Effects of short-term detraining on postprandial metabolism, endothelial function, and inflammation in endurance-trained men:dissociation between changes in triglyceride metabolism and endothelial function

Endurance-trained athletes experience a low level of postprandial lipaemia, but this rapidly increases with detraining. We sought to determine whether detraining-induced changes to postprandial metabolism influenced endothelial function and inflammation. Eight endurance-trained men each undertook two oral fat tolerance tests [blood taken fasted and for 6 h following a high-fat test meal (80 g fat, 80 g carbohydrate)]: one during a period of their normal training (trained) and one after 1 wk of no exercise (detrained). Endothelial function in the cutaneous microcirculation was assessed using laser Doppler imaging with iontophoresis in the fasted state and 4 h postprandially during each test. Fasting plasma triglyceride (TG) concentrations increased by 35% with detraining (P = 0.002), as did postprandial plasma (by 53%, P = 0.002), chylomicron (by 68%, P = 0.02) and very low-density lipoprotein (by 51%, P = 0.005) TG concentrations. Endothelial function decreased postprandially in both the trained (by 17%, P = 0.03) and detrained (by 22%, P = 0.03) conditions but did not differ significantly between the trained and detrained conditions in either the fasted or the postprandial states. These results suggest that, although fat ingestion induces endothelial dysfunction, interventions that alter postprandial TG metabolism will not necessarily concomitantly influence endothelial function.

Advanced tilted fiber gratings and their applications

This thesis presents a detailed numerical analysis, fabrication method and experimental investigation on 45º tilted fiber gratings (45º-TFGs) and excessively tilted fiber gratings (Ex-TFGs), and their applications in fiber laser and sensing systems. The one of the most significant contributions of the work reported in this thesis is that the 45º-TFGs with high polarization extinction ratio (PER) have been fabricated in single mode telecom and polarization maintaining (PM) fibers with spectral response covering three prominent optic communication and central wavelength ranges at 1060nm, 1310nm and 1550nm. The most achieved PERs for the 45º-TFGs are up to and greater than 35-50dB, which have reached and even exceeded many commercial in-fiber polarizers. It has been proposed that the 45º-TFGs of high PER can be used as ideal in-fiber polarizers for a wide range of fiber systems and applications. In addition, in-depth detailed theoretical models and analysis have been developed and systematic experimental evaluation has been conducted producing results in excellent agreement with theoretical modeling. Another important outcome of the research work is the proposal and demonstration of all fiber Lyot filters (AFLFs) implemented by utilizing two (for a single stage type) and more (for multi-stage) 45º-TFGs in PM fiber cavity structure. The detailed theoretical analysis and modelling of such AFLFs have also been carried out giving design guidance for the practical implementation. The unique function advantages of 45º-TFG based AFLFs have been revealed, showing high finesse multi-wavelength transmission of single polarization and wide range of tuneability. The temperature tuning results of AFLFs have shown that the AFLFs have 60 times higher thermal sensitivity than the normal FBGs, thus permitting thermal tuning rate of ~8nm/10ºC. By using an intra-cavity AFLF, an all fiber soliton mode locking laser with almost total suppression of siliton sidebands, single polarization output and single/multi-wavelength switchable operation has been demonstrated. The final significant contribution is the theoretical analysis and experimental verification on the design, fabrication and sensing application of Ex-TFGs. The Ex-TFG sensitivity model to the surrounding medium refractive index (SRI) has been developed for the first time, and the factors that affect the thermal and SRI sensitivity in relation to the wavelength range, tilt angle, and the size of cladding have been investigated. As a practical SRI sensor, an 81º-TFG UV-inscribed in the fiber with small (40μm) cladding radius has shown an SRI sensitivity up to 1180nm/RIU in the index of 1.345 range. Finally, to ensure single polarization detection in such an SRI sensor, a hybrid configuration by UV-inscribing a 45º-TFG and an 81º-TFG closely on the same piece of fiber has been demonstrated as a more advanced SRI sensing system.

Commuting Nonselfadjoint Operators and their Characteristic Operator-Functions

* Partially supported by Grant MM-428/94 of MESC.

Randomized Push-Out Mechanisms in Priority Queuing and their Probability Characteristics

2002 Mathematics Subject Classification: 60K25.

(G, λ)-Extremal Processes and Their Relationship with Max-Stable Processes

2000 Mathematics Subject Classification: 60G70, 60G18.

Piecewise Convex Curves and their Integral Representation

2000 Mathematics Subject Classification: 52A10.

UV-Inscribed optical fiber gratings in Mid-IR range and their laser applications

We have UV-inscribed fiber Bragg gratings (FBGs), long-period gratings (LPGs), and tilted fiber gratings (TFGs) into mid-IR 2μm range using three common optical fiber grating fabrication techniques (two-beam holographic, phase mask, and point-by-point). The fabricated FBGs have been evaluated for thermal and strain response. It has been revealed that the FBG devices with responses in mid-IR range are much more sensitive to temperature than that in near-IR range. To explore the unique cladding mode coupling function, we have investigated the thermal and refractive index sensitivities of LPGs and identified that the coupled cladding modes in mid-IR range are also much more sensitive to temperature and surrounding medium refractive index change. The 45° tilted fiber gratings (45°-TFGs) as polarizing devices in mid-IR have been investigated for their polarization extinction characteristics. As efficient reflection filters and in-cavity polarizers, the mid-IR FBGs and 45°-TFGs have been employed in fiber laser cavity to realize multi-wavelength 2 μm Tm-doped CW and mode locked fiber lasers, respectively.

Stressors, mediators, and health care outcomes of home enteral nutrition in family caregivers and their older adult care recipients

Family caregivers manage home enteral nutrition (HEN) for over 77% of an estimated 1 of every 400 Medicare recipients. Increasing usage of HEN in older adults combined with reliance on family caregivers raises concerns for the quality, outcomes, and costs of care. These concerns are relevant in light of Medicare limitations on nursing assistance and non-reimbursement for nutrition services, despite annual costs of over $600 million. This study applied stress process theories to assess stressor, mediator, and outcome variables salient to HEN and caregiving. In-home structured interviews occurred with a multi-ethnic sample of 30 caregiving dyads at 1–3 months after discharge on HEN. Care recipients were aged ≥60 (M = 68.4 years) and did not have dementia. Caregivers were aged ≥21, unpaid, and lived within 45 minutes of care recipients. Caregivers performed an average of 19.7 tasks daily for 61.9 hours weekly. Training needs were identified for 33 functional, care management, technical, and nutritional tasks. Preparedness scores were low (M = 1.73/4.0), and positively correlated with competence, self-rated quality of care and positive feelings, and negatively with overload, role captivity, and negative feelings (Ps < .05). Caregivers had multiple changes in lifestyle and dietary behaviors. Lifestyle changes positively correlated with overload, and negatively with preparedness and positive feelings. Dietary changes positively correlated with number of tasks, overload, role captivity and negative feelings, and negatively with preparedness (Ps < .01). Fifty-seven percent of caregivers aged >50 were at nutrition risk. Care recipients fared worse. Average weight change was −4.35 pounds (P < .001). Physical complications interrupted daily enteral infusions. Water intake was half of fluid need and associated with signs of dehydration (P < .001). Physical and social function was poor, with older subjects more impaired ( P < .04). Those with better prepared or less overloaded caregivers had higher functionality and QOL (P < .002). Complications, type of feeding tube, and caregiver preparedness correlated with frequency of health care utilization (Ps < .05). Efficacy of HEN in older adults requires specialized caregiver training, attention to caregivers' needs, and frequent monitoring from a highly skilled multidisciplinary team including dietitians. ^

Improving the scientific reliability of biological detection of explosives by Canis familiaris: Active odour signatures and their implications

The use of canines as a method of detection of explosives is well established worldwide and those applying this technology range from police forces and law enforcement to humanitarian agencies in the developing world. Despite the recent surge in publication of novel instrumental sensors for explosives detection, canines are still regarded by many to be the most effective real-time field method of explosives detection. However, unlike instrumental methods, currently it is difficult to determine detection levels, perform calibration of the canines' ability or produce scientifically valid quality control checks. Accordingly, amongst increasingly strict requirements regarding forensic evidence admission such as Frye and Daubert, there is a need for better scientific understanding of the process of canine detection. ^ When translated to the field of canine detection, just like any instrumental technique, peer reviewed publication of the reliability, success and error rates, is required for admissibility. Commonly training is focussed towards high explosives such as TNT and Composition 4, and the low explosives such as Black and Smokeless Powders are added often only for completeness. ^ Headspace analyses of explosive samples, performed by Solid Phase Microextraction (SPME) paired with Gas Chromatography - Mass Spectrometry (GC-MS), and Gas Chromatography - Electron Capture Detection (GC-ECD) was conducted, highlighting common odour chemicals. The odour chemicals detected were then presented to previously trained and certified explosives detection canines, and the activity/inactivity of the odour determined through field trials and experiments. ^ It was demonstrated that TNT and cast explosives share a common odour signature, and the same may be said for plasticized explosives such as Composition C-4 and Deta Sheet. Conversely, smokeless powders were demonstrated not to share common odours. An evaluation of the effectiveness of commercially available pseudo aids reported limited success. The implications of the explosive odour studies upon canine training then led to the development of novel inert training aids based upon the active odours determined. ^

Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Capabilities of Police and Military Forces in Central America -- A Comparative Analysis of Guatemala, El Salvador, Honduras an d Nicaragua

A difficult transition to a new paradigm of Democratic Security and the subsequent process of military restructuring during the nineties led El Salvador, Honduras, Guatemala and Nicaragua to re-consider their old structures and functions of their armed forces and police agencies. This study compares the institutions in the four countries mentioned above to assess their current condition and response capacity in view of the contemporary security challenges in Central America. This report reveals that the original intention of limiting armies to defend and protect borders has been threatened by the increasing participation of armies in public security. While the strength of armies has been consolidated in terms of numbers, air and naval forces have failed to become strengthened or sufficiently developed to effectively combat organized crime and drug trafficking and are barely able to conduct air and sea operations. Honduras has been the only country that has maintained a proportional distribution of its armed forces. However, security has been in the hands of a Judicial Police, supervised by the Public Ministry. The Honduran Judicial Police has been limited to exercising preventive police duties, prohibited from carrying out criminal investigations. Nicaragua, meanwhile, possesses a successful police force, socially recognized for maintaining satisfactory levels of security surpassing the Guatemalan and El Salvadoran police, which have not achieved similar results despite of having set up a civilian police force separate from the military. El Salvador meanwhile, has excelled in promoting a Police Academy and career professional education, even while not having military attachés in other countries. Regarding budgetary issues, the four countries allocate almost twice the amount of funding on their security budgets in comparison to what is allocated to their defense budgets. However, spending in both areas is low when taking into account each country's GDP as well as their high crime rates. Regional security challenges must be accompanied by a professionalization of the regional armies focused on protecting and defending borders. Therefore, strong institutional frameworks to support the fight against crime and drug trafficking are required. It will require the strengthening of customs, greater control of illicit arms trafficking, investment in education initiatives, creating employment opportunities and facilitating significant improvements in the judicial system, as well as its accessibility to the average citizen.

A phenomenological exploration of Black male law enforcement officers' perspectives of racial profiling and their law enforcement career exploration and commitment

This phenomenological study explored Black male law enforcement officers' perspectives of how racial profiling shaped their decisions to explore and commit to a law enforcement career. Criterion and snow ball sampling was used to obtain the 17 participants for this study. Super's (1990) archway model was used as the theoretical framework. The archway model "is designed to bring out the segmented but unified and developmental nature of career development, to highlight the segments, and to make their origin clear" (Super, 1990, p. 201). Interview data were analyzed using inductive, deductive, and comparative analyses. Three themes emerged from the inductive analysis of the data: (a) color and/or race does matter, (b) putting on the badge, and (c) too black to be blue and too blue to be black. The deductive analysis used a priori coding that was based on Super's (1990) archway model. The deductive analysis revealed the participants' career exploration was influenced by their knowledge of racial profiling and how others view them. The comparative analysis between the inductive themes and deductive findings found the theme "color and/or race does matter" was present in the relationships between and within all segments of Super's (1990) model. The comparative analysis also revealed an expanded notion of self-concept for Black males – marginalized and/or oppressed individuals. Self-concepts, "such as self-efficacy, self-esteem, and role self-concepts, being combinations of traits ascribed to oneself" (Super, 1990, p. 202) do not completely address the self-concept of marginalized and/or oppressed individuals. The self-concept of marginalized and/or oppressed individuals is self-efficacy, self-esteem, traits ascribed to oneself expanded by their awareness of how others view them. (DuBois, 1995; Freire, 1970; Sheared, 1990; Super, 1990; Young, 1990). Ultimately, self-concept is utilized to make career and life decisions. Current human resource policies and practices do not take into consideration that negative police contact could be the result of racial profiling. Current human resource hiring guidelines penalize individuals who have had negative police contact. Therefore, racial profiling is a discriminatory act that can effectively circumvent U.S. Equal Employment Opportunities Commission laws and serve as a boundary mechanism to employment (Rocco & Gallagher, 2004).

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.