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IMPACT OF IMATINIB MESYLATE ON SLC22A1 GENE EXPRESSION IN CHRONIC MYELOID LEUKAEMIA CELL LINE, K562Sreenivasan, Sandhya 21 November 2013 (has links)
Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder
characterised by the BCR-ABL fusion gene. The BCR-ABL fusion gene encodes a
constitutively active BCR-ABL tyrosine kinase, which is the driving force of the
malignancy. Otherwise fatal, the use of imatinib mesylate has proved highly
effective in the treatment of this disease in up to 85% of CML patients. However,
approximately 25% of CML patients appear to respond suboptimally or experience
treatment failure with imatinib. Suboptimal response in CML patients has been
attributed to inadequate BCR-ABL kinase inhibition as a result of reduced
intracellular accumulation of imatinib in target leukemic cells. The cellular influx of
imatinib is mediated by the influx transport protein, SLC22A1. Therefore, its
activity is considered a clinical determinant of imatinib uptake, and hence patients
response to therapy.
A number of studies use levels of SLC22A1 mRNA as a measure of SLC22A1
activity. It has been reported that cells over expressing levels of SLC22A1 mRNA
showed significantly increased uptake of imatinib, thus, suggesting that levels of
SLC22A1 mRNA can be used as a measure of SLC22A1 activity. However, there
is a concern that imatinib may affect SLC22A1 expression. This consideration,
however, is based on two studies involving a limited patient cohort and although
widely accepted, has not been proven conclusively. Should it be proven that
imatinib does influence SLC22A1 expression, levels of SLC22A1 mRNA may not be a reliable indicator of SLC22A1 activity. It is therefore important to understand
the effect of treatment with imatinib on SLC22A1 gene expression.
The data from this study demonstrated that imatinib induces expression of
SLC22A1 mRNA in a non-linear dose dependent manner. It was also observed
that expression of SLC22A1 was not dependent on time of exposure to imatinib.
These results explain the differential expression of SLC22A1 mRNA reported in
CML patients on a standard dose of 400 mg/day of imatinib. The trough plasma
levels of imatinib achieved between patients after 24 hours of exposure to the
same dose of imatinib may vary owing to inter individual differences. Since
SLC22A1 expression is dependent on plasma levels of imatinib, therefore, patients
administered the same dose of imatinib may show differential expression of
SLC22A1. These findings suggest that imatinib does affect SLC22A1 mRNA
expression and that the change in SLC22A1 expression observed at any particular
time is dependent on the intracellular levels of imatinib achieved in CML patients
within 24 hours of exposure to the drug.
One of the challenges in this study was the availability of suitably qualified
SLC22A1 antibodies for use in the Taqman protein assay to quantify SLC22A1
protein. Antibodies used in the Taqman assay have to fulfil specific criteria and
out of 55 commercially available antibodies, only three SLC22A1 antibodies met
the minimum requirements for use in the assay. However, despite various efforts
focused at optimising the assay, the range of the assay was very limited and
hence it was not possible to quantify SLC22A1 protein. We hypothesize that one of the reasons for assay failure could be as a result of antibodies not binding to the
target protein at the required spatial distance to facilitate amplification by real-time
PCR. Since the antibodies used in the assay have not been epitope mapped, it is
uncertain whether they fulfil this requirement. Future research will be aimed at
antibody production for manufacturing SLC22A1 antibodies suitable for use in the
Taqman protein assay to enable successful quantification of SLC22A1 protein.
In conclusion, this is the first study which specifically aimed to investigate the
influence of imatinib on SLC22A1 expression. This is also the first study to
demonstrate that expression of SLC22A1 is not time dependent, but follows a nonlinear
correlation to imatinib concentration. Although it would have been useful to
investigate the effect of increasing levels of SLC22A1 mRNA on intracellular
uptake of imatinib in K562 cells, unfortunately, the latter technique requires the use
of radio-labelled imatinib and specialized equipment which made it a limiting factor
for use in this study. While this study does not invalidate the use of levels of
SLC22A1 mRNA as a prognostic marker for treatment outcome, these findings
suggest that levels of SLC22A1 mRNA as a measure of SLC22A1 activity is only
applicable to newly diagnosed imatinib naive or previously untreated CML patients.
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A case study of practical work in a cell biology course at the Eduardo Mondlane University in Mozambique.Cossa, Eugenia Flora Rosa. January 2007 (has links)
<p>This study was carried out with the assumption that practical work does contribute to the teaching and learning of cell biology at Eduardo Mondlane University in Mozambique. In this regard, the main purpose of this study was to investigate the impact of practical work in the teaching and learning of cell biology concepts, specifically focussing on cell divisions concepts. It also aimed at determining the students' perceptions of the role of practical work in the learning of cell biology. On the other hand, the study sought also to understand the lecturers' practical work teaching experiences and views regarding the cell biology practical work.</p>
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Cellular and molecular analysis of chromosomal passenger complex in vertebrate cellsXu, Zhenjie January 2009 (has links)
Sucessful cell division is primarily controlled by temporal and spatial regulation of two critical events in the cell cycle: the faithful replication of chromosomes and the accurate segregation of the replicated chromosomes. Of all the cellular proteins involved in regulating these processes, the chromosomal passenger complex displays some of the most visually striking patterns of behaviours during mitosis. The chromosomal passenger proteins associate with chromosomes as cells prepare to segregate them during mitosis. Subsequently, when chromosomes are aligned on the spindle microtubules that do the mechanical work of segregation, the passenger proteins become concentrated in distinct foci between paired kinetochores—the protein-based structures on chromosomes where the microtubules attach. Then, as chromosomes start to segregate, the passenger proteins abruptly dissociate from the kinetochores and instead localize to the central spindle in the area midway between the separating chromosomes. This ultimately becomes the midbody during cytokinesis. The first protein found to display such dramatic changes in localization during nuclear division was named INCENP. Three further proteins—Aurora B, Survivin and Borealin were later found to show localization patterns similar to INCENP. Structural analyses revealed that these four proteins form a well conserved chromosomal passenger complex throughout mitosis, in which INCENP acts as a scaffold to recruit Survivin and Borealin to its N-terminus, while Aurora B associates with its C-terminus. The function of this complex parallels its dynamic localization. These include roles in spindle assembly, correcting erroneous kinetochore microtubule interactions that are inappropriate for chromosome alignment, signaling to the spindle checkpoint, and the completion of cytokinesis. Functional analysis of kinetochore proteins in vertebrates using DT40 cells has greatly improved our understanding of kinetochore assembly, kinetochore structure and functions in guiding cell division. Following the identification of chicken Aurora B, Borealin/Dasra B and Dasra A, we are now able to address the pleiotropic functions of the conserved chromosomal passenger complex in DT40 cells using gene disruption. Gene disruption of INCENP using a novel promoter hijack technique in DT40 cells has demonstrated that accurate cell division requires coordination of spindle dynamics, chromosome segregation and cell cleavage and that INCENP integrates these diverse processes. Recent concentrated efforts on elucidating the precise contribution of each subunit in localizing the complex have provided great insights of how CPC executes its mitotic function. Here, through introducing a series of INCENP structural mutants into this sophisticated INCENP knockout system, my in vivo data presented here indicated that interaction between INCENP-Aurora B is essential for cell division. Remarkably, dissociation of Aurora B from INCENP does not disturb the other passengers targeting to the centromere, but Aurora B activity through binding and activation by INCENP plays a direct role in guiding the transfer of the complex to the spindle midzone. Furthermore, a feed-back loop involving phosphorylation of INCENP at its TSS motif by Aurora B is essential for cell viability, even though its disruption by mutation to TAA does not prevent the passengers eventually transfer to spindle midzone and midbody. Thus, this final step to maximize the activity of Aurora B is a critical function of INCENP. The outstanding question I have addressed in my study is why the passenger proteins are interdependent. More importantly, my work reveals that the intrinsic modulation within chromosomal passenger complex directed by the interactions between INCENP and Aurora B regulates the transfer of the complex to the spindle midzone and midbody. Thus, the results here with previous study suggest that regulation of the centromere and spindle midzone targeting of the complex involves two distinctly intrinsic mechanisms directed by two targeting sub-complexes within CPC, in which INCENP, Borealin and Survivin form the core sub-complex for centromere targeting, and Aurora B regulated by INCENP forms the sub-complex serving to direct the CPC transfer to spindle midzone by modulation of Aurora B activity.
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UNDERSTANDING AND MANIPULATING ENDOCYTOSIS-DEPENDENT SIGNALING CIRCUITSWen-Chieh Hsieh (6824807) 13 August 2019 (has links)
<p>Signal transductions
are essential processes for living cells to react to environmental stimuli
adequately, and they need to be tightly regulated as they can affect cell
survival and cell fate determination. Since many of these signaling events rely
on the presence of receptors on the cell membrane, members of endocytic
proteins play critical regulatory roles in signaling via changing the
localization of the receptors. In particular, endocytic adaptors are the linkers that connect membrane cargo and other members of
endocytic machinery to accomplish the
process. We focused on the roles of the endocytic adaptors epsins and their
cargoes in signaling, as both epsins’ transmembrane and cytosolic cargoes
participate in signaling pathways.</p>
<p>We investigated
the molecular mechanism of how epsins recognize specific
ubiquitinated membrane cargoes among other ubiquitinated membrane
proteins. Through genetic, biochemical, and cell biological approaches, we identified the first yeast transmembrane
cargo, Ena1, a P-type ATPase sodium pump. We report that the simultaneous presence of phosphorylation
and ubiquitination on the Ena1
are required for epsin-specific recognition. We also demonstrated that post-translational modifications are Yck1/2
and Art3-Rsp5 dependent, and the spatial arrangement of the modifications is essential.
</p>
<p>In addition to the regulation
of signaling pathways through internalizing
transmembrane cargoes, epsins
are also involved in the regulation of
Rho GTPase signaling
pathways. Through direct interaction,
epsins inhibit activities of their cytosolic cargoes, Rho GTPase activating
proteins (RhoGAPs). Ocrl1 is one of the epsin interacting RhoGAP domain-containing
proteins. The deficiency of Ocrl1
leads to a lethal developmental disease
called Lowe syndrome (LS).
While the patients display developmental problems affecting the brain and eyes,
they also suffer from kidney dysfunction that results in death. The
pathological mechanism is currently obscure and no cure, partly due to the lack of an adequate cell model from the
affected tissues. We generated the first iPSC model from fibroblasts of LS
patients and normal individuals and further generated kidney cells from
these iPSCs. Consistent with observations obtained from LS fibroblasts,
the LS iPSC derived kidney
cells from patient cells also have a deficiency in
ciliogenesis.</p>
Further, we discovered that
Six2, a crucial transcriptional factor in kidney development, is mislocalized
to the Golgi-apparatus in patient iPSC-derived kidney cells as well as in an <i>OC</i><i>RL1</i> K.O. proximal tubular cell line. Disproportional cell
lineage differentiation is also observed in the patient group. The iPSC model
provides an opportunity to investigate the differences between normal and
disease cell differentiation in all the affected tissues, generate organoids,
and develop cell replacement therapies.
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Multiple Functions Of The Striated Rootlet Proteins Of The Paramecium Basal BodyNabi, Md Ashikun 01 January 2018 (has links)
Paramecium ciliary basal bodies align in straight rows from posterior to anterior. Each basal body is connected to three rootlets ((Post Ciliary Rootlet (PCR), Transverse Rootlet (TR) and Striated Rootlet (SR)). The SR, the longest, projects from the basal body toward the anterior past several more anterior basal bodies. The depletion of Meckelin (MKS3) misaligns SRs, disorganizes basal body rows and makes the SRs appear ragged and serpentine. In this study we clarify the composition of the Paramecium ciliary basal body’s SR and demonstrate that the SR plays a critical role in creating the orderly array of basal bodies in rows that run from pole to pole of the cell, likely through the interactions with centrins and other cytoskeletal elements underlying the cell surface. Here in this study we first report the reciprocal relationship between the SR and centrin related infraciliary lattice (ICL) protein that can dictate the cell surface morphology.
The SR of Chlamydomonas is the best studied. Using the single SR Chlamydomonas gene SF-assemblin to search in Paramecium DB, we found thirty Paramecium genes in thirteen Paralog Groups. Proteins from 13 paralog groups were confirmed to be in the SR structure using immunofluorescence. LC-MS/MS analyses of density fractions from SRs isolation show all thirty SR members are within the same density fraction. We further categorized all 30 SR genes in five Structural Groups based on their ability to form coiled coil domain and evaluate the function of all five Structural Group using RNA interference (RNAi). Silencing the transcripts of the any of the Structural Group showed misaligned basal body rows and the disordered organization of the SRs with abnormal appearance of SRs all over the cell surface. Silencing of Paralog Group showed normal phenotype except for the two Paralog Group (Paralog Group 1 or Paralog Group 7) which themselves constitute Structural Group individually. Isolated SRs from the control or Paralog Group depleted cells show a characteristic striation pattern that includes characteristic major and minor striations. Isolated SRs from any of the Structural Group depleted cells demonstrate abnormal shapes and striation periodicity. There is a correlation between the SR Structural Group RNAi surface misalignment phenotype and the isolated SR Structural Group RNAi phenotype for shape and periodicity of the SR. Strikingly our study of SR clearly demonstrates the role of SRs in shaping the other cytoskeleton structures of the cell cortex e.g., ICL, epiplasm territory and cortical unit territory.
In another follow up study of MKS3 (Picariello et al., 2014), we depleted the transcripts of MKS5 gene in Paramecium tetraurelia. Depletion of MKS5 transcripts in Paramecium causes cilia loss all over the cell surface. Unlike MKS3 depletion, MKS5 depletion does not affect the straight basal body rows and the ordered organization of SRs. Moreover, data presented in this study clearly demonstrates depletion of MKS5 transcripts somehow affect the localization of another transition zone protein, B9D2.
It appears when lacking any of the SR Structural Group, the rest fail to interact properly with each other to maintain the SRs structure and directionality toward the anterior. As a result, abnormal SRs appear to lose the interaction with other cytoskeleton structures such as ICL network complex, which eventually results in misaligned basal body rows and altered swimming behavior. From the data presented in this study it is reasonable to postulate ICL1e subfamily and SRs are in a reciprocal relationship to maintain the straight basal body rows and the highly ordered organization of the SRs all over the cell surface.
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Inflammation Of The Taste Sensory System: Cyclophosphamide And AmifostineSarkar, Anish Ali 01 January 2019 (has links)
Chemotherapeutics are used extensively to treat cancer patients and often induce adverse effects, including taste dysfunctions. Disturbances in taste are detrimental to the overall well-being of cancer patients, causing malnutrition and weight loss that aggravate their condition even further. Inflammation due to an infection of the taste sensory system as previously shown, has detrimental effects on the taste sensation. Our study focused on if chemotherapy induced an inflammatory response in the taste buds using cyclophosphamide (CYP), a pro-drug. Once metabolized by the P450 enzyme complex, its primary metabolite functions as an alkylating agent, involved in inhibiting cell replication cycle and cell death. We used immunohistochemistry and fluorescent microscopy to analyze and observe the expression of the pro-inflammatory cytokine TNF-α in different types of taste sensory cells. Previously we found that a sulfhydryl cytoprotective drug, amifostine, can prevent taste bud damage and therefore we asked if it could prevent an inflammatory response. Our research observed an inflammatory response in both Type II and Type III cells in taste buds of fungiform and circumvallate papilla. Type II cells showed a peak response at 8- and 24-hour post-CYP injection whereas Type III cells had a peak expression at 24-hour post-CYP injection in the circumvallate papillae. Pre-treatment with amifostine appeared to prevent an inflammatory response within taste buds from CYP induced cytokine response. Identifying inflammation as a potential factor in taste related disorders could help clinicians develop better treatment modalities such as cytoprotective drugs preventing chemotherapy induced long term adverse effects such as malnutrition. In the future, we would like to expand our research to investigate expression of other pro-inflammatory cytokines and possible signaling mechanisms that could be responsible for CYP-induced inflammatory response in Type III cells.
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Cell-Cell Interactions in the Development of the Vascular System of XenopusStimson, Krista Marie 01 January 1998 (has links)
No description available.
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The Pax-5 Gene is Alternatively Spliced in Trout B CellsTalauliker, Raaj Mahendra 01 January 2010 (has links)
No description available.
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Cloning and Functional Characterization of Hypoxia-Inducible Factor 1alpha Upstream Regions in Xenopus laevisSipe, Conor W. 01 January 2003 (has links)
No description available.
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Endosomal regulation of DAF-16FOXO transcription factors by the insulin signaling pathway in «Caenorhabditis elegans»Meras, Icten January 2018 (has links)
No description available.
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