性) 二：管理的必要性三：管理的概念第二节管理的职能与性质一：管理的职能（计划、组织、领导、控制、创新）二：管理的自然属性三：管理的社会属性第三节管理者的角色与职能一：管理者的角色（人际角色、信息角色、决策角色）二：管理者的职能罗伯特卡次的研究，管理者必须具备三种技能：（技术技能、人际技能、概念机能）第四节管理学的对象与方法一：管理学的研究对象二：管理学的研究方法（一）归纳法（二）试验法（三）演绎法第二章管理思想的发展第一节中国传统管理思想一：中国传统思想形成的社会文化背景二：中国传统管理思想的要点第二节西方传统管理思想一：西方早期管理思想的产生 1：亚当斯密《国富论》1776（英国） 2：查理巴贝奇（英国） 3：罗伯特。欧文（英国的空想主义家）二：科学管理理论的产生和发展（19世纪末20世纪初）（一）“泰罗”的科学管理理论——科学管理之父亨利。甘特：布雷斯及他的妻子：（二）对“泰罗制”的评价（三）法约尔的“组织管理理论” 第三节西方现代管理思想的发展一：行为科学学派霍桑试验： 1：需求层次理论——马斯洛 2：双因素理论——赫茨伯格 3：X、Y理论 4：Z理论——威廉。大内二：“管理科学”学派三：“决策理论”学派四：对现代管理理论的思考五：新经济时代管理思想的变革

（一）管理思想的创新（二）管理原则的创新（三）经营目标创新（四）经营战略创新（五）生产系统创新（六）企业组织创新第三节中国现代管理思想的发展一：中国现代管理思想形成的历史背景（一）中国官僚资本企业和民族资本企业的管理（二）我国革命根据地公营企业的管理（三）全面学习西方的管理模式（四）探索中国现在管理模式二：社会主义经济管理体制改革（一）由国内管理向国际化管理转化（二）由科学管理向信息化管理转化（三）由首长管理向人性化管理转化（四）由政府管理向民营化管理转化（五）由封闭式实体管理向开放式虚拟管理转化第三章管理的基本原理第四章第一节管理原理的特征第五章一：管理原理的主要特征第六章二：研究管理原理的意义第七章第二节系统原理第八章一：系统的概念第九章二：系统的特征第十章三：系统原理要点第十一章第三节人本原理第十二章一：职工是企业的主体第十三章二：有效管理的关键是职工参与第十四章三：现代管理的核心是使人性得到最完美的发展第十五章四：管理是为人服务的第十六章第四节责任原理第十七章一：明确每个人的职责第十八章二：职位设计和权限委任要合理第十九章三：奖惩要分明，公正而及时第二十章第五节效益原理第二十一章一：效益的概念第二十二章二：效益的评价第二十三章三：效益的追求第四章信息化管理第一节信息与信息化一、信息的含义二、信息化的内涵三、信息化的影响

经典层序地层学的原理与方法

第二章经典层序地层学的原理与方法经典层序地层学为分析沉积地层和岩石关系提供了有力的方法手段，其原理和实践已被大多数地质学家所接受。理论上，层序地层学特别重视海平面升降周期对地层层序形成的重要影响；实践上，它通过年代地层格架的建立，对地层分布模式作出解释和同时代成因地层体系域的划分，为含油气盆地地层分析和盆地规模的储层预测提供坚实的理论和油气勘探的有效手段，有力的推动了地质学，特别是石油地质学的发展，它的推广与应用标志着隐蔽油气藏勘探研究进入了一个全新的精细描述、精细预测阶段。第一节经典层序地层学中的两种层序边界 Vail等在硅质碎屑岩层系中已经识别出两类不同的层序，即Ⅰ类层序和Ⅱ类层序，这两类层序在碳酸盐岩研究中得到了广泛应用。以下详细论述这两类层序边界的含义、特征和识别标志。一、Ⅰ型层序边界及其特征和识别标志当海平面迅速下降且速率大于碳酸盐台地或滩边缘盆地沉降速率、海平面位置低于台地或滩边缘时，就形成了碳酸盐岩的Ⅰ型层序界面。Ⅰ型层序界面以台地或滩的暴露和侵蚀、斜坡前缘侵蚀、区域性淡水透镜体向海方向的运动以及上覆地层上超、海岸上超向下迁移为特征(图1-2-1)。图1-2-1碳酸盐岩Ⅰ型层序边界特征(据Sarg，1988) 1．碳酸盐台地或滩边缘暴露侵蚀的岩溶特征碳酸盐台地广泛的陆上暴露和合适的气候条件为形成Ⅰ型层序界面提供了地质条件，层

序界面以下的沉积物具有明显的暴露、溶蚀等特征，碳酸盐台地或陆棚沉积背景上的陆上暴露，可通过古岩溶特征来识别，因此，风化壳岩溶是识别碳酸盐台地碳酸盐岩Ⅰ型层序的重要特征。 ①古岩溶面常是不规则的，纵向起伏几十至几百米。岩溶地貌常表现为岩溶斜坡和岩溶凹地。如我国鄂尔多斯盆地奥陶系顶部、新疆奥陶系顶部、川东石炭系黄龙组顶部等发育的古岩溶。 ②地表岩溶主要特征为出现紫红色泥岩、灰绿色铝土质泥岩以及覆盖的角砾灰岩、角砾白云岩的古土壤。风化壳顶部的岩溶角砾岩往往成分单一，分选和磨圆差。碎屑灰岩和碎屑如鲕粒、生物碎屑常被溶解形成铸模孔等。 ③古岩溶存在明显的分带性，自上而下可分为垂直渗流岩溶带、水平潜流岩溶带和深部缓流岩溶带。 ④岩溶表面和岩溶带中出现各种岩溶刻痕和溶洞，如细溶沟、阶状溶坑、起伏几十米至几百米的夷平面、落水洞、溶洞以及均一的中小型蜂窝状溶孔洞等。 ⑤溶孔内存在特征充填物，可充填不规则层状且分选差的角砾岩、泥岩或白云质泥的示底沉积，隙间或溶洞内充填氧化铁粘土和石英粉砂以及淡水淋虑形成的淡水方解石和白云岩。 ⑥具有钙质壳、溶解后扩大的并可被粘土充填的解理、分布广泛的选择性溶解空隙。 ⑦岩溶地层具有明显的电测响应，如明显的低电阻率、相对较高的声波时差、较高的中子孔隙度、较明显的扩径、杂乱的地层倾角模式和典型的成像测井响应。 ⑧古岩溶面响应于起伏较明显的不规则地震反射，古岩溶带常对应于明显的低速异常带。此外，古岩溶面上下地层的产状、古生物组合、微量元素及地化特征也有明显的差别。 2．斜坡前缘的侵蚀作用在Ⅰ型层序界面形成时，常发生明显的斜坡前缘的侵蚀，导致台地和滩缘斜坡上部大量沉积物被侵蚀掉，结果造成大量碳酸盐砾屑的向下滑塌堆积作用和碳酸盐砂的碎屑流、浊流沉积作用和碳酸盐砂砾的密度流沉积作用(图1-2-1)。斜坡前缘侵蚀作用可以是局部性或区域性的，向上可延伸到陆棚区形成发育良好的海底峡谷，滩前沉积物可被侵蚀掉几十至几百米。在碳酸盐缓坡和碳酸盐台地边缘出现的水道充填砾屑灰岩，以及向陆方向由河流回春作用引起的由海相到陆相、碳酸盐岩到碎屑岩的相变沉积物以及向上变浅的沉积序列也是Ⅰ型层序边界的标志。 3．淡水透镜体向海的方向运动 Ⅰ型层序界面形成时发生的另一种作用，就是淡水透镜体向海或向盆地方向的区域性迁移(图1-2-1)。淡水透镜体渗入碳酸盐岩剖面的程度与海平面下降速率、下降幅度和海平面保持在低于台地或滩边缘的时间长短有关。在大规模Ⅰ型层序边界形成时期，当海平面下降75～100米或更多并保持相当长的时间时，在陆棚上就会长期地产生淡水透镜体，它的影响会充分地深入到地下，并可能深入到下伏层序。若降雨量大，剖面浅部就会发生明显的淋滤、溶解作用，潜流带出现大量的淡水胶结物，如不稳定的文石、高镁方解石可能被溶解，形成低镁方解石沉淀(Sarg，1998)。Vail的海平面升降曲线表明，在全球海平面下降中，少见大规模的Ⅰ型海平面下降。一般的海平面下降幅度不超过70～100m。也就是说，在小规模Ⅰ型层序边界形成时期，淡水透镜体未被充分建立起来，只滞留在陆架地层的浅部，没有造成广泛的溶解和地下潜水胶结物的沉淀。在Ⅰ型层序边界形成时期，在适宜的构造、气候和时间条件下可能发育风化壳。同时，伴随Ⅰ型界面形成期间，可发生不同规模的混合水白云化和强烈蒸发作用而引起的白云化。二、Ⅱ型层序界面及其特征、识别标志

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八年级下学期全部长篇课文 Unit 1 3a P6 In ten years , I think I'll be a reporter . I'll live in Shanghai, because I went to Shanfhai last year and fell in love with it. I think it's really a beautiful city . As a reporter, I think I will meet lots of interesting people. I think I'll live in an apartment with my best friends, because I don' like living alone. I'll have pets. I can't have an pets now because my mother hates them, and our apartment is too small . So in ten yers I'll have mny different pets. I might even keep a pet parrot!I'll probably go skating and swimming every day. During the week I'll look smart, and probably will wear a suit. On the weekend , I'll be able to dress more casully. I think I'll go to Hong Kong vacation , and one day I might even visit Australia. P8 Do you think you will have your own robot In some science fiction movies, people in the future have their own robots. These robots are just like humans. They help with the housework and do most unpleasant jobs. Some scientists believe that there will be such robots in the future. However, they agree it may take hundreds of years. Scientist ae now trying to make robots look like people and do the same things as us. Janpanese companies have already made robts walk and dance. This kond of roots will also be fun to watch. But robot scientist James White disagrees. He thinks that it will be difficult fo a robot to do the same rhings as a person. For example, it's easy for a child to wake up and know where he or she is. Mr White thinks that robots won't be able to do this. But other scientists disagree. They think thast robots will be able t walk to people in 25 to 50tars. Robots scientists are not just trying to make robots look like people . For example, there are already robots working in factories . These robots look more like huge arms. They do simple jobs over and over again. People would not like to do such as jobs and would get bored. But robots will never bored. In the futhre, there will be more robots everwhere, and humans will have less work to do. New robots will have different shapes. Some will look like humans, and others might look like snakes. After an earthquake, a snake robot could help look for people under buildings. That may not seem possibe now, but computers, space rockets and even electric toothbrushes seemed

ARoseforEmily英文分析及简评

is divided into five secti ons. A Rose for Emily The first sect ion ope ns with a descripti on of the Griers on house in Jeffers on. The n arrator men ti ons that over the past 100 years, Miss Emily Griers on ' s home has fall into disrepair and become “ an eyesore among eyesores. ” The first sentence of the story sets the tone of how the citize ns of Jeffers on felt about Emily: “ When Miss Emily Griers on died, our whole town went to the funeral: the men through a sort of respectful affection for a fallen monument, the wome n mostly out of curiosity to see the in side of her house, which no one save an old man serva nt -a comb ined garde ner and cook - had see n in at least ten years. It is known around tow n that Emily Griers on has not had guests in her home for the past decade, except her black serva nt who runs errands for her to and from the market. When a new city council takes over, however, they begi n to tax her once aga in. She refuses to pay the taxes and appear before the sheriff, so the city authorities invite themselves into her house. When confron ted on her tax evasi on, Emily reminds them that she does n't have to pay taxes in Jeffers on and to speak to Colonel Sartoris, although he had died 10 years before. In section two, the narrator explains that the Griersons had always been a very proud Souther n family. Mr. Griers on, Emily ' s father, believes no man is suitable for his daughter and does n't allow her to date. Emily is largely depe nden t upon her father, and is left foun deri ng whe n he dies. After Mr. Griers on's death, Emily does not allow the authorities to remove his body for three days, claiming he is still alive. She breaks down and allows authorities to take the body away for a quick burial. Section three introduces Emily ' s beau, Homer Barron, a foreman fronthe north. Homer comes to Jeffers on with a crew of men to build sidewalks outside the Griers on home. After Emily and Homer are seen driving through town several times, Emily visits a druggist. There, she asks to purchase arse nic. The druggist asks what the arse nic is for since it was required of him to ask by law. Emily does not respond and coldly stares him down un til he looks away and gives her the arse nic. When Emily ope ns the package, underneath the skull and bones sign is written, "For Rats." Citize ns of Jeffers on believe that Miss Emily is going to commit suicide si nce Homer has not yet proposed in the beg inning of sect ion four. The tow nspeople con tact and in vite Emily's two cousins to comfort her. Shortly after their arrival, Homer leaves and then retur ns after the cous ins leave Jeffers on. After stay ing in Jeffers on for one ni ght, Homer is never seen again. After Homer ' s disappearance, Emily begins to age, gain weight, and is rarely see n outside of her home. Soon, Miss Emily passes away.

A rose for Emily中译本赏析,以杨岂深的译本为例

“A Rose for Emily”中译本赏析 ——以杨岂深的译本为例摘要：A Rose for Emily 《献给埃米莉的玫瑰》讲述了美国南北战争后南方小镇-----杰弗逊镇上没落的格尔森贵族家庭中埃米莉的悲剧故事。作者威廉·卡斯伯特·福克纳（William Cuthbert Faulkner）为美国文学史上最具影响力的作家之一，美国“南方文艺复兴”时期成就最显著的南方作家和现代主义作家。以杨岂深先生的译本为例，对小说分别从忠实的标准，形似与神似的矛盾，主人公对话语言的描写三个方面进行翻译研究，有益于提高读者的文学素养，提升文学翻译实践能力。关键词：埃米莉；译本；忠实；形似与神似；对话 Abstract: A Rose for Emily tells the tragic story of Emily Grierson, the daughter of a noble declining family, which happened in a small southern American town-----Jefferson after American Civil War. The author, William Cuthbert Faulkner, is one of the most influential writers in the American literary history, the most significant Southern writer and modernism writer in the American "Northern Renaissance" period. This paper is based on the version of translator Yang Qishen. Mainly discusses the translation study from three aspects——faithful standard, the contradictions between appearance and soul , description of the heroine's conversation, which will be helpful for the improvement of readers '

a rose for Emily 分析报告

A Rose for Emily 的评析 (2010-06-21 23:49:34)转载▼ 标签：文化威廉.福克纳和他的《献给爱米丽的玫瑰》摘要：福克纳把南方的历史和现实社会作为自己创作源泉而成为美国南方文学的代表。《献给爱米丽的玫瑰》通过爱米丽的爱情悲剧揭示了新旧秩序的斗争及没落贵族阶级的守旧心态，福克纳运用神秘、暗语、象征、时序颠倒等写作手法来揭示这一主题。关键词：威廉·福克纳；献给爱米丽的玫瑰；南方小说一、威廉·福克纳的南方情结威廉·福克纳(William Faulkner，1897-1962)是美国文学史上久负盛名的作家之一，生于密西西比州一个在内战中失去财富和地位的没落的南方种植园家庭。福克纳的大多数作品都以美国南方为背景，强调南方主题和南方意识。在他19部长篇小说和75篇短篇小说中，绝大多数小说的故事都发生在他虚构的美国的约克纳帕塔法县(Yoknapatawpha county）和杰弗生镇。这些作品所展示的生活画卷和人物形象构成了福克纳笔下的“约克纳帕塔法世系”。 “约克纳帕塔法世系”是以该县家族的兴衰、变迁为主题，故事所跨越的时间上起自印地安人与早期殖民者交往的岁月，止于第二次世界大战后，长约二百年。他的世系小说依南方家系人物的生活而展开，以南方浓郁的泥土气息伴随着因工业文明而带来的焦虑、惶惑、无奈，把一百多年来即从1800年到第二次世界大战之后社会发展过程中，南方人所独有的情感和心态通过独特的艺术方式展示出来，可谓一部“南方生活的史诗”。在这部史诗的字里行间，留下了作家的血与泪之痕：割不断爱恋南方古老精神的一片深情，可又抵御不了现代文明进程的必然性。正如福克纳所说：“我爱南方，也憎恨它。这里有些东西我本不喜欢。但是我生在这里，这是我的家。因此，我愿意继续维护它，即便是怀着憎恨。”这种矛盾恰好构成了福克纳情感意识及其小说世界的无穷魅力。结果，约克纳帕塔法县成了旧南方的象征，而福克纳也借此成功地表现了整个南方社会的历史和意识。二、《献给爱米丽的玫瑰》暗示南方的腐朽没落福克纳素以长篇小说著称于世，但其短篇小说，无论艺术构思、意境创造、人物塑造抑或语言风格、结构艺术等，均可与其长篇小说互论短长，而其最著名的短篇之一《献给爱米丽的玫瑰》 A rose for Emily则对理解、研究福克纳的主要作品，即“约克纳帕塔法世系”具有十分重要的意义。在这篇小说中，作者以凝练的笔触、独特的结构成功地塑造了爱米丽·格里尔森Emily Grieson这个艺术典型。《献给爱米丽的玫瑰》是福克纳1930年4月发表的被誉为最负盛名的短篇小说。故事发生在约克纳帕塔法县的杰弗生镇，小说分为五小部分，在这五个写作空间里，运用回忆来描写爱米丽·格里尔森（Emily Grieson)神秘的一生，讲述了一位被剥夺了与他人建立正常人际关系的妇女如何逃避现实以至于精神失常的故事，表现了新旧南方价值观念之间的冲突。爱米丽的个性冲突、所做所为源于她南方古老而辉煌的家庭背景，福克纳用神秘、暗语、象征等写作方法来揭示爱米丽是南方腐朽传统的象征。故事的叙述采用了时序颠倒的手法，这一手法不仅使小说结构奇突，情节跳跃，更为重要的是其具有深化主题思想的艺术功效。作者用这种手法打破时空界限，把过去和现在直接放在一起，在它们之间形成鲜明的对照，从而使读者深深地感到时代的变迁和传统价值观念的沦丧。小说一开始就告诉读者爱米丽死了，全镇的人都去送丧。她的死象征着南方古老传统、价值观念、生活方式的彻底灭亡和消失。为展示新旧双方的矛盾和以爱米丽为代表的贵族阶级虽大势已去却拒不接受社会变革的心态，作者选取了一个极为典型的事件——纳税事件。

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1.以指定教材为中心，重视基础，善于总结 2.在理解的基础上记忆，并能联系实际 3.答题要有逻辑，条理要清楚讲义分为两部分：宋体部分是教材内容的结构和要点，应以此为复习框架；楷体部分是对教材内容的补充，对于完善答题内容，充实知识结构有帮助；讲义最后另附有重要理论的补充，这是教材上没有详述但是作为管理类学生必须掌握的知识。祝学员金榜题名，马到成功！

目录第一章管理与管理学................................................................................................................ - 1 - 第一节人类的管理活动.................................................................................................... - 1 - 第二节管理的职能与性质................................................................................................ - 2 - 第三节管理者的角色与职能............................................................................................ - 2 - 第四节管理学的对象与方法............................................................................................ - 3 - 第二章管理思想的发展............................................................................................................ - 4 - 一、管理实践、管理思想与管理理论三者之间的关系.......................................................... - 4 - 二、管理学形成的阶段划分...................................................................................................... - 4 - 三、美国出现“管理运动”的必然性 ...................................................................................... - 5 - 第一节中国传统管理思想................................................................................................ - 5 - 第二节西方传统管理思想................................................................................................ - 5 - 第三节西方现代管理思想的发展.................................................................................... - 7 - 第三节中国现代管理思想的发展.................................................................................. - 12 - 第三章管理的基本原理.......................................................................................................... - 13 - 第一节管理原理的特征.................................................................................................. - 13 - 第二节系统原理.............................................................................................................. - 13 - 第三节人本原理.............................................................................................................. - 13 - 第四节责任原理............................................................................................................. - 14 - 第五节效益原理.............................................................................................................. - 14 - 第六节伦理原理.............................................................................................................. - 15 - 第四章管理的基本方法.......................................................................................................... - 16 - 第一节管理的方法论...................................................................................................... - 16 - 第二节管理的法律方法.................................................................................................. - 16 - 第三节管理的行政方法.................................................................................................. - 16 - 第四节管理的经济方法.................................................................................................. - 17 - 第五节管理的教育方法.................................................................................................. - 17 - 第六节管理的技术方法.................................................................................................. - 18 - 第五章管理伦理...................................................................................................................... - 19 - 第一节有关伦理的几种观点.......................................................................................... - 19 - 第二节伦理管理的特征和影响伦理的因素.................................................................. - 19 - 第三节改善伦理行为的途经........................................................................................ - 20 - 第四节伦理行为的具体表现.......................................................................................... - 20 - 第六章组织文化...................................................................................................................... - 22 - 第一节组织文化的概念和基本特征.............................................................................. - 22 - 第二节组织文化的基本要素.......................................................................................... - 22 - 第三节组织文化的功能.................................................................................................. - 22 - 第四节塑造组织文化的主要途经.................................................................................. - 23 - 2：全面归纳.............................................................................................................................. - 23 - 补充：企业文化的四种类型............................................................................................ - 23 - 第七章管理信息...................................................................................................................... - 25 - 第一节信息概述.............................................................................................................. - 25 -