Fandom

VroniPlag Wiki

Quelle:Pak/Ahmed 2007

< Quelle:Pak

31.373Seiten in
diesem Wiki
Seite hinzufügen
Diskussion0

Störung durch Adblocker erkannt!


Wikia ist eine gebührenfreie Seite, die sich durch Werbung finanziert. Benutzer, die Adblocker einsetzen, haben eine modifizierte Ansicht der Seite.

Wikia ist nicht verfügbar, wenn du weitere Modifikationen in dem Adblocker-Programm gemacht hast. Wenn du sie entfernst, dann wird die Seite ohne Probleme geladen.

Angaben zur Quelle [Bearbeiten]

Autor     Farid Ahmed
Titel    Die funktionelle Bedeutung des ATP–bindenden Transportproteins ABCG2 für die Hämatopoese
Ort    Munich
Jahr    2007
Anmerkung    Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München
URL    http://edoc.ub.uni-muenchen.de/7360/1/Ahmed_Farid.pdf

Literaturverz.   

no
Fußnoten    no
Fragmente    10


Fragmente der Quelle:
[1.] Pak/Fragment 021 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:48:59 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
Hindemith
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 21, Zeilen: 1-11
Quelle: Ahmed_2007
Seite(n): 6, 7, Zeilen: 6: 12 ff.; 7: 4 ff.
The production of mature blood cells from HSC requires three distinct genetic programs. These include: (a) the specification of HSC, (b) their self-renewal and c) their commitment/ proliferation/ differentiation. Most of the studies leading to the knowledge of genes involved in HSC genetic programs have been carried by assaying haematopoietic cells from animals deficient for the gene of interest. More recently, expression profiling strategies have been used to determine genetic and molecular signatures of HSC.68,69 Genes involved in deciding fate of HSCs during early embryogenesis include: SCL and Rbtn2/Lmo-2, which are necessary for primitive and definitive haematopoiesis.70,71 GATA-2 and AML1 are specifically required for definitive haematopoiesis.72

68.Ivanova M, Rozemuller E, Tyufekchiev N, Michailova A, Tilanus M, Naumova E. HLA polymorphism in Bulgarians defined by high-resolution typing methods in comparison with other populations. Tissue Antigens. 2002;60:496-504.

69.Georgantas RW, 3rd, Tanadve V, Malehorn M, et al. Microarray and serial analysis of gene expression analyses identify known and novel transcripts overexpressed in hematopoietic stem cells. Cancer Res. 2004;64:4434-4441.

70.Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature. 1995;373:432-434.

71.Warren HS, Kinnear BF, Skipsey LJ, Pembrey RG. Differential expression of CD45R0 on natural killer (NK) cells in patients with an NK lymphocytosis. Immunol Cell Biol. 1994;72:500-507.

72.Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 1996;84:321-330.

The production of mature blood cells from HSC requires three distinct genetic programs. These include: a) the specification of HSC, b) their self-renewal and c) their commitment/proliferation/differentiation (fig 1.2).

[page 7]

Most of the studies leading to the knowledge of genes involved in HSC genetic programs have been carried by assaying haematopoietic cells from animals deficient for the gene of interest. More recently, expression profiling strategies have been used to determine genetic and molecular signatures of HSC (Ivanova et al., 2002).

Genes involved in specifying HSC during early embryogenesis include: SCL and Rbtn2/Lmo-2 which are necessary for primitive and definitive haematopoiesis (Shivdasani et al., 1995; Warren et al., 1994). GATA-2 and AML1 are specifically required for definitive haematopoiesis (Okuda et al., 1996).


Ivanova,N.B., Dimos,J.T., Schaniel,C., Hackney,J.A., Moore,K.A., and Lemischka,I.R. (2002). A stem cell molecular signature. Science 298, 601-604.

Okuda,T., van Deursen,J., Hiebert,S.W., Grosveld,G., and Downing,J.R. (1996). AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84, 321-330.

Shivdasani,R.A., Mayer,E.L., and Orkin,S.H. (1995). Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373, 432-434.

Warren,A.J., Colledge,W.H., Carlton,M.B., Evans,M.J., Smith,A.J., and Rabbitts,T.H. (1994). The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 78, 45-57.

Anmerkungen

The source is not mentioned. Interestingly, the author gives different publications of Warren et al. (1994) and Ivanova et al. (2002) compared to the source.

Sichter
(Hindemith), SleepyHollow02

[2.] Pak/Fragment 022 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:49:03 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
Hindemith
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 22, Zeilen: 1-25
Quelle: Ahmed_2007
Seite(n): 7, 8, Zeilen: 7: 12 ff.; 8: 1 ff.
Some other factors appear to be more lineage-specific in action such as GATA-3, Ikaros, PU.1, GATA-1, CBP, Atf4, c-myb, and E2A, and their absence affects specific haematopoietic lineages. 73-77 The genetic factors involved in regulating fetal liver HSC are: Meis1, which is highly expressed in fetal liver Sca-1 +Lin- cells that are enriched for HSC activity78 and Hoxb4, which causes in vivo and ex vivo expansion of HSC when constitutively expressed. 79,80 Hox proteins interact with another transcription factor Pbx1, which itself interacts with Meis1 and forms a trimeric nuclear complex which is involved in target gene regulation81,82 (Fig.1.3.4a).

HSC self- renewal maintenance in adult BM is regulated by a different set of genes. A number of recent studies point out to nuclear factors such as the Polycomb (PcG) genes Bmi-1 and Rae-28, GATA-2 and TEL for potentially regulating this process. It has been observed that Bmi-1 levels decline during haematopoietic development, and that Bmi-1 deficient mice develop hypocellular BM and die at less than 2 months of age. This led to the speculation that Bmi-1 is involved in maintenance of the HSC pool.83 Rae-28, a known nuclear partner of Bmi-1, also plays a crucial role in maintaining the activity of HSCs during fetal haematopoiesis. 84

The zinc-finger transcription factor GATA-2, a member of GATA family, plays a critical role in maintaining the pool of multipotent progenitors and HSCs, both during embryogenesis and in the adult.85 The zinc-finger transcription factors GATA-1 and its transcriptional cofactor called Friend of GATA-1 (FOG-1), have been found to be essential for erythroid and megakaryocytic differentiation.86,87 PU.1 is a member of the Ets family of transcription factors and is essential in the development of cells of the monocytic, granulocytic and lymphoid lineages.88


73.Bain G, Maandag EC, Izon DJ, et al. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell. 1994;79:885-892.

74.Wang H, Xie Z, Scott RE. JunD phosphorylation, and expression of AP-1 DNA binding activity modulated by serum growth factors in quiescent murine 3T3T cells. Oncogene. 1996;13:2639-2647.

75.Scott G, Ewing J, Ryan D, Abboud C. Stem cell factor regulates human melanocyte-matrix interactions. Pigment Cell Res. 1994;7:44-51.

76.Emambokus N, Vegiopoulos A, Harman B, Jenkinson E, Anderson G, Frampton J. Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb. EMBO J. 2003;22:4478-4488.

77.Lessard J, Faubert A, Sauvageau G. Genetic programs regulating HSC specification, maintenance and expansion. Oncogene. 2004;23:7199-7209.

78.Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol. 2002;30:49-57.

79.Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell. 2002;109:39-45.

80.Buske C, Feuring-Buske M, Antonchuk J, et al. Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and in vivo. Blood. 2001;97:2286-2292.

81.Liu JP, Laufer E, Jessell TM. Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron. 2001;32:997-1012.

82. Mann RS, Affolter M. Hox proteins meet more partners. Curr Opin Genet Dev. 1998;8:423-429.

83.Lessard J, Schumacher A, Thorsteinsdottir U, van Lohuizen M, Magnuson T, Sauvageau G. Functional antagonism of the Polycomb-Group genes eed and Bmi1 in hemopoietic cell proliferation. Genes Dev. 1999;13:2691-2703.

84.Ohta H, Sawada A, Kim JY, et al. Polycomb group gene rae28 is required for sustaining activity of hematopoietic stem cells. J Exp Med. 2002;195:759-770.

85.Tsai FY, Orkin SH. Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation. Blood. 1997;89:3636-3643.

86.Pevny L, Simon MC, Robertson E, et al. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991;349:257-260.

87.Vyas P, McDevitt MA, Cantor AB, Katz SG, Fujiwara Y, Orkin SH. Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA- 1 gene. Development. 1999;126:2799-2811.

88.Scott EW, Simon MC, Anastasi J, Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science. 1994;265:1573-1577.

Some other factors appear to be more lineage-specific in action such as GATA-3, Ikaros, PU.1, GATA-1, CBP, Atf4, c-myb, and E2A, and their absence affects specific haematopoietic lineages (Bain et al 1994, Scott 1994, Wang et al 1996 Mauoka 2002, Wang 1997, Emambokus 2003). [...] The genetic factors involved in regulating fetal liver HSC are: Meis1, which is highly expressed in fetal liver Sca-1 + Lin- cells that are enriched for HSC activity (Pineault et al., 2002) and Hoxb4, the overexpression of which causes in vivo and ex vivo expansion of HSC (Antonchuk et al., 2001; Buske et al., 2002). Hox proteins interact with another transcription factor Pbx which itself interacts with Meis1 and forms a trimeric nuclear complex which is involved in target gene regulation (Liu et al., 2001; Swift et al., 1998). HSC self-renewal maintenance in adult BM is regulated by a different set of genes. A number of recent studies point out to nuclear factors such as the Polycomb-Group (PcG) genes Bmi-1 and Rae-28, GATA-2 and TEL for potentially regulating this process. It has been observed that Bmi-1 levels decline during haematopoietic development, and that Bmi-1 deficient mice develop hypocellular BM and die at less than 2 months of age. This led to the speculation that Bmi-1 is involved in maintenance of the HSC pool (Lessard et al., 2004). Rae-28, a known nuclear partner of Bmi-1 also plays a crucial role in maintaining the activity of HSC during fetal haematopoiesis (Ohta et al., 2002). The zinc-finger transcription factor GATA-2, a member of GATA family, plays a critical role in

[page 8]

maintaining the pool of multipotent progenitors and HSC, both during embryogenesis and in the adult (Tsai et al., 1994).

[...] The zinc-finger transcription factor GATA-1 and its transcriptional cofactor called Friend of GATA-1 (FOG-1), have been found to be essential for erythroid and megakaryocytic differentiation (Pevny et al., 1991; Tsang et al., 1998; Vyas et al., 1999). PU.1 is a member of the Ets family of transcription factors and is essential in the development of cells of the monocytic, granulocytic and lymphoid lineages (Scott et al., 1994).


Antonchuk,J., Sauvageau,G., and Humphries,R.K. (2001). HOXB4 overexpression mediates very rapid stem cell regeneration and competitive haematopoietic repopulation. Exp. Hematol. 29, 1125-1134.

Buske,C., Feuring-Buske,M., Abramovich,C., Spiekermann,K., Eaves,C.J., Coulombel,L., Sauvageau,G., Hogge,D.E., and Humphries,R.K. (2002). Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells. Blood 100, 862-868.

Lessard,J., Faubert,A., and Sauvageau,G. (2004). Genetic programs regulating HSC specification, maintenance and expansion. Oncogene 23, 7199-7209.

Liu,Y., MacDonald,R.J., and Swift,G.H. (2001). DNA binding and transcriptional activation by a PDX1.PBX1b.MEIS2b trimer and cooperation with a pancreas-specific basic helix-loop-helix complex. J Biol. Chem. 276, 17985-17993.

Ohta,H., Sawada,A., Kim,J.Y., Tokimasa,S., Nishiguchi,S., Humphries,R.K., Hara,J., and Takihara,Y. (2002). Polycomb group gene rae28 is required for sustaining activity of hematopoietic stem cells. J Exp. Med. 195, 759-770.

Pevny,L., Simon,M.C., Robertson,E., Klein,W.H., Tsai,S.F., D'Agati,V., Orkin,S.H., and Costantini,F. (1991). Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349, 257-260.

Pineault,N., Helgason,C.D., Lawrence,H.J., and Humphries,R.K. (2002). Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp. Hematol. 30, 49-57.

Scott,E.W., Simon,M.C., Anastasi,J., and Singh,H. (1994). Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573-1577.

Swift,G.H., Liu,Y., Rose,S.D., Bischof,L.J., Steelman,S., Buchberg,A.M., Wright,C.V., and MacDonald,R.J. (1998). An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2). Mol. Cell Biol. 18, 5109-5120.

Tsai,F.Y., Keller,G., Kuo,F.C., Weiss,M., Chen,J., Rosenblatt,M., Alt,F.W., and Orkin,S.H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371, 221-226.

Tsang,A.P., Fujiwara,Y., Hom,D.B., and Orkin,S.H. (1998). Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG. Genes Dev. 12, 1176-1188.

Vyas,P., Ault,K., Jackson,C.W., Orkin,S.H., and Shivdasani,R.A. (1999). Consequences of GATA-1 deficiency in megakaryocytes and platelets. Blood 93, 2867-2875.

Wang,J.C., Doedens,M., and Dick,J.E. (1997). Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 89, 3919-3924.

Anmerkungen

The source is not given.

Note: Bain et al (1994) is not listed in the list of references of the source, neither is Wang et al. (1996), Mauoka (2002) and Emambokus (2003).

Sichter
(Hindemith), SleepyHollow02

[3.] Pak/Fragment 032 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:49:07 Hindemith
Ahmed 2007, Fragment, Gesichtet, KomplettPlagiat, Pak, SMWFragment, Schutzlevel sysop

Typus
KomplettPlagiat
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 32, Zeilen: 1-14, 17-29
Quelle: Ahmed 2007
Seite(n): 32, 33, Zeilen: 32: 7-20; 33: 3-14
[Introduction of retroviral vectors into PG13] cells results in the production of retrovirus virions capable of infecting cells from many species excluding mice.

K562: An erythroleukemia cell line derived from a chronic myeloid leukaemia patient in blast crisis. K562 cells were purchased from ATCC.

M2-10B4 j-GCSF-tkneo j-IL-3-hytk: Murine M2-10B4 fibroblasts engineered to produce high levels of both human granulocyte colony-stimulating factor (GCSF) and interleukin-3 (IL-3; 190 and 4 ng/mll, respectively), referred henceforth as M2-10B4 G-CSF / IL-3, were provided courteously provided by Connie Eaves (Terry Fox Laboratory, Vancouver, Canada).

Sl/Sl j-SF-tkneo j-IL-3-hytk: SI/SI fibroblasts engineered to produce high levels of soluble Steel factor (SF), with or without production of the transmembrane form of SF (60 and 4 ng/ ml, respectively), referred henceforth as Sl/Sl SF / IL-3, were provided kindly by Connie Eaves (Terry Fox Laboratory, Vancouver, Canada).

[• MS-5: Mouse stromal cells established by irradiation of the adherent cells in long-term bone marrow cultures derived from C3H/HeNSlc strain mice.]

3.1.4. The NOD-SCID Mice

The NOD/LtSz-scid strain was generated by crossing the SCID mutation from C.B-17- SCID mice onto the NOD background. C.B-17-scid mice lack functional T & B lymphocytes. The NOD strain mouse is an animal model of spontaneous autoimmune T-cell mediated insulin dependent diabetes mellitus (IDDM); however they have multiple defects in innate immunity. They are deficient in NK cell activity; display defects in myeloid development and function, and cannot generate either the classical or alternative pathways of haemolytic complement activation.

The NOD/LtSz-scid lacks an adaptive immune system; due to the absence of T cells, they do not develop autoimmune IDDM and remain insulitis- and diabetes free throughout life. However they carry the innate immune defects present in the parental NOD/Lt stock of mice.109


109.Lowry PA, Shultz LD, Greiner DL, et al. Improved engraftment of human cord blood stem cells in NOD/LtSz-scid/scid mice after irradiation or multiple-day injections into unirradiated recipients. Biol Blood Marrow Transplant. 1996;2:15-23.

[page 32]

Introduction of retroviral vectors into PG13 cells results in the production of retrovirus virions capable of infecting cells from many species excluding mice.

K562: An erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis. K562 cells were purchased from ATCC.

M2-10B4 j-GCSF-tkneo j-IL-3-hytk: Murine M2-10B4 fibroblasts engineered to produce high levels of both human granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3; 190 and 4 ng/mll, respectively), referred henceforth as M2-10B4 G-CSF / IL-3, were provided courteously provided by Connie Eaves (Terry Fox Laboratory, Vancouver, Canada).

Sl/Sl j-SF-tkneo j-IL-3-hytk: SI/SI fibroblasts engineered to produce high levels of soluble Steel factor (SF), with or without production of the transmembrane form of SF (60 and 4 ng/ ml, respectively), referred henceforth as Sl/Sl SF / IL-3, were provided kindly by Connie Eaves (Terry Fox Laboratory, Vancouver, Canada).

[page 33]

3.1.6 The NOD-SCID Mice:

The NOD/LtSz-scid strain was generated by crossing the SCID mutation from C.B-17-scid mice onto the NOD background. C.B-17-scid mice lack functional T & B lymphocytes. The NOD strain mouse is an animal model of spontaneous autoimmune T-cell mediated insulin dependent diabetes mellitus (IDDM); however they have multiple defects in innate immunity. They are deficient in NK cell activity; display defects in myeloid development and function, and cannot generate either the classical or alternative pathways of haemolytic complement activation. The NOD/LtSz-scid lacks an adaptive immune system; due to the absence of T cells, they do not develop autoimmune IDDM and remain insulitis- and diabetes free throughout life. However they carry the innate immune defects present in the parental NOD/Lt stock of mice (Lowry et al., 1996).


Lowry,P.A., Shultz,L.D., Greiner,D.L., Hesselton,R.M., Kittler,E.L., Tiarks,C.Y., Rao,S.S., Reilly,J., Leif,J.H., Ramshaw,H., Stewart,F.M., and Quesenberry,P.J. (1996). Improved engraftment of human cord blood stem cells in NOD/LtSz-scid/scid mice after irradiation or multiple-day injections into unirradiated recipients. Biol. Blood Marrow Transplant. 2, 15-23.

Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

[4.] Pak/Fragment 033 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:49:11 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 33, Zeilen: 1-3, 6-21
Quelle: Ahmed 2007
Seite(n): 33-34, Zeilen: 33: 1-2,14-18, 26-30 - 34: 1-8
[NOD/SCID mice were bred from the breeding] pairs originally obtained from Taconic Bomholt, Denmark and maintained in the animal facility located at the GSF-Haematology, Munich. All animals were handled under sterile conditions and maintained under micro isolators. [xenografts [sic] into immunodeficient NOD-SCID mice are the gold standard readout for human long-term repopulating hematopoietic cells.110-112]
3.1.5. Mice related reagents and equipment

Sterile Syringes: BD Plastipak 1 ml syringe (BD Biosciences, Palo Alto, CA) for injection of cells in mice and Kendall Monoject 3 ml syringes (Tyco Healthcare, UK) for bone marrow flushing and plating of CFC.

Sterile needles: 0.5 x 25 mm for intra venous injection of cells in mice and 0.55 x 25 mm (BD Microlance, Drogheda, Ireland) for bone marrow aspiration from living mice and flushing of bone marrow from extracted bones. 16 X 1.5 inch needles for dispensing and plating Methocult (CFC) media (Stem Cell Technologies, Vancouver, Canada).

Ammonium Chloride solution: For erythrocyte lysis 0.8% NH4Cl with 0.1 mM EDTA (Stem Cell Technologies, Vancouver, Canada).

Heparinized capillaries: (Microvette CB 300) plastic capillaries for collection of blood, containing 15 units (I.E) Lithium heparin per ml of blood (Sarstedt, Numbrecht, Germany).

3.1.6. Bacterial strain

• E. coli DH5á [sic]

3.1.5 Bacterial strain

• E. coli DH5α

[...]

NOD/SCID mice were bred from the breeding pairs originally obtained from Taconic Europe and maintained in the animal facility located at the GSF-Haematology, Munich. All animals were handled under sterile conditions and maintained under micro isolators.

3.1.7 Mice related reagents and equipment:

[...]

Sterile Syringes: BD Plastipak 1 ml syringe (BD Biosciences, Palo Alto, CA) for injection of cells in mice and Kendall Monoject 3 ml syringes (Tyco Healthcare, UK) for bone marrow flushing and plating of CFC.

Sterile needles: 0.5 x 25 mm for intra venous injection of cells in mice and 0.55 x 25 mm (BD Microlance, Drogheda, Ireland) for bone marrow aspiration from living

[page 34]

mice and flushing of bone marrow from extracted bones. 16 X 1.5 inch needles for dispensing and plating Methocult (CFC) media (Stem Cell Technologies, Vancouver, Canada)

Ammonium Chloride solution: For erythrocyte lysis 0.8% NH4Cl with 0.1 mM EDTA (Stem Cell Technologies, Vancouver, Canada)

Heparinized capillaries: (Microvette CB 300) plastic capillaries for collection of blood, containing 15 units (I.E) Lithium heparin per ml of blood (Sarstedt, Numbrecht, Germany).

Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

[5.] Pak/Fragment 048 03 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:50:22 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 48, Zeilen: 3-27
Quelle: Ahmed 2007
Seite(n): 40 f., Zeilen: 40: 18-34 - 41: 1-7
3.2.6. Purification of umbilical cord blood CD34+ cells (CB CD34+) from mononuclear umbilical cord blood (UCB).

Umbilical cord blood was collected in heparinised syringes according to institutional guidelines following normal full-term deliveries. Informed consent was obtained in all cases. Mononuclear cells (MNC) were separated using density gradient centrifugation. Fresh umbilical cord blood, not older than 12 hours, was diluted with 2 volumes of PBS and layered over Pancoll. Usually 35 ml of diluted blood was layered over 15 ml Pancoll in a 50 ml conical tube. This was centrifuged at 400x g for 30 minutes at 20°C in a swinging-bucket rotor without brakes. The upper layer was aspirated and discarded, leaving the interphase undisturbed. The interphase containing MNC such as lymphocytes, monocytes and thrombocytes was then transferred to a new 50 ml tube, washed twice with large volumes of PBS, and then counted before labelling with magnetic bead or fluorochrome conjugated antibodies.

hCB CD34+ cell purification was conducted using MACS CD34+ Cell Isolation Kit that uses positive selection method. Cells were resuspended in a volume of 300 ml per 1x108 MNCs These were blocked with 100 ml of FcR Blocking Reagent and labelled with 100 ml of CD34 Microbeads. When working with higher cell number, all the reagent volumes & the total volume was scaled up accordingly. This was followed by incubation for 30 minutes at 4-8°C. Cells were then washed twice by adding 10x the labelling volume of buffer and centrifuged at 300 x g for 15 minutes. The resultant cell pellet was then resuspended in 500 ml of MACS buffer and loaded into MS Column mounted on magnetic separator. The negative cells were allowed to pass through and the column was washed at least three times with 2 ml buffer.

3.2.6 Purification of human UCB CD133+ and CD34+ cells

Density Gradient Centrifugation:

Umbilical cord blood was collected in heparinised syringes according to institutional guidelines following normal full-term deliveries. Informed consent was obtained in all cases. Mononuclear cells (MNC) were separated using density gradient centrifugation. Fresh umbilical cord blood, not older than 12 hours, was diluted with 2 volumes of PBS and layered over Pancoll. Usually 35 ml of diluted blood was layered over 15 ml Pancoll in a 50 ml conical tube. This was centrifuged at 400x g for 30 minutes at 20°C in a swinging-bucket rotor without brakes. The upper layer was aspirated and discarded, leaving the interphase undisturbed. The interphase containing MNC such as lymphocytes, monocytes and thrombocytes was then transferred to a new 50 ml tube, washed twice with large volumes of PBS, and then counted before labelling with magnetic bead or fluorochrome conjugated antibodies.

Magnetic Separation:

CD133+ cell purification was conducted using MACS CD133 Cell Isolation Kit that uses positive selection method. Cells were resuspended in a volume of 300 μl per 108 cells, blocked with 100 μl of FcR Blocking Reagent and labelled with 100 μl of

[page 41]

Materials & Methods

CD133 Microbeads. When working with higher cell number, all the reagent volumes & the total volume was scaled up accordingly. This was followed by incubation for 30 minutes at 4-8°C. Cells were then washed twice by adding 10x the labelling volume of buffer and centrifuged at 300 x g for 15 minutes. The resultant cell pellet was then resuspended in 500 μl of MACS buffer and loaded into MS Column mounted on magnetic separator. The negative cells were allowed to pass through and the column was washed at least three times with 2 ml buffer.

Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

[6.] Pak/Fragment 049 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:50:19 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 49, Zeilen: 1-8
Quelle: Ahmed 2007
Seite(n): 41, Zeilen: 7-19
[The column was then removed from the separator, placed on a collection tube, loaded with fresh buffer, and the magnetically labelled cells flushed out using the] plunger. The magnetic separation was usually repeated to get a purity of more than 95%. Purified cells were then frozen in FBS with 10% DMSO and thawed when needed for pre-stimulation and transduction. hCB CD34+ cell enrichment was done by FACS. For separation by FACS, MNCs were thawed from frozen stocks or prepared freshly from UCB and labelled using anti CD34-PE antibody (100 ml per 108 cells), for 30 minutes on ice. Labelled cells were then washed twice with PBS, resuspended in FACS buffer and sorted. The sorted cells with purity above 95 % were used for 48 hour pre-stimulation followed by transduction. The column was then removed from the separator, placed on a collection tube, loaded with fresh buffer, and the magnetically labelled cells flushed out using the plunger. The magnetic separation was usually repeated to get a purity of more than 95%. Purified cells were then frozen in FBS with 10% DMSO and thawed when needed for pre-stimulation and transduction.

FACS sorting:

CD34+ cell enrichment was done either by MACS as done for CD133+ cells or by FACS. For separation by FACS, MNCs were thawed from frozen stocks or prepared freshly from UCB and labelled using anti CD34-PE antibody (100 μL per 108 cells), for 30 minutes on ice. Labelled cells were then washed twice with PBS, resuspended in FACS buffer and sorted. The sorted cells with purity above 95% were used for 48 hour pre-stimulation followed by transduction.

Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

[7.] Pak/Fragment 053 05 - Diskussion
Zuletzt bearbeitet: 2014-04-06 09:00:17 Hindemith
Ahmed 2007, Fragment, Gesichtet, KomplettPlagiat, Pak, SMWFragment, Schutzlevel sysop

Typus
KomplettPlagiat
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 53, Zeilen: 5-29
Quelle: Ahmed 2007
Seite(n): 42-43, Zeilen: 10ff.;1ff.
3.2.11. Colony forming cell assay

Haematopoietic colony-forming cells (CFC) were assayed using methylcellulose-based medium (MethoCult H 4434) (fig. 3.2.11). Required number of pre-aliquoted tubes of MethoCult medium was thawed overnight under refrigeration (2-8°C) or at room temperature. The cells were diluted with IMDM + 2% FBS to 10X the final concentration(s) required for plating. For a duplicate assay 0.3 ml of diluted cells was added to 3 ml MethoCult tube and the contents vortexed vigorously. After about 5 minutes, 1.1 ml of cell: methylcellulose mixture was dispensed into 35 mm culture dishes using sterile a 3 ml syringe and16-gauge bunt-end needle. The 35 mm culture dishes were placed into 10 cm petri dish along with an extra 35 mm dish containing sterile water to maintain humidity and placed in a CO2 incubator at 37°C and >95% humidity.

CFC numbers were evaluated after an incubation period of 12-14 days and distinguished into following classes: Colony-forming unit-erythroid (CFU-E): Produces 1-2 cell clusters containing a total of 8-200 erythroblasts. A CFU-E consists of mature erythroid progenitors that require erythropoietin (EPO) for differentiation. Burst-forming unit-erythroid (BFU-E): Produces a colony containing >200 erythroblasts in a single or multiple clusters. A BFU-E consists of more immature progenitors than CFU-E and requires EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem Cell Factor (SCF) for optimal colony growth.

Colony-forming unit-granulocyte, macrophage (CFU-GM): Produces a colony containing at least 20 granulocyte cells (CFU-G), macrophages (CFU-M) or cells of both lineages (CFU-GM). CFU-GM colonies arising from primitive progenitors may contain thousands of cells in single or multiple clusters. Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM):

3.2.8 Human CFC Assay

Haematopoietic colony-forming cells (CFC) were assayed using methylcellulose-based medium (MethoCult; H 4434) (fig 3.3). Required number of pre-aliquoted tubes of MethoCult medium was thawed overnight under refrigeration (2-8°C) or at room temperature. The cells were diluted with IMDM + 2% FBS to 10X the final concentration(s) required for plating. For a duplicate assay 0.3 ml of diluted cells was added to 3 ml MethoCult tube and the contents vortexed vigorously. After about 5 minutes, 1.1 ml of cell: methylcellulose mixture was dispensed into 35 mm culture dishes using sterile a 3 ml syringe and16-gauge bunt-end needle. The 35 mm culture dishes were placed into 10 cm petri dish along with an extra 35 mm dish containing sterile water to maintain humidity and placed in a CO2 incubator at 37°C and >95%

[Page 43]

humidity. CFC numbers were evaluated after an incubation period of 12-14 days and distinguished into following classes:

Colony-forming unit-erythroid (CFU-E): Produces 1-2 cell clusters containing a total of 8-200 erythroblasts. A CFU-E consists of mature erythroid progenitors that require erythropoietin (EPO) for differentiation.

Burst-forming unit-erythroid (BFU-E): Produces a colony containing >200 erythroblasts in a single or multiple clusters. A BFU-E consists of more immature progenitors than CFU-E and require [sic] EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem Cell Factor (SCF) for optimal colony growth.

Colony-forming unit-granulocyte, macrophage (CFU-GM): Produces a colony containing at least 20 granulocyte cells (CFU-G), macrophages (CFU-M) or cells of both lineages (CFU-GM). CFU-GM colonies arising from primitive progenitors may contain thousands of cells in single or multiple clusters.

Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM):

Anmerkungen

A literal, unattributed copy that continues on teh next page.

Sichter
(SleepyHollow02) Agrippina1

[8.] Pak/Fragment 054 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 08:28:35 Hindemith
Ahmed 2007, Fragment, Gesichtet, KomplettPlagiat, Pak, SMWFragment, Schutzlevel sysop

Typus
KomplettPlagiat
Bearbeiter
Agrippina1
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 54, Zeilen: 1, 4-5
Quelle: Ahmed 2007
Seite(n): 43, Zeilen: 15-17
[A multi-] potential progenitor that produces a colony containing erythroblasts and cells of at least two other recognizable lineages. Due to their primitive nature, CFU-GEMM tend to produce large colonies of >500 cells. A multi-potential progenitor that produces a colony containing erythroblasts and cells of at least two other recognizable lineages. Due to their primitive nature, CFU-GEMM tend to produce large colonies of >500 cells.
Anmerkungen

Fortsetzung von der Seite davor.

Sichter
(Aggripina1) SleepyHollow02

[9.] Pak/Fragment 056 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 07:42:20 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 56, Zeilen: 1-14
Quelle: Ahmed 2007
Seite(n): 460, Zeilen: 3-18
[Wells were rinsed once with 1 ml PBS and added to tube. 1 ml Trypsin-EDTA was added to each well and incubated for 3 to 5 minutes] and examined for detached cells. Once the adherent cells are detached, the wells are washed with more PBS and the medium collected in the appropriated tube.

The wells are finally washed with 1 ml IMDM containing 2 % FBS and transferred to the specific tube. The tubes were centrifuged at 1200 rpm for 10 minutes and the supernatant was removed without disturbing the cell pellet. Approximately 200ìL of medium was left along with the cell pellet and vortexed. To this 3ml of Methocult (H4434) methylcellulose medium was added and vortexed again. Each tube (contents of one well) was plated individually into 2 different (1.5 ml/dish) 35 mm petri dish with 1 ml syringe (without needles attached). Different dishes (6-8) were placed in a 15 cm petri-dish along with an additional 60 mm open dish containing 5 ml sterile water to maintain humidity. The dishes are incubated at 37°C in humidified incubator (>95 %) with 5% CO2 in air for 16-20 days. Colonies were scored as positive if one or more BFU-E, CFU-GM or CFU-GEMM were detected or scored as negative if no colonies were present.

[The number of LTC-IC for the test cell population was calculated by dividing the total number of CFC detected in the culture by the average number of clonogenic progenitors per LTC-IC for the standard conditions used. Alternatively the values were expressed as LTC-IC derived CFC per number of test cells.115]


[115.Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ. Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor. Blood. 1996;88:3765-3773.]

Wells were rinsed once with 0.2 ml PBS and added to tube. 0.1 ml Trypsin-EDTA was added to each well and incubated for 3 to 5 minutes and examined for detached cells. Once the adherent cells are detached, the wells are washed with more PBS and the medium collected in the appropriated tube. The wells are finally washed with 0.2 ml IMDM containing 2% FBS and transferred to the appropriate tube. The tubes were centrifuged at 1200 rpm for 10 minutes and the supernatant removed without disturbing cell pellet. Approximately 0.1 ml of medium was left along with the cell pellet and vortexed. To this 1 ml of Methocult (H4435) methylcellulose medium was added and vortexed again. Each tube (contents of one well) was plated individually into 35 mm petri dish with 1 ml syringe (without needles attached). Several dishes (6-8) were placed in a 15 cm petri-dish along with an additional 60 mm open dish containing 5 ml sterile water to maintain humidity. The dishes are incubated at 37°C in humidified incubator (>95%) with 5% CO2 in air for 12 to 16 days. Colonies were counted and a well scored as positive if one or more BFU-E, CFU-GM or CFU-GEMM were detected or scored as negative if no colonies were present. [The LTC-IC frequency in the test cell population was calculated from the proportion of negative wells (no CFC present) and the method of maximum likelihood. Statistical analysis was performed using L-Calc™ software for limiting dilution analyses.]
Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

[10.] Pak/Fragment 068 06 - Diskussion
Zuletzt bearbeitet: 2014-04-06 06:50:26 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 68, Zeilen: 6-11
Quelle: Ahmed 2007
Seite(n): 54, Zeilen: 1-8
Clonogenic haematopoietic assays detect progenitor cells committed to a specific lineage by seeding candidate populations into semi-solid methylcellulose media. Progenitors identified by this assay are retrospectively classified as colony forming cells (CFC) and can be quantitatively subdivided into lineage restricted subtypes by examining the composition of the resulting progeny (methods, p-51)(Fig. 4.5.1a). 4.4 Constitutive expression of ABCG2 increases the production of CFC in vitro

Clonogenic haematopoietic assays detect progenitor cells committed to a specific lineage by seeding candidate populations into semi-solid methylcellulose media. Progenitors identified by this assay are retrospectively classified as colony forming cells (CFC) and can be quantitatively subdivided into lineage restricted subtypes by examining the composition of the resulting progeny (described in the methods section).

Anmerkungen

The source is not given.

Sichter
(SleepyHollow02) Schumann

Auch bei Fandom

Zufälliges Wiki