Current Search: Honors Student Theses (x) » Faris, Andrew (x) » Quintyne, Nicholas (x)
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- Title
- Anchors aweigh: using shRNA TO MAP p150Glued functionality in anchoring microtubules at the centrosome.
- Creator
- Hazellief, Kristal, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Dynactin is a multisubunit protein complex required for proper functioning of the microtubule motor, cytoplasmic dynein. Dynactin serves as a processivity factor for the motor as well as a cargo adaptor, allowing dynein to function in a wide array of cellular processes. Additionally, dynactin serves as a microtubule anchor. The p150Glued subunit of dynactin is of particular importance to these processes, as it possesses dynactin’s microtubule binding sequences, termed the CAP-Gly and Basic...
Show moreDynactin is a multisubunit protein complex required for proper functioning of the microtubule motor, cytoplasmic dynein. Dynactin serves as a processivity factor for the motor as well as a cargo adaptor, allowing dynein to function in a wide array of cellular processes. Additionally, dynactin serves as a microtubule anchor. The p150Glued subunit of dynactin is of particular importance to these processes, as it possesses dynactin’s microtubule binding sequences, termed the CAP-Gly and Basic domains. These domains have differential affinities for microtubules, with CAP-Gly binding to microtubules with a higher affinity than Basic. By testing a set of shRNA plasmids with antisense sequences to the untranslated region of p150Glued we have found effective knockdown of the protein in COS-7 cells; future researchers can then introduce plasmids for p150Glued lacking either the CAP-Gly or Basic domains, or both, potentially showing a differential effect on anchoring, dependent upon which domain is present.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003516
- Format
- Document (PDF)
- Title
- Cellular effects of knocking down expression of dynactin's p150Glued subunit.
- Creator
- Praver, Joseph, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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Dynactin is made up of 11 different peptide units with a distinct ultrastructure consisting of a shoulder/sidearm complex and a rod-like domain. It functions as a cofactor for cytoplasmic dynein, assisting in the processes of long-range vesicle movements, microtubule anchoring, endomembrane localization, and mitotic progression. Previous studies have shown that dynactin binds to microtubules at the centrosome, keeping a radial arrangement during interphase. The p150Glued subunit contains two...
Show moreDynactin is made up of 11 different peptide units with a distinct ultrastructure consisting of a shoulder/sidearm complex and a rod-like domain. It functions as a cofactor for cytoplasmic dynein, assisting in the processes of long-range vesicle movements, microtubule anchoring, endomembrane localization, and mitotic progression. Previous studies have shown that dynactin binds to microtubules at the centrosome, keeping a radial arrangement during interphase. The p150Glued subunit contains two distinct microtubule-binding domains: CAP-Gly and Basic. While both of these sequences can interact with microtubules, CAP-Gly has a much greater affinity for binding to microtubules, suggesting that the two domains may be active for different functions within the cell. Using shRNA, I looked at the overexpression and knockdown of p150Glued and examined the effect that had on the cell. Knockdown has been shown to cause defects in centrosome organization and mitotic index.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003599
- Format
- Document (PDF)
- Title
- Correlation between Specific Carcinogens and Mitotic Defects in Uterine Cancer.
- Creator
- Zlotcavitch, Leonid, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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The development of uterine cancer and its progression can be linked to external elements such as carcinogens, natural hormones and chemotherapeutic drugs. The goal of this project was to investigate the possible correlation between exposure to specific chemical and an increase of mitotic defects. These defects manifest themselves as lagging chromosomes, multipolar spindles, and anaphase bridges. Using the uterine cancer cell line MES-SA as a model, we established a baseline for the mitotic...
Show moreThe development of uterine cancer and its progression can be linked to external elements such as carcinogens, natural hormones and chemotherapeutic drugs. The goal of this project was to investigate the possible correlation between exposure to specific chemical and an increase of mitotic defects. These defects manifest themselves as lagging chromosomes, multipolar spindles, and anaphase bridges. Using the uterine cancer cell line MES-SA as a model, we established a baseline for the mitotic defects in the absence and presence of various chemicals using γ-tubulin and DAPI-immune stained fixed cells. The cells were treated with varying concentrations of Vinyl Chloride, β-estradiol, Fluvestrant, and were examined using Immunofluorescent microscopy. We found that an exposure to Vinyl Chloride, β-estradiol and Fulvestrant have increased the number of mitotic defects in this cell line.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003611_0
- Format
- Document (PDF)
- Title
- Development of cell-based functional assays to identify and characterize novel GABAb receptor allosteric modulators.
- Creator
- Aitken, Maria de Lourdes, McDonald, Patricia, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Dysfunction of GABAB-receptor (GABAB-R)-mediated synaptic transmission underlies various nervous system disorders including epilepsy, depression, schizophrenia, and addiction. Currently, only one GABAB-R orthosteric ligand is in clinical use. However, side effects such as sedation, tolerance, and motor impairment limit its use. A dissociation of the therapeutic effects from the side effects may be achievable with drugs enhancing the endogenous physiological cellular response. The development...
Show moreDysfunction of GABAB-receptor (GABAB-R)-mediated synaptic transmission underlies various nervous system disorders including epilepsy, depression, schizophrenia, and addiction. Currently, only one GABAB-R orthosteric ligand is in clinical use. However, side effects such as sedation, tolerance, and motor impairment limit its use. A dissociation of the therapeutic effects from the side effects may be achievable with drugs enhancing the endogenous physiological cellular response. The development of GABAB-R allosteric modulators has provided new modes of efficacy that may facilitate the development of novel therapeutic agents. In the present study, we investigated the effects of novel, newly synthesized GABAB-R allosteric ligands using a HEK-293 cell line stably expressing human GABAB1(b)/human GABAB(2) subunits using a cell-based cAMP HTRF assay. One compound was identified and characterized as a potential novel GABAB-R positive allosteric modulator (PAM), and will be subjected to further in vitro and in vivo analyses.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003502
- Format
- Document (PDF)
- Title
- Disrupting methamphetamine associated memory by targeting synaptic dynamics.
- Creator
- Zigmond, Zachary, Miller, Courtney, Young, Erica, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Methamphetamine (METH) is addictive and associated with a high rate of relapse. One relapse trigger is re-experiencing drug-associated contextual associations. Therefore it is possible that, by targeting METH-associated contextual memories, drug seeking behavior can be inhibited. Recent evidence has suggested that memory formation relies on actin polymerization, which allows dendritic spines to undergo structural and functional plasticity, key components of memory. To see if actin...
Show moreMethamphetamine (METH) is addictive and associated with a high rate of relapse. One relapse trigger is re-experiencing drug-associated contextual associations. Therefore it is possible that, by targeting METH-associated contextual memories, drug seeking behavior can be inhibited. Recent evidence has suggested that memory formation relies on actin polymerization, which allows dendritic spines to undergo structural and functional plasticity, key components of memory. To see if actin polymerization could be a target for the extinction of METH seeking memories we inhibited actin polymerization in animals that had been trained in either METH or food associated conditioned place preference. Pretest inhibition of actin cycling in the basolateral amygdala complex produced immediate and persistent extinction of METH seeking behavior. Additionally, inhibiting actin polymerization 24hrs before testing disrupted seeking behavior for METH but not food. These results indicate that METH-associated memories are selectively vulnerable to disruption through inhibition of actin dynamics.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003542
- Format
- Document (PDF)
- Title
- Keep it together: knockdowns of dynactin subunits demonstrate varying levels of importance in dynactin structure and microtubule organization.
- Creator
- Turn, Christina, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Dynactin, a multisubunit protein complex, serves as a processivity factor and cargo adaptor for the microtubule motor cytoplasmic dynein. Together, these proteins permit mitotic progression, intracellular trafficking, and endomembrane organization. Dynactin also independently anchors microtubules at the centrosome during interphase. In this study, we use shRNAs to knock down expression of the p24, p27, p62, and Arp1 subunits of dynactin to determine those components’ roles in anchoring....
Show moreDynactin, a multisubunit protein complex, serves as a processivity factor and cargo adaptor for the microtubule motor cytoplasmic dynein. Together, these proteins permit mitotic progression, intracellular trafficking, and endomembrane organization. Dynactin also independently anchors microtubules at the centrosome during interphase. In this study, we use shRNAs to knock down expression of the p24, p27, p62, and Arp1 subunits of dynactin to determine those components’ roles in anchoring. Recent work has shown p62’s importance to dynactin integrity (Yeh et al., 2012), and we confirm that its loss disrupts microtubule organization. The peripheral p27 and binding partner p25 seem to contribute to membrane selectivity and have little effect on dynactin structure and anchoring. We find that an intact dynactin complex is necessary to maintain microtubule organization via the anchoring complex. While not directly bound to microtubules, these subunits provide crucial structural support to facilitate interactions among dynactin and other centrosomal anchoring components.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003537
- Format
- Document (PDF)
- Title
- Mechanisms of Neuroprotection Mediated by Ghrelin in Neuronal Cells.
- Creator
- Metzner, Michael John, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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Ghrelin is a 28-amino acid peptide secreted by the stomach. Over the past decade, studies have shown ghrelin to have broad effects on growth hormone release, appetite regulation, and glucose metabolism. These effects are explained largely by the high expression of the ghrelin receptor (GHS-R1a) in both the hypothalamus and pituitary. Recently, ghrelin has been shown to have possible neuroprotective effects in other brain regions expressing GHS-R1a, specifically stimulating anti-apoptotic and...
Show moreGhrelin is a 28-amino acid peptide secreted by the stomach. Over the past decade, studies have shown ghrelin to have broad effects on growth hormone release, appetite regulation, and glucose metabolism. These effects are explained largely by the high expression of the ghrelin receptor (GHS-R1a) in both the hypothalamus and pituitary. Recently, ghrelin has been shown to have possible neuroprotective effects in other brain regions expressing GHS-R1a, specifically stimulating anti-apoptotic and antiinflammatory pathways. The endoplasmic reticulum (ER) is a cellular organelle responsible for processing proteins. When an abundance of misfolded proteins accumulate in the ER, cellular stress pathways ensue, and are linked to a number of neurodegenerative diseases. This project will examine the effects of ghrelin during ER stress in a human neuronal cell line. Expression of genes and proteins involved in potential neuroprotective pathways will be examined to learn how ghrelin mediates its anti-apoptotic effects in neurons.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003595
- Format
- Document (PDF)
- Title
- MYC-induced repression of Tristetraprolin alters the expression of are-containing genes in prostate cancer.
- Creator
- Altman, Evan, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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Prostate cancer has the second highest mortality rate of all cancers in men. The Myc oncoprotein is misregulated in 70% of all cancers, including 70% of prostate cancers, and affects several cancer mechanisms. Myc is able to directly repress the expression of Tristetraprolin (TTP). TTP regulates mRNA stability by binding to select mRNAs. Furthermore, TTP is able to suppress Myc‐driven B cell lymphoma in mice. In these studies, cell culture models were used to access the role of Myc-induced...
Show moreProstate cancer has the second highest mortality rate of all cancers in men. The Myc oncoprotein is misregulated in 70% of all cancers, including 70% of prostate cancers, and affects several cancer mechanisms. Myc is able to directly repress the expression of Tristetraprolin (TTP). TTP regulates mRNA stability by binding to select mRNAs. Furthermore, TTP is able to suppress Myc‐driven B cell lymphoma in mice. In these studies, cell culture models were used to access the role of Myc-induced repression of TTP in prostate cancer. Prostate cancer cells lines were identified with inverse expression of Myc and TTP. Additionally, ARE‐containing genes with roles in various cancer mechanisms were differentially expressed in these models. These findings suggest that Myc’s ability to downregulate TTP is important in prostate cancer and provide new avenues for treating Myc‐driven prostate cancer.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003554
- Format
- Document (PDF)
- Title
- Regulation of dynactin-mediated microtubule anchoring by the p24 subunit.
- Creator
- McCullough, Lindsay, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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Dynactin is a 1.2 MDa multisubunit complex that is involved in a variety of cellular functions including vesicle motility, microtubule organization, and cell division. The function of most dynactin subunits are at least partially characterized, with the exception of p24. Very few things are known about p24: it has a molecular weight of ~20,800 Da, is mostly α-helical, is only found in dynactin and binds directly to p150Glued. We are using overexpression, shRNA-mediated knockdown, and...
Show moreDynactin is a 1.2 MDa multisubunit complex that is involved in a variety of cellular functions including vesicle motility, microtubule organization, and cell division. The function of most dynactin subunits are at least partially characterized, with the exception of p24. Very few things are known about p24: it has a molecular weight of ~20,800 Da, is mostly α-helical, is only found in dynactin and binds directly to p150Glued. We are using overexpression, shRNA-mediated knockdown, and fluorescent microscopy, to determine a role for p24. When p24 is overexpressed, p150Glued is lost from the centrosome and Arp 1 remains, suggesting disruption of the complex. In addition, microtubule organization is disrupted. When p24 is knocked down, we retain p150Glued at the centrosome, along with Arp 1 and again see microtubule disorganization. These data suggest that p24 may act as an anchor to stabilize p150Glued when it binds to microtubules.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003594
- Format
- Document (PDF)
- Title
- Regulation of mitosis by the motor Protein KIF9.
- Creator
- Billow, Alexa M., Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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The kinesin superfamily of microtubule motors is subdivided based upon structure and function. KIF9 is a member of the largely uncharacterized Kinesin-9 family. It was originally identified by sequence homology to other kinesins. Subsequent studies have indicated that KIF9 interacts with proteins involved in cell shape remodeling and cell migration. We have examined KIF9 function in mammalian cells using siRNA-mediated knockdown. By knocking down KIF9 expression in these cells, we have seen...
Show moreThe kinesin superfamily of microtubule motors is subdivided based upon structure and function. KIF9 is a member of the largely uncharacterized Kinesin-9 family. It was originally identified by sequence homology to other kinesins. Subsequent studies have indicated that KIF9 interacts with proteins involved in cell shape remodeling and cell migration. We have examined KIF9 function in mammalian cells using siRNA-mediated knockdown. By knocking down KIF9 expression in these cells, we have seen several effects on normal cell cycle progression. We have observed a decreased mitotic index, suggesting cells either fail to enter mitosis or abort the process prior to completion. Of the cells in that population that enter mitosis, we see a decrease in the number of cells in anaphase and telophase. Multinuclearity is greatly increased, indicating failure of cytokinesis. Interphase cells show decreased microtubule organization. We propose that KIF9 is important for normal completion of mitosis by regulating the contractile ring.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003559
- Format
- Document (PDF)
- Title
- Septins: one ring to bind them.
- Creator
- Kain, Anthony, Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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The cytoskeleton is composed of dynamic polymers of actin, tubulin, and intermediate filaments. These proteins are responsible for maintaining cell shape, intracellular organization and transport, cell division, and many other cellular processes. Among the cytoskeletal interacting proteins are septins, a conserved family of GTP binding proteins that polymerize into higher ordered filaments. Septins interact with and regulate the dynamics of both actin and microtubule cytoskeletons. Septins...
Show moreThe cytoskeleton is composed of dynamic polymers of actin, tubulin, and intermediate filaments. These proteins are responsible for maintaining cell shape, intracellular organization and transport, cell division, and many other cellular processes. Among the cytoskeletal interacting proteins are septins, a conserved family of GTP binding proteins that polymerize into higher ordered filaments. Septins interact with and regulate the dynamics of both actin and microtubule cytoskeletons. Septins also regulate the processes of cytokinesis and exocytosis. In this study we use COS-7 cells as a model for studying the localization, interaction, and dynamics of SEPT2 and SEPT7. We demonstrate that septins form filaments that colocalize with actin filaments in the cell periphery. Further, we show that actin filaments, but not microtubules, are required for formation of septin filaments in vivo.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003588
- Format
- Document (PDF)
- Title
- The determination of KIF9's possible involvement in vesicular transport.
- Creator
- Cabibi, Lewis J., Quintyne, Nicholas, Harriet L. Wilkes Honors College, Florida Atlantic University
- Abstract/Description
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Vesicular transport is vital to the living cell. Motor proteins, such as the kinesin superfamily proteins (KIFs), travel along microtubules with attached vesicles to achieve vesicular transport (Hirokawa 2008). However, not all kinesins participate in vesicular transport, some are required for other cellular functions. A recent study showed that the KIF9 motor is involved in keeping the microtubule organization center (MTOC) correctly positioned and oriented in Dictyostelium (Tikhonenko et al...
Show moreVesicular transport is vital to the living cell. Motor proteins, such as the kinesin superfamily proteins (KIFs), travel along microtubules with attached vesicles to achieve vesicular transport (Hirokawa 2008). However, not all kinesins participate in vesicular transport, some are required for other cellular functions. A recent study showed that the KIF9 motor is involved in keeping the microtubule organization center (MTOC) correctly positioned and oriented in Dictyostelium (Tikhonenko et al., 2009), suggesting it may be involved in non-transport functions. By modulating protein expression levels of KIF9 in COS7 cells, this study aims to determine whether or not KIF9 contributes to vesicular motility. Using a fluorescent dye, we have been able to track lysosome movements in living cells. Our analysis has shown that decreasing KIF9 expression has no effect on the velocity of vesicle motility, but does slightly affect processivity. This suggests that KIF9 is not directly involved in cell motility, and serves another function within the cell which may serve to decrease overall processivity.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/fau/fd/FA00003566
- Format
- Document (PDF)
- Title
- Tracking mitotic defects via time-lapse photography.
- Creator
- Williams, Joseph, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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As tumors generate, there is a progression in genomic instability derived from chromosomal rearrangement and instability. Often, these manifest themselves as defects in mitosis, frequently as lagging chromosomes, multipolar spindles, and anaphase bridges. Lagging chromosomes are the result of inaccurate chromosomal division in mitosis, thus jeopardizing the genome of an organism’s offspring; they derive from several errors, such as failure of a chromosome to attach to the mitotic spindle. The...
Show moreAs tumors generate, there is a progression in genomic instability derived from chromosomal rearrangement and instability. Often, these manifest themselves as defects in mitosis, frequently as lagging chromosomes, multipolar spindles, and anaphase bridges. Lagging chromosomes are the result of inaccurate chromosomal division in mitosis, thus jeopardizing the genome of an organism’s offspring; they derive from several errors, such as failure of a chromosome to attach to the mitotic spindle. The goal of this project has been to characterize the mechanisms of lagging chromosomes in the cancer cell line UPCI:SCC103. Our laboratory’s work has shown that treatment with certain carcinogens increase the rate of mitotic defect. To further our understanding these defects, we are monitoring the progression of lagging chromosomes in UPCI:SCC103 cells with live cell analysis, using GFP-tagged histone H2B to track their appearance and fate, so to distinguish between the possible causes and resolutions of this mitotic defect.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003539
- Format
- Document (PDF)
- Title
- Using oral cancer cell line UPCI: SCC078 to purify NuMA protein.
- Creator
- Rodrigues, Ana, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Formation of multipolar spindles is closely linked to increased genomic instability and tumor progression. Centrosome hyperamplification is insufficient to initiate this mitotic defect, centrosome coalescence must be interrupted. Studies have indicated that cytoplasmic dynein is a key factor in preventing multipolarity, and overexpression of the NuMA protein is sufficient to mislocalize dynein from the spindle and abrogate the coalescence machinery. Because the mechanism by which NuMA can...
Show moreFormation of multipolar spindles is closely linked to increased genomic instability and tumor progression. Centrosome hyperamplification is insufficient to initiate this mitotic defect, centrosome coalescence must be interrupted. Studies have indicated that cytoplasmic dynein is a key factor in preventing multipolarity, and overexpression of the NuMA protein is sufficient to mislocalize dynein from the spindle and abrogate the coalescence machinery. Because the mechanism by which NuMA can inhibit dynein is unclear, we are purifying NuMA to use in in vitro studies, to better understand how NuMA blocks dynein activity. Purifying NuMA from recombinant sources has not been successful; therefore we are utilizing a native source. We are using the oral cancer cell line UPCI:SCC078 as the source because it has nine copies of the NUMA1 gene. With modifications to the protocols used previously, our goal is to yield sufficient quantities of NuMA for biochemical analysis with purified NuMA.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003532
- Format
- Document (PDF)
- Title
- You’ve got to keep ‘em separated: characterizing lagging chromosome prevention in oral cancer cells.
- Creator
- Beltran, Rosa Nathalie, Quintyne, Nicholas, Harriet L. Wilkes Honors College
- Abstract/Description
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Mitosis is the separation of duplicated chromosomes into two daughter cells in order to create viable offspring. There are many checks in mitosis to ensure the inherited chromosome number is correct. Sometimes, these checkpoints are overcome and daughter cells inherit defects which can lead to cancer. One defect is the appearance of lagging chromosomes, the result of inaccurate chromosomal separation which leads to incorrect chromosome number termed aneuploidy. Aneuploidy is one of the...
Show moreMitosis is the separation of duplicated chromosomes into two daughter cells in order to create viable offspring. There are many checks in mitosis to ensure the inherited chromosome number is correct. Sometimes, these checkpoints are overcome and daughter cells inherit defects which can lead to cancer. One defect is the appearance of lagging chromosomes, the result of inaccurate chromosomal separation which leads to incorrect chromosome number termed aneuploidy. Aneuploidy is one of the defining traits of cancerous cells. The potential mechanism of lagging chromosomes in the cancerous cell line UPCI:OSCC070 is investigated in this study. siRNA-induced knockdown of KIFC1, a protein that is involved in the centrosomal clustering to prevent multipolar spindles, was used in the cells. Examining both levels of knockdown and time of exposure, we saw that the loss of KIFC1 led to a significant increase in lagging chromosomes, indicating this protein is critical to proper mitotic progression.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/fau/fd/FA00003505
- Format
- Document (PDF)