Spelling suggestions: "subject:"animal well"" "subject:"animal cell""
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Lethal and sub-lethal effects of hydrodynamic forces on animal cell cultureGodoy, Ruben D. 21 August 2008 (has links)
No description available.
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Cilia Associated Signaling In Adult Energy HomeostasisRuchi Bansal (12476844) 28 April 2022 (has links)
<p> </p>
<p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>
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利用新式生物反應器培養動物細胞生產日本腦炎病毒 / Using a novel bioreactor to cultivate animal cell for Japanese encephalitis virus production王琪婷, Chi-ting Wang January 1994 (has links)
摘 要
本研究主要是探討利用新式生物反應器以固定化細胞培養技術生產日本腦炎病毒(Japanese encephalitis virus;JEV)之研究,首先根據所培養細胞的生長特性與原有生物反應器之缺點,利用已改良設計之新式生物反應器,評估此新式生物反應器適用性、效能,以及所培養細胞之生長代謝情形與病毒力價。整個實驗過程大致分為幾個階段,第一個階段探討細胞固定化培養之最適化培養條件與生長代謝情形,第二個階段找出細胞固定化培養於此新式生物反應器中最佳生長狀態,最後一個階段為病毒的培養。實驗後發現Vero細胞經固定化貼附於FIBRA-CEL®載體上,可擴大培養於新式生物反應器,Vero細胞最佳生長量達到6.6×106cells/mL。希望藉由此改良之新式生物反應器提供細胞與病毒一個良好之生長培養環境,獲得高產量、品質穩定一致之細胞生物製品,以提供ㄧ設備簡單與製程操作容易、低成本、低能源消耗之細胞製品生產基座。 / Abstract
In this study, we investigated the production of Japanese encephalitis virus (JEV) by the immobilized cell technology in a novel bioreactor. According to the disadvantages of original bioreactor and growth characteristics of cell culture, we evaluated the suitability and efficiency of a design-improved novel bioreactor as well as the growth and metabolic situation of cultured cells and titers of JEV. All studies including three major stages: (1) investigation of the optimal conditions and metabolic situation for the growth of immobilized cells, (2) finding the optimal conditions for the growth of immobilized cells in this novel bioreactor, and (3) growth of JEV using immobilized cells in this novel bioreactor. Our results showed that after immobilization on the FIBRA-CEL® carries, Vero cells can grow on the novel bioreactor up to the density of 6.6 × 106 cells/mL. Hopefully, the improvement of the novel bioreactor will provide an optimal growth condition for both the cells and viruses. Furthermore, it will also provide the basis for the production of cell products with advantages of simple-equipped, easy-to-operate, low cost, and low energy consumption. / 目 錄
誌謝------------------------------------------------- i
中文摘要 -------------------------------------------- ii
英文摘要 -------------------------------------------- iii
目錄 -------------------------------------------- iv
表目錄 -------------------------------------------- v
圖目錄 -------------------------------------------- vi
第一章 緒論---------------------------------------- 1
第二章 文獻探討------------------------------------ 3
第一節 日本腦炎病毒疫苗---------------------------- 3
第二節 動物細胞的培養------------------------------ 4
第三節 載體上動物細胞的培養------------------------ 5
第四節 動物細胞培養於生物反應器-------------------- 7
第三章 材料與方法---------------------------------- 10
一 細胞株的培養-------------------------------- 10
二 細胞冷凍保存與解凍培養---------------------- 10
三 細胞滾瓶培養-------------------------------- 11
四 病毒株的培養-------------------------------- 12
五 固定化載體材料製備-------------------------- 12
六 載體上細胞數的測定-------------------------- 12
七 細胞貼壁率的計算---------------------------- 13
八 生物反應器結構特性與固定化細胞培養---------- 13
九 日本腦炎病毒力價測定------------------------ 19
十 葡萄糖的測定-------------------------------- 19
第四章 結果與討論---------------------------------- 20
一 固定化載體材料比例對Vero細胞生長的影響------ 20
二 細胞貼附固定化時間對Vero細胞生長的影響------ 23
三 細胞接種量對Vero細胞生長的影響-------------- 24
四 生物反應器培養系統對Vero細胞生長的影響------ 25
五 新鮮培養基更換對Vero細胞生長的影響---------- 27
六 最適化細胞生長條件培養日本腦炎病毒---------- 29
第五章 結論與建議---------------------------------- 30
參考文獻 -------------------------------------------- 31
附錄一 PBS配製方法--------------------------------- 62
附錄二 Medium 199配製方法-------------------------- 62
附錄三 MEM medium配製方法-------------------------- 62
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Procédés de cultures de cellules VERO en milieu sans sérum : contributions au développement d'une stratégie PAT / Vero cell culture processes in serum-free medium : contributions to the development of the PAT strategyPetiot, Emma 06 November 2009 (has links)
Ce travail apporte une contribution au développement de la stratégie PAT pour les procédés de culture de cellules animales. Il propose l'amélioration de la compréhension et de la maîtrise de la culture de cellules Vero, dédiées à la production de vaccins viraux, et cultivées sur microporteurs dans un milieu sans sérum. Une première partie a permis de cribler les effets de certains groupes de composés du milieu de culture par le suivi de la croissance en microplaques. Puis, des études cinétiques et métaboliques plus approfondies, réalisées en spinners, ont montré que le métabolisme carboné des cellules Vero est saturé par l'accumulation intracellulaire de pyruvate et qu’il est peu orienté vers la croissance. Alors que le renouvellement du milieu ou l'ajout ponctuel de glutamine amélioraient la croissance cellulaire sans ré-équilibrer le métabolisme, la substitution du glucose et de la glutamine a permis de réduire l’apoptose et d'améliorer les performances métaboliques et de croissance. Par ailleurs, les spectroscopies diélectrique et proche-infrarouge ont été évaluées pour le contrôle en-ligne du procédé, en prenant en compte les particularités des cellules adhérentes. Nous avons montré leur capacité à évaluer les concentrations de cellules, de composés du milieu, et à détecter l'apoptose. Enfin, les principales améliorations par substitution de la glutamine ont été appliquées en bioréacteurs, à la production d'un vaccin prototype contre la dengue, dans des conditions proches de celles d'un procédé industriel. Ceci a permis de limiter les renouvellements de milieu pendant l’expansion cellulaire, sans compromettre la production de particules virales infectieuses / This work contributes to the development of the PAT strategy for animal cell culture processes. The aim of this study was to improve the understanding and the control of Vero cell culture, dedicated to the production of viral vaccines, and grown on microcarriers in serum-free medium. An initial study was performed to screen the effects of certain groups of compounds of the culture medium, by the cell growth monitoring in microplates. Then, kinetic and metabolic studies conducted in spinners flasks allowed to go further and to show that the Vero cell metabolism is saturated through the pyruvate intracellular accumulation and that it is not oriented toward growth. While media renewal or punctual addition of glutamine improve the cell growth without improving the metabolism balance, the substitution of glucose and glutamine allowed to reduce apoptosis and to improve growth and metabolic performances.Furthermore, dielectric and near-infrared spectroscopies have been evaluated for the in-line process monitoring, taking into account the particularities of adherent cells. We have demonstrated their ability to quantify cell concentrations, medium component concentrations, and to detect apoptosis. Finally, major improvements by substitution of glutamine have been applied to bioreactor culture to produce a dengue vaccine prototype, with culture conditions close to industrial process. In these cases, medium renewal during the cell expansion was removed without compromising the production of infectious viral particles
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MRI-TRACKABLE MURINE MODEL OF CEREBRAL RADIATION NECROSISAndrew J. Boria (8703303) 17 April 2020 (has links)
<p>Cerebral radiation necrosis as a
consequence of radiation therapy is often observed in patients several months
to years after treatment. Complications include painful headaches, seizures,
and in the worst-case death. Radiation necrosis is an irreversible condition
with the options available to manage it all having noticeable downsides. As
such, there is a critical need for better ways of either preventing the onset
of necrosis and/or managing its symptoms. As radiation necrosis cannot be
induced in humans for ethical reasons, a mouse model that mirrors the features
of radiation necrosis observed in patients would allow for new techniques to be
tested before being used in human clinical trials. This thesis will explain how
our lab designed a murine model of cerebral radiation necrosis that uses a
320 keV cabinet irradiator to produce radiation necrosis and MRI and histology
to evaluate the development of radiation necrosis at multiple time points.</p><p><br></p>
<p> </p>
<p>Our model required the development
of a mouse positioning apparatus that could be used in the cabinet irradiator
used as well as the machining of lead shields so that focal semi-hemispheric
irradiations could be conducted with other critical structures spared. The MRI
scans used as well as the algorithm used to draw radiation necrosis lesions
were based off what has been used in previous Gamma Knife models of radiation
necrosis. Our initial work showed that since the cabinet irradiator has a
relatively flat dose distribution unlike the Gamma Knife, the radiation lesion
volumes produced in the former either plateaued or decreased, unlike in the
case of the latter where lesion volumes tended to decrease over time. Further
work analyzed the effects of fractionation and found minimal sparing using four
different fractionation schemes. The effects of strain and sex on the
development of radiation necrosis were also analyzed, with strain being found
to be a statistically significant parameter while sex was not. Future research
should focus on testing the effects of new drugs and techniques for better
dealing with radiation necrosis.<b></b></p>
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<b>Investigation of odorant receptors associated with nestmate recognition in the Argentine ant, </b><b><i>L</i></b><b><i>inepithema humile</i></b>Mathew A. Dittmann (5930612) 18 April 2024 (has links)
<p dir="ltr">Given the relatively poor visual acuity of compound eyes, many insects have developed alternative means for navigating their environment. For example, insects often rely on chemosensation to find food, mates, and inter- and intraspecific communication. Eusocial insects in particular have developed complex systems of pheromone communication to organize their colonies, enabling them to partition labor for foraging, brood care, and colony defense tasks to different portions of the colony. A variety of genes coding for proteins are involved in detecting these chemicals, including gustatory receptors, ionotropic receptors, and odorant receptors (ORs). Eusocial insects, and especially ants, have evolved an expanded clade of ORs in their genome, likely due to an increased reliance on pheromones compared to other insects. The ability to recognize nestmates from non-nestmates is one of the vital functions performed by these ORs, which detect hydrocarbons present on the cuticle to distinguish friend from foe. However, research into the details of nestmate recognition has been stymied due to difficulties in manipulating OR genes. Despite advances in genetic sequencing and manipulation technologies, strict reproductive divisions within most ant lineages make generating transgenic ants nearly impossible, and so we have been left with limited options to further investigate these receptors. To narrow down the ORs that could be involved in nestmate recognition in the Argentine ant (Mayr, 1868), I took a multi-pronged approach of generating tissue transcriptomes to identify ORs that are selectively upregulated in the antennae, as well as conducting a phylostratigraphic analysis to identify which OR genes arose more recently in the Argentine ant genome. While conducting these analyses, it became necessary to reannotate the set of Argentine ant OR genes, due to current published annotations not containing the full breadth of <i>L. humile</i> ORs. Finally, I orally administered fluorescently-labelled dsRNA to workers, and tracked the extent to which ingested dsRNA is capable of traversing the tissues of ant workers, to investigate whether RNAi is a viable method for investigating gene function for genes showing tissue-selective expression. I discovered a subset of OR genes that are highly expressed in the antennae and confirmed that dsRNA is able to reach the antennae and knock down OR gene expression through ingestion, meaning that RNA interference is a viable method for the practical study of ant OR genes and can be used to further explore how individual ORs regulate nestmate recognition.</p>
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DYRK1A-RELATED TRABECULAR DEFECTS IN MALE TS65DN MICE EMERGE DURING A CRITICAL DEVELOPMENTAL WINDOWJonathan Mark LaCombe (11022450) 06 August 2021 (has links)
<p> Down syndrome (DS) is a complex genetic disorder caused by
the triplication of human chromosome 21 (Hsa21). The presence of an extra copy
of an entire chromosome greatly disrupts the copy number and expression of over
350 protein coding genes. This gene dosage imbalance has far-reaching effects on
normal development and aging, leading to cognitive and skeletal defects that
emerge earlier in life than the general population.</p>
<p> The present
study begins by characterizing skeletal development in young male Ts65Dn mice to
test the hypothesis that skeletal defects in male Ts65Dn mice are developmental
in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of
age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical
defects were present generally throughout development, but trabecular defects emerged
at P30 and persisted until P42. </p>
<p> The gene <i>Dual-specificity
tyrosine-regulated kinase 1a </i>(<i>Dyrk1a</i>) is triplicated in both
DS and in Ts65Dn mice and has been implicated as a putative cause of both
cognitive and skeletal defects. To test the hypothesis that trisomic <i>Dyrk1a</i>
is related to the emergence of trabecular defects at P30, expression of <i>Dyrk1a</i>
in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown
to fluctuate throughout development and overexpression generally aligned with
the emergence of trabecular defects at P30.</p>
<p> The growth
rate in trabecular measures between male Ts65Dn and euploid littermates was
similar between P30 and P42, suggesting a closer look into cellular mechanisms
at P42. Assessment of proliferation of BMSCs, differentiation and activity of
osteoblasts showed no significant differences between Ts65Dn and euploid
cellular activity, suggesting that the cellular microenvironment has a greater
influence on cellular activity than genetic background.</p>
These
data led to the hypothesis that reduction of <i>Dyrk1a</i> gene expression and
pharmacological inhibition of DYRK1A could be executed during a critical period
to prevent the emergence of trabecular defects at P30. To tests this hypothesis,
doxycycline-induced cre-lox recombination to reduce <i>Dyrk1a</i> gene copy
number or the DYRK1A inhibitor CX-4945 began at P21. The results of both
genetic and pharmacological interventions suggest that trisomic <i>Dyrk1a</i>
does not influence the emergence of trabecular defects up to P30. Instead, data
suggest that the critical window for the rescue of trabecular defects lies
between P30 and P42.
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Investigating TRPV4 Signaling in Choroid Plexus Culture ModelsLouise Susannah Hulme (12456711) 12 July 2022 (has links)
<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p>
<p><br></p>
<p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p>
<p><br></p>
<p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>
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Identification and characterization of microRNAs which moderate neutrophil migration and acute inflammationAlan Y Hsu (8912033) 09 September 2022 (has links)
<p>Neutrophils are the first cells recruited to an immune
stimulus stemming from infection or sterile injuries via a mixture of
chemoattractant cues. In addition to eliminating pathogens, neutrophils
coordinate the overall inflammation by activating and producing inflammatory
signals in the tissue while modulating the activation of other immune cells
which in some cases leads to adverse tissue damage. Over amplified or chronic
neutrophil recruitment directly leads to autoimmune diseases including
rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer.
Dampening neutrophil recruitment is a strategy to intervene in
neutrophil-orchestrated chronic inflammation. Despite intensive research over
the past several decades, clinical studies targeting neutrophil migration have
been largely unsuccessful, possibly due to the prominent redundancy of adhesion
receptors and chemokines. Additional challenges lie in the balance of dampening
detrimental inflammation while preserving immunity. Neutrophils are terminally
differentiated cells that are hard to study in cell culture. Mouse models are
often used to study hematopoiesis, migration, and chemotaxis of neutrophils but
is very labor intensive. To discover novel therapeutic targets that modulate
neutrophil migration, we performed a neutrophil-specific microRNA (miRNA)
overexpression screen in zebrafish and identified eight miRNAs as potent
suppressors of neutrophil migration. We have generated transgenic zebrafish
lines that overexpresses these candidate miRNAs where we recapitulated the
mitigation in neutrophil motility and chemotaxis to tissue injury or infection.
Among those we further characterized two miRNAs which have not been reported to
regulate neutrophil migration, namely miR-722 and miR-199.</p>
<p> </p>
<p>MiR-722 downregulates the transcript level of <i>rac2</i> through binding to the <i>rac2</i> 3'UTR. Furthermore, miR-722-overexpressing
larvae display improved outcomes in both sterile and bacterial systemic models,
which correlates with a robust upregulation of the anti-inflammatory cytokines
in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide
challenge. In addition, overexpression of mir-722 reduced chemotaxis of human
neutrophil like cells, indicating that miR-722
is a potential agent to reduce inflammation in humans. </p>
<p>MiR-199<i>,</i> decreases neutrophil chemotaxis in zebrafish
and human neutrophil-like cells. Intriguingly, in terminally differentiated
neutrophils, miR-199 alters the cell cycle-related pathways and
directly suppresses cyclin-dependent kinase 2 (<i>cdk2</i>), whose known
activity is restricted to cell cycle progression and cell differentiation.
Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis
of zebrafish neutrophils without inducing cell death. Human neutrophil-like
cells deficient in CDK2 fail to polarize and display altered signaling
downstream of the formyl peptide receptor. Chemotaxis of primary human
neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression
or CDK2 inhibition significantly improves the outcome of lethal systemic
inflammation challenges in zebrafish. </p>
<p> </p>
<p>In summary, our results reveal previously unknown functions
of these miRNAs, and
provide potential avenues to modulate neutrophil migration as well as lead to
discoveries of novel factors which can regulate this process. We have also
discovered a non-classical role of CDK2 in regulating neutrophil migration
which provides directions for alleviating systemic inflammation and a better
understanding of neutrophil biology. </p>
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Inferencing Gene Regulatory Networks for Drosophila Eye Development Using an Ensemble Machine Learning ApproachAbdul Jawad Mohammed (18437874) 29 April 2024 (has links)
<p dir="ltr">The primary purpose of this thesis is to propose and demonstrate BioGRNsemble, a modular and flexible approach for inferencing gene regulatory networks from RNA-Seq data. Integrating the GENIE3 and GRNBoost2 algorithms, this ensembles-of-ensembles method attempts to balance the outputs of both models through averaging, before providing a trimmed-down gene regulatory network consisting of transcription and target genes. Using a Drosophila Eye Dataset, we were able to successfully test this novel methodology, and our validation analysis using an online database determined over 3500 gene links correctly detected, albeit out of almost 530,000 predictions, leaving plenty of room for improvement in the future.</p>
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