text:
Frequently Asked Questions
Cyber risk is actually a group of risks that impact a company's computer, network and data security. The definition is always evolving but right now "cyber risk" encompasses the risk of PII, PHI and PCI; lost revenue and/or intellectual property and trade secrets; the risk of reputational loss; and the risk of legal action and/or regulatory fines and penalties for failure to comply with best security or privacy practices.
- Have an internal discussion between senior management and IT staff about cyber risk management. Look at what cyber security practices are in place to safeguard private information and the integrity of your systems. Are they reasonable, given the size and scope of the organization, and is there the proper support from management to sustain these practices? If not, how can these practices be improved? Do you have an inventory of vital personal data from customers and network components and do you know where it resides?
- Conduct an enterprise-level cyber risk assessment—this can be through a third-party consultant or it can be an internal self-assessment. The point is to determine and document how cyber security practices measure up to recognized privacy and security standards, note any weak spots and document them in a remediation plan.
- Revisit your insurance coverage portfolio to determine whether there is cyber liability insurance coverage and, if not, consider either purchasing a cyber policy or choose to cede residual risk through contractual defense/indemnity service-level agreement provisions with third party providers (although this is often difficult).
Assess your current security/privacy posture. Then get cyber liability insurance if you don't have it. That's technically two things which, in our view, are equally important, but they are also interdependent. You might not be able to get coverage unless you have a risk assessment that demonstrates your computer security practices and policies, and even then you may still have significant residual risk which might only be offset with a solid cyber liability policy.
In this day and age of corporate governance and state and federal regulations, it's paramount to demonstrate proactiveness and that you have reasonable cyber security practices in place. This can be accomplished through an enterprise-level cyber risk assessment, preferably by a third party. You want an independent view of how you measure up to industry standards, compare against your peers, and having that information allows you to make strategic decisions about investing in additional security, including finding the right insurance. An assessment gives you both a plan for internal cyber risk management and a document that demonstrates that you've taken prudent steps to protect against these risks—proof that any external partners and insurance companies will want to see.
A security assessment looks at traditional security practices, making sure, for example, you have the right firewall controls and backup procedures in place. Typically security assessments adhere to industry standards such as ISO 27001 and NIST. A cyber risk assessment, on the other hand, is broader, covering not only cyber security practices but also privacy violations and other types of network-emanating risk such as media liability (copyright or trademark infringement). You can have privacy liability without a data breach so it's critical to understand those weaknesses as well.
Some insurers are willing to take on a client without it but there are many advantages to completing a cyber risk assessment. Even if they don't require it, an insurance company might reward an assessment by offering greater insurance limits or broader coverage.
In theory, it can. However, an insurance company won't advertise that in writing. We have seen that a favorable cyber risk assessment underscoring a strong computer network/data protection security posture can be taken into consideration in the overall underwriting process and may result in a favorable quote. On the other hand, we have seen that a cyber risk assessment that reveals too many weaknesses or "burning plank" issues can work against you, too. An underwriter might choose not to insure you, or suggest you correct those weaknesses before they will insure you. Or they might choose to insure you but at a higher premium level and insist on revisiting the assessment in a year's time to see how those issues have been remediated.
An insurance customer can access the Hub through an access pass (or registration instructions) provided by their cyber liability insurance carrier or their insurance broker. Because we allow license holders to customize and brand the Hub, it may appear under a different name and reflect the insurer's brand, but much of the content—the latest cyber risk news, data breach best practices and insight about the regulatory and legal landscape and often including access to the top cyber security companies and vendors—is maintained by NetDiligence on a daily basis.
The Breach Coach® role is still evolving, but essentially, this invaluable team member is a cyber security expert—most typically, a lawyer—who will act as a crisis manager and walk your organization through the process of immediately responding to a data breach incident. This lawyer is very knowledgeable on state and federal data breach compliance duties. Some Breach Coaches are in demand and addressing new data breach incidents every day. The Breach Coach will also review the response plan, keep everyone calm and begin taking the first steps toward recovery. The Breach Coach also acts as quarterback, engaging experts from computer forensics to credit monitoring to a PR team and assisting the client on a daily basis to keep data breach response in compliance with all regulations. Your organization might retain a Breach Coach or, depending on your coverage, your insurance company might retain one on your behalf.
The cliched but deadly accurate saying in our industry is "not if but when." Pretending a breach event or cyber attack will never happen to you is not effective risk management nor is it remotely realistic. No matter the size of your company you need to engage in data breach planning to ensure that your internal staff and engaged experts are following the proper protocols so that when that inevitable event occurs you can respond in a rapid and timely manner and hopefully recover while keeping your reputation intact and minimizing any future liability exposure.
A good IRP should be very granular, going step by step through the data breach response process and, of course, it should be in compliance with state and federal laws. Having said that, it should also include an abbreviated checklist view and access to action steps to facilitate an urgent review. Depending on the breach scenario it can include internal IT staff, business managers, and/or counsel but it should also include an external 'tiger team' of experts such as a Breach Coach or lawyer, computer forensics, PR, victim notification and others. The IRP should be vetted by a privacy lawyer or security expert who typically acts as a first responder. The document itself should be concise and accessible—a 100-page plan that sits on a shelf will be difficult to use on a Saturday night when a breach is detected. Our Breach Plan ConnectÔ service helps companies build, securely store and easily access their IRP from any computer or device and presents the information in a user-friendly checklist format.
An important component of cyber risk planning and security training, a tabletop exercise allows you to walk through a data breach event before it happens. Typically, the exercise is facilitated annually by an outside expert such as a Breach Coach or forensics expert who goes through the IRP step by step in real time. This exercise effectively tests the IRP and makes sure your staff is acclimated to the data breach response process.
We recommend the following practices for vendor security management:
- Procuring testimonials from past customers
- Asking vendors for insurance certification, particularly a cyber liability policy if the vendor will be touching your network or data in any way
- Requesting proof of the vendor's security posture in the form of a cyber risk assessment from a third party. You can also ask the vendor to complete a self-assessment using your own security questionnaire. Our QuietAuditÒ service is a web-based self-assessment that produces a summary scorecard that details cyber risk preparedness.
- One-sided service-level agreements
- Lack of knowledge about security in the face of changing technology and third party dependencies.
- Lack of awareness about where your data actually resides and who has access to it
In essence, cloud risk is often tied to service-level agreement-based contractual risk. Make sure you have a service-level agreement in place that has been reviewed by counsel. Often there are caveats in these agreements that make no assurances or guarantees about the safety of your data in a cloud network, and they rarely agree to defend and indemnify you for their mistakes. It is also important to look for outside intelligence, such as security experts' assessments of the contractor or service provider. When in doubt, ask a potential cloud vendor to participate in the cyber risk assessment that you conduct for your own company.


text:
Hotel Cabin, Iceland (or similar)
Hotel Cabin is a modern hotel ideally located in the heart of Iceland's capital city, Reykjavik. It offers a wide range of facilities including a bar and restaurant with the option of packed lunches being available for day excursions, a tour desk and souvenir shop, 24 hour reception and a conference and reception room. The bedrooms are clean and comfortable with all of the mod cons you would expect, including wireless internet access and tv in room.

subset: commoncrawl


text:
When I returned to school more than two decades after getting my master's degree in mechanical engineering, it was with the intent of teaching at the university level. I had previously taught engineering technology classes, early in my career, at one of Purdue's extension campuses, and I enjoyed the experience. But I had studied to […]
Linked In
Google +
RSS Feed


text:
Dr. Michael Muzzin, DC, RCCSS(C)
Founder and Partner
Through his experience and passion for helping people achieve active and healthy lifestyles, Dr. Muzzin has identified Five Key Aspects for Optimal Health: Exercise, Nutrition, Rest, Mental Health, and Spinal Health. The human body (infant to senior) has an amazing ability to heal itself when just given the chance. Each of the five aspects of optimal health is like a vital link that forms a strong chain; and of course a chain is only as strong as its weakest link. Dr. Muzzin emphasizes each of these aspects, and as a Chiropractor he focuses in an area known as NeuroStructural Correction.
Dr. Muzzin first started practice in Whitby Ontario just outside of Toronto in 1988. While there he completed an additional three-year post Doctorate in Sports Injuries and earned the Specialty Fellowship of the Royal College of Chiropractic Sports Science (Canada), RCCSS(C). In 1994 he moved back to B.C. and settled in Kelowna where he founded Active Care Health.
Dr. Michael Muzzin has an established brand identity with a well-respected reputation in the community for over 20 years. He is the past president of the Canadian Cancer Society (Kelowna), the Kelowna Chiropractic Association, the official Chiropractor for the Canadian Junior Hockey Championship's Memorial Cup, the World Junior Hockey Championships, and one of the first Chiropractor's in history to be an Official Medical Staff Member of the Olympic Games (Vancouver 2010).
Dr. Muzzin has also worked with Kelowna Falcons Semi-Pro Baseball Team, Okanagan Sun Junior Football Team, Ogopogo Triathlon Club, and is currently the Team Chiropractor for both the Kelowna Chiefs Junior B Hockey Club & the Kelowna Rockets WHL Junior Hockey Club.
When he's not at the office, Dr. Muzzin can be found skiing with his daughter Jenna on either the lakes or slopes (depending on the season) in the beautiful Okanagan Valley.


text:
Our Story
Custom Cuts & Color opened in 1987, owned by Teresa Owens. In '89 Sandy joined our team, Lisa in '91, Janice in '05, and Melody in '18. In '03 ownership was handed to Lisa. In those thirty years, we have moved buildings three times, but stayed on the original property.
Lisa Melton
Lisa Melton is the owner of Custom Cuts & Color. She received her education in cosmetology from Haywood Community College. Her strengths as a hairstylist are short cuts, men's cutting and creative coloring. She loves fun challenges and creating bold styles. She is an Asheville native and enjoys spending her free time with family, being outdoors and playing music in her church.
Teresa Owens
Teresa Owens has been educating clients on healthy hair since 1986. She loves working with her clients to find the right product to suit their hair and lifestyles. She also enjoys getting creative with cuts and hair color. Teresa is our resident OPI expert and can even tell you what color you're wearing without looking at any bottles. Outside of the salon she enjoys spending time with her family and dog in her log cabin her family built.
Sandy McDaniel
Sandy McDaniel has been designing beautiful hair since 1981. She enjoys all aspects of hairstyling including razor cutting, dimensional color, and special occasion hair. Sandy enjoys expressing her creativity in a quiet atmosphere and with a gentle touch. When not in the salon, you'll find her designing costumes and hairstyles for local theater, volunteering with the Girl Scouts, and spending time with her family.
Melody Sorrentino
Born and raised on the New Jersey shore, Melody joins us with 18 years of work under her belt. Her daughter went to Western, and Melody and her husband fell in love with Asheville and decided to make a new home here. Melody loves seeing her clients leave looking and feeling beautiful.
Janice Duke
Janice Duke has been doing hair for 25 years. She specializes in products for those with gluten sensitivities. When not in the salon she spends time with her family, grandchildren and her dogs. Janice is always on the lookout for an odd teapot to add to her collection.
Commonly Asked Questions
Our busy times change day by day, but we do get regular rushes the week or two before major holidays. If you wish to have an appointment during a quieter time, please ask when making an appointment. We'd be glad to help.
We strive to be on top of our schedule and ready for your appointment and respectfully ask you to do the same. We have a general 15 minute grace period, but if you know you're going to be late we'd love if you gave us a call so we can make sure you'll have time for your full service.
We ask that Cancellations be made at least 24 hours before your scheduled appointment.


text: Toddler Sleep How can I help my 2-year-old adjust to a bed? Answered by: Linda Jonides, R.N., C.P.N.P.Question:What are some easy suggestions to help our 2-year-old adjust to her first bed? She wants nothing to do with it, only her crib. She cries so hard every timewe attempt to put her in the bed.Answer:There's no specific age when children are ready to move to a big bed. As with all other developmental changes, children make this transition at atremendous range of ages. If she's not climbing out of the crib, I'd just wait and try again in a few months. Many toddlers feel very secure in that bed with rails around, even if your new bed for herhas guardrails. One approach that sometimes works is to put the mattress of the new bed on the floor in her room, and let her get used to that, maybe justfor naps at first. That way if she rolls, she won't go far or hurt herself. You may have to put a gate at her doorway so she can't wander through the houseand hurt herself. Also, gradually keep talking about the big bed, and make sure she knows she can take her favorite blanket or stuffed animals with her tothe new bed. Good luck! You might also like:


text:
Radiation pneumonitis is an inflammation of the lung tissue. Radiation pneumonitis often appears between 6 and 24 weeks after radiation therapy.
Air moves through the airways into air sacs of the lungs called alveoli. The alveoli have several blood vessels that allow for gas exchange between the lungs and blood. Oxygen can pass from the lungs to the blood and carbon dioxide can pass from the blood to the lungs. Radiation pneumonitis causes inflammation in these alveoli. The inflammation can decrease the amount of oxygen that enters your body. If it remains untreated, pneumonitis can also lead to permanent tissue damage.
In most cases, radiation pneumonitis is caused by radiation therapy to the lungs.
Lung tissue is sensitive to irritants like radiation. Sometimes after exposure to high doses of radiation the tissue becomes inflamed. Inflamed tissue swells and produces extra fluid which blocks gas exchange between the lungs and the bloodstream. The body will have less oxygen available in the blood. In addition, carbon dioxide (a waste product) builds up in the blood because it is harder for the gas to pass from the blood to the lungs.
Over time, if left untreated, the inflammation gradually wears down the lung tissue and leads to scarring. This scarring is permanent and can lead to future lung or breathing complications.
Risk Factors
Radiation pneumonitis is most common in people who have radiation therapy for lung cancer.
Treatment factors that increase the risk of radiation pneumonitis:
- High dosage or duration of treatment
- Larger amount of lung tissue exposed to radiation
- Previous radiation therapy or lung injury
- Other recent or current treatments, such as chemotherapy
Other factors that may increase the chances of radiation pneumonitis:
- Increasing age, especially over 65 years old
- Other lung disorders which inhibit gas exchange, such as chronic obstructive lung disease (COPD)
Some people with radiation pneumonitis do not have apparent symptoms. In those that have them, the most common is shortness of breath with activity or rest, which worsens with time.
Radiation pneumonitis may also cause:
- Persistent dry cough, which may range from mild to severe
- Chest pain
- Increasing amounts of sputum, which may or not have blood
- Fever
- Muscle aches
- Increasing weakness
The doctor will ask about any symptoms and medical history including recent treatment. A physical exam will be done including listening to the lungs. Symptoms of radiation pneumonitis are similar to many other lung disorders. It is generally diagnosed by eliminating other causes of your symptoms. Tests may include:
- Blood tests—to look for signs of infection or imbalance in blood
- Pulmonary function tests—to assess function of the lungs
- Swabs to test secretions in the throat—to look for infection
- Sputum culture—to look for infection
Imaging tests to view the lungs and/or surrounding structures:
When caught early the goal of treatment is to stop inflammation before it causes damage. The course of treatment depends on the severity of the radiation pneumonitis. Prompt treatment may lead to a full recovery in most people.
Radiation pneumonitis is treated with:
- Corticosteroids to reduce lung inflammation
- Medications to suppress the immune system
Other medication or medical treatment may be needed to ease symptoms until the pneumonitis can be resolved. Options include:
- Antitussives to control cough
- Oxygen therapy to increase the amount of available oxygen
Some medication of chemotherapy drugs can make pneumonitis worse. Your current medications or treatment plan may need to be altered.
Lung function will also be regularly monitored to look for changes.
Radiation is carefully measured to deliver the least amount of radiation to the smallest area of tissue while being effective.
The earlier symptoms are recognized and treated, the better the outcomes. It is important to monitor any side effects from radiation therapy.
Revision Information
- Reviewer: EBSCO Medical Review Board Mohei Abouzied, MD, FACP
- Review Date: 03/2018 -
- Update Date: 10/03/2016 -


text:
S-Chassis Polyethylene Transmission MountThis Polyethylene Transmission mount will work on all S13 and S14 KA/SR/CA models.
With easy to access bolts, this mount allows easy installation and removal of your transmission as well as reduces and eliminates shaking and vibration from your transmission. Designed to be long lasting, able to take abuse, and be used on track applications.
- Year: 89-98
- Material Type: Polyethylene
- Nissan Model: S-Chassis


text:
Leasing commercial space is more complex than renting an apartment, and in the current economy, both parties have more at stake. The experienced real estate lawyers of Scura, Wigfield, Heyer & Stevens, LLP, can protect your interests on the front end of commercial lease agreements or when conflicts arise regarding renewal, termination or violations. You don't have to deal with your business disputes alone.
New Jersey Commercial Lease Agreement Attorneys
Our lawyers are known for their acumen in business transactions, real property law and civil litigation. Thus we are attuned to the important considerations in commercial leasing, especially those issues which are most likely to create friction and result in future litigation. In drafting or reviewing a commercial lease, we can knowledgeably address:
- Long-term versus short-term lease
- Right of first refusal
- Option to buy
- Terms and conditions
- Forums for dispute resolution
- Rent increases
- Insurance, utilities and other obligations of each party
- Environmental compliance
Newark Commercial Lease Dispute Lawyers
Proper planning can avoid many conflicts, but perhaps you did not have that luxury when you entered or inherited your current lease. We represent tenant businesses and commercial property owners in all types of lease disputes, from rent and eviction disputes to litigation for damage, fraud or breach of contract.
We are sensitive to our clients' bottom line and work to resolve conflicts as efficiently as possible through out-of-court mediation or negotiation. When no such solution is feasible, we have experience in arbitration and litigation of commercial property leases.
To arrange a consultation to discuss your business dispute with one of our knowledgeable real estate lawyers, call 973-870-0434 or contact us online.


text:
This Book Set Contains 4 books that will make your next camping trip the best tasting ever! Camping Food will Never Taste So Good and Be So Easy to Prepare!
This Camping Cookbook is full of easy-to-prepare recipes that take you off the beaten grill path to all sorts of taste adventures. Here we want to connect you with true outdoor cooking. That means no pots and pans, just straight-up grilling over a fire pit or grill.
Find inside:
•The how to of food preparation for camping trips including food preparation, safety and packing for camping
•Techniques for cooking over the campfire
•Camp food hacks
•Mouth-watering poultry recipes like Chicken Bacon Tootsies
•Flavorful beef dishes such as Flintstone Steak and Lemony Asparagus
•Easy and colorful pork and lamb meals like the Rosemary Lavender Pork Chops
•Light quick and easy fish and seafood recipes such as the Ying and Yang Salmon
•Heavenly desserts your family will love such as the Deconstructed Peach Cheesecake
Foil packet cooking is a food preparation method that involves placing all of the ingredients inside a piece of aluminum foil that has been reconstructed into a sealed packet.
With this book, learn everything there is to know about foil packet cooking including:
•the folding techniques,
•the cooking methods,
•the tips for successful foil packet preparation and cooking,
•how to prepare at home to enjoy your camping trip,
•food safety and cooking guidelines.
•Breakfast foil packet recipes like the Orange Choco Nut Energy Muffins
•Beef foil packet recipes like the Hearty Foil Packet Pot Roast
•Pork foil packet recipes such as the Honey Mustard Chops with potatoes
•Chicken foil packet recipes including the Tex-Mex Chicken Pockets
•Fish and seafood like the Fish Tacos with Roasted Corn Salsa
•Side dish and snack recipes like the Garlicky Cheese Bread or Rustic Whole Carrots
•Fabulous foil packet dessert recipes such as Gooey Banana Boat
Use a cast iron Dutch oven to cook fabulous-tasting food on your next camping trip. You'll never want to leave home without it ever again!
In this book, your will learn to cook like the pioneer did and know all there is to know about cooking with a Dutch oven in the great outdoors!
Inside find:
•How to choose your cast iron Dutch oven
•How to season, clean and cook in a Dutch oven at the camp site
•How to prep early to make camping more fun
•Energizing breakfast recipes like sausage and wild mushroom quiche
•Delicious beef recipes like the Steak Enchilada Casserole
•Easy chicken recipes like the Chicken and Leek Rustic Pizza
•Luscious pork recipes like Pulled Pork Hoagies
•Delightful fish and seafood dishes like the Fish and Corn Chowder
•Amazing vegetables and side recipes like the Dutch Oven Style Risotto
•Yummy snack recipes like the Overloaded Potato Skins
•Sinfully good desserts like Gooey Bottom Chocolate Brownies
Camping recipes that sacrifice the kitchen, not the taste!
Inside find the essentials to plan your camping trip and what to bring to make it the most memorable ever. You will also get an overview of the quickest, easiest, and most effective cooking techniques such as:
• Using foil packets,
• Grilling over the campfire,
• Working with an open flame,
• Garbage can cooking.


text:
The Owen Sound Camera Club is hosting a presentation by Willy Waterton on Tuesday, November 13. Titled Salt of the Earth:People of Bruce Grey, it will cover both his B&W and colour work over his extensive career and will delve into the making of some of his portraits which capture the character of the local folk.
Willy's tagline reads: "photographing the land and people for over 40 years", and after having a taste of the 'people' portion, you can get a taste of the 'land' side at the Griffin Gallery (at the Roxy Theatre) until Jan. 13, 2019. Beautifully presented landscapes and wildlife, you'll find inspiring canvases and prints from the polar regions and closer to home.
Salt of the Earth:People of Bruce Grey
November 13 2018
Doors open 6:30 pm Meeting 7pm
First Christian Reformed Church of Owen Sound
201 4th Ave West Owen Sound
Open to the public. Donations welcome.


text:
College students and others at George Mason University were dumbstruck by the tedious nature of an elementary level Common Core problem during a short series of interviews conducted byCampus Reform last week.
The problem, 32-12, was demonstrated to those on campus the traditional way and juxtaposed with the Common Core method.
"That was extra difficult for no reason" one interviewee told Campus Reform.
"Make it simple, cause that's confusing."
Last week the SAT, America's leading college entrance exam, was revamped to closely align with the Common Core curriculum according to an announcement by the President of the College Board and "architect" of Common Core, David Coleman.
Did you know that lead Common Core Standards writer, David Coleman, became President of the College Board in 2012? Why is that significant? Well because in 2013, the College Board began to simplify the material on the SAT exams and all AP courses to match the Common Core.


text:
Check out our past events below.
IFPMA ("we", "us") owns and operates www.ifpma.org ("the/our website"). Any person accessing and/or using the website ("the visitor/user") agrees to the "Terms of Use", containing the conditions, rules and other provisions with respect to the access and/or use of our website. If you have questions about the "Terms of Use", please feel free to contact us at firstname.lastname@example.com.
The material contained on this website is for information only. The website is entitled to be served as an information resource for the internal and external stakeholders of IFPMA, including the general public.
Third-Party Websites
Links to other websites are provided solely as a convenience and therefore imply neither IFPMA's responsibility for, nor its approval of the information contained in these websites.
Intellectual Property
Any use of information from the website should have an acknowledgement of IFPMA/Third-Party as a source respectively, citing the corresponding uniform resource locator (URL).
Logo and Name
The use of IFPMA's name and/or logo is prohibited without prior express authorization from IFPMA. The access and/or use of the website does not permit to copy, reproduce, modify, distribute or elsehow exploit IFPMA's name and/or logo except with the prior explicit permission.
Abuse and Violation
Any abuse or violations of the Terms of Use and/or Privacy Policy should be reported to firstname.lastname@example.com.
Please be aware that other websites to which links may be provided, including social media, may collect visitor's personally identifiable information when accessed. The information collection practices of other websites linked to IFPMA's website are not covered by this Privacy Policy.


text:
I was watching the YouTube video Top 5 Features of Ice Cream Sandwich with Mono for Android 4.0, and I tried to follow along with the part about creating tabbed applications with the action bar. However, I cannot use the code from the video; the ActionBar property doesn't exist, neither does the ActionBarNavigationMode enum, or the Fragment class. I've double checked this in the assembly browser. I've re-downloaded the Mono for Android installer to make sure I have the most recent version, but when I look at the references, the Mono.Android version is 22.214.171.124. How do I get access to the new features in Android 4.x?
the ActionBar property doesn't exist
The Activity.ActionBar property was added in Android 3.0 (API 11). The default Android version that Mono for Android targets is Android 2.2 (API 8); you will thus need to change your target Android version in the Project Properties window:
Within Visual Studio, you will want Project Options > Application tab > Minimum Android to target drop-down.
Thanks, it's working now.
But this widget is present into the tool window ?
in UI design ?
@MassimilianoFabbri: The ActionBar is not something you can insert in your Layout files, so no.
There is an 'how to' for implementation and use and maybe to customize it in graphics ?
For graphics customization you can use this. It's a part of android asset studio. Very useful tool. As for how to implement it, you can easily find some samples.


text:
A new concept for separation of particles based on repetitive dielectrophoretic trapping and release in a flow system is proposed. Calculations using the finite element method have been performed to envision the particle behavior and the separation effectiveness of the proposed method. As a model system, polystyrene beads in deionized water and a micro-flow channel with arrays of interdigited electrodes have been used. Results show that the resolution increases as a direct function of the number of trap-and-release steps, and that a difference in size will have a larger influence on the separation than a difference in other dielectrophoretic properties. About 200 trap-and-release steps would be required to separate particles with a size difference of 0.2%. The enhanced separation power of dielectrophoresis with multiple steps could be of great importance, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity, or for separations based on combinations of difference in size and dielectric properties.
One of the major applications for dielectrophoresis is selective trapping and fractionation of particles. If the surrounding medium is of low conductivity, the trapping force is high, but if the conductivity increases, the attraction decreases and may even become negative. However, high-conductivity media are essential when working with biological material such as living cells. In this paper, some basic calculations have been performed, and a model has been developed which employs both positive and negative dielectrophoresis in a channel with interdigitated electrodes. The finite element method was utilized to predict the trajectories of Escherichia coli bacteria in the superpositioned electrical fields. It is shown that a drastic improvement of trapping efficiency can be obtained in this way, when a high conductivity medium is employed.
A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.
Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and 'off-target' binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process.
The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available.
In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed.
The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.
Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoE-SI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.
We demonstrate wavelength locking of a diode laser at 760 nm with feedback from an elastic transmission grating in the Littrow configuration. The laser was in a single longitudinal mode with a side-mode suppression of 20 dB. By stretching the grating the laser could be tuned over a few nm. The grating was fabricated in a silicone elastomer ( polydimethylsiloxane) by a moulding technique, and coated by a thin layer of Ti and Au to achieve an increased diffraction efficiency needed for efficient locking.
In this paper the feasibility of modeling strength and optical pulp properties from length distribution curves extracted from acoustic data using continuous wavelet transform-fiber length extraction, CWT-FLE (A Björk and L-G Danielsson, 'Extraction of Distribution Curves from Process Acoustic Measurements on a TMP-Process', Pulp and Paper Canada 105 No. 11 (2004), T260-T264) by use of Partial Least Squares (PLS) have been tested. The curves used have earlier been validated against length distribution curves obtained by analyzing pulp samples with a commercial analyzer (FiberMaster). The curves were extracted from acoustic data without any "calibration" against fiber length analyses. The acoustic measurements were performed using an accelerometer affixed to the refiner blow-line during a full-scale trial with a Sunds Defibrator double disc refiner at SCA Ortviken, Sweden. Pulp samples were collected concurrently with the acoustic measurements and extensive physical testing has been made on these samples. For each trial point three pulp samples were collected. PLS1 and PLS2 models were successfully made linking the distribution curves obtained using CWT-FLE to pulp tensile strength properties as well as optical properties. The resulting Root Mean Square Error of Prediction (RMSEP) for all parameters is comparable to what can be obtained by pooling the standard deviations of reference measurements from the different trial points. The results obtained are compared to FiberMaster data modeled in the same fashion, yielding lower prediction errors than the CWT-FLE data. However, this can be partly due to the five-year storage of pulp samples between pulp sampling/acoustic measurement and FiberMaster analyses/sheet testing. The acoustic method is fast and produces results without dead time and could constitute a new tool for improving process control and optimizing the fiber characteristics in a specific process and for a specific purpose. The technique could be implemented in a PC-environment at a fairly low cost.
Sampling and sample introduction are two key steps in quantitative gas chromatography. In this thesis, a development of a previously described sampling technique as well as a novel concept for sample introduction in gas chromatography are presented. The thesis is based on two papers.
Paper I describes a method for preparing physically mixed polymers for use as sorbent phases in open tubular trapping of gaseous analytes. The concept is based on mechanical disintegration and mixing of solid or liquid poly(ethylene glycol), PEG, into poly(dimethylsiloxane), PDMS, in a straightforward manner. The resulting mixture exhibits a higher affinity towards polar analytes, as compared to pure PDMS.
Paper II describes a novel approach to liquid sample introduction with the split/splitless inlet, used in gas chromatography. Classical injection techniques struggle with discrimination of high boiling analytes and poor repeatability of the injected amount of analytes. The presented injection technique utilizes high voltage to obtain a spraying effect of the injected liquid. The spraying effect can be achieved with a cold needle, which is unprecedented for gas chromatographic injections. The cold needle spraying results in highly repeatable injections, free from discrimination of high boiling analytes.
The split/splitless inlet system has basically the same fundamental drawbacks it had when it was introduced: poor repeatability of the injected amount of sample and discrimination of high-boiling analytes. Hot needle injection improves the repeatability of the sample transfer but suffers from in-needle discrimination. Injection with a fast autosampler, resulting in minimal heating of the needle, solves this problem but usually requires a glass wool packing in the inlet liner to assist in vaporization of the sample. As glass wool has been reported to cause degradation of labile analytes, it cannot be applied as a general remedy for improving incomplete vaporization. In this paper, a novel concept, based on electrostatic nebulization of the injected sample, is presented. The resulting fine droplets promote a more effective heat transfer and a rapid vaporization. Evaluation of the electrospray inlet in the split mode, using a straight, empty glass liner and a cold needle, showed an improvement in peak area repeatability by about 1 order of magnitude, compared with the results obtained when no electrostatic field was applied, Splitless injection of a series of hydrocarbons up to C-28 in the electrospray inlet with an empty, tapered liner, using a cold needle, showed no measurable analyte discrimination. The relative standard deviation in terms of area count for the largest hydrocarbon (C-28) was < 1.5%, compared to similar to 30% for injections where no high voltage was applied.
In this work, we present a technique for the preparation of tailor-made sorbent phases for thick film open tubular traps. Solid or liquid polymers are dispersed in a polydimethylsiloxane (PDMS) pre-polymer, which is cross-linked in situ after coating. The technique is evaluated by preparing thick film open tubular traps with PDMS containing solid or liquid poly(ethylene glycol) (PEG). A significant increase in retention for polar analytes is observed, even when only 7.5% PEG is present. The increase in retention for 3-chloro-1,2-propanediol is more than tenfold. The preparation method is simple and no solvents are required. Also, the concept provides great flexibility in terms of phase composition.
We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.
Ovarian cancer is one of the leading causes of death from cancer in women. The lifetime risk is around 1.5%, which makes it the second most common gynecologic malignancy (the first one being breast cancer). To have a definitive diagnose, a surgical procedure is generally required and suspicious areas (samples) will be removed and sent for microscopic and other analysis. This paper describes the result of a pilot study in which an electronic nose is used to "smell" the aforementioned samples, analyze the multi-sensor signals and have a close to real-time answer on the detection of cancer. Besides being fast, the detection method is inexpensive and simple. Experimental analysis using real ovarian carcinoma samples shows that the use of proper algorithms for analysis of the multi-sensor data from the electronic nose yielded surprisingly good results with more than 77% classification rate. The electronic nose used in this pilot study was originally developed to be used as a "bomb dog" and can distinguish between e.g. TNT, Dynamex, Prillit. However, it was constructed to be a flexible multi-sensor device and the individual (16) sensors can easily be replaced/exchanged. This is suggestive for further investigations to obtain even better results with new, specific sensors. In another pilot experiment, headspace of an ovarian carcinoma sample and a control sample were analyzed using gas chromatography-mass spectrometry. Significant differences in chemical composition and compound levels were recorded, which would explain the different response obtained with the electronic nose.
In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers.
All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width.
In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls.
In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting <100> crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls.
For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis.
In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques.
In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap.
In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer's disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized.
In this paper, we describe a new method for fabrication of nanoelectrospray emitters. The needles were pulled from fused silica capillary tubing, which was melted by means of a plasma, formed by electrical discharges between two pointed platinum electrodes. A key feature of the pulling device is a rotating configuration of the electrodes, which results in an even radial heating of the capillary. The construction of the setup is straightforward, and needles with a variety of shapes can be fabricated, including orifices of submicrometer dimensions. Pulled needles with long tapered tips and an orifice of 0.5 mu m were utilized for electrospray ionization mass spectrometry (ESI-MS) of discrete sample volumes down to 275 pL. The picoliter-sized samples were transferred into the tip of the needle from a silicon microchip by aspiration. To avoid a rapid evaporation of the sample, all manipulations were performed under a cover of a fluorocarbon liquid. The limit of detection was measured to be ca. 20 attomole for insulin (chain B, oxidized).
In this paper, a silicon chip - based electrospray emitter with a variable orifice size is presented. The device consists of two chips, with a thin beam elevating from the center of each of the chips. The chips are individually mounted to form an open gap of a narrow, uniform width between the top areas of the beams. The electrospray is generated at the endpoint of the gap, where the spray point is formed by the very sharp intersection between the crystal planes of the < 100 > silicon chips. Sample solution is applied to the rear end of the gap from a capillary via a liquid bridge, and capillary forces ensure a spontaneous imbibition of the gap. The sample solution is confined to the gap by means of a hydrophobic treatment of the surfaces surrounding the gap, as well as the geometrical boundaries formed by the edges of the gap walls. The gap width could be adjusted between 1 and 25 μm during electrospray experiments without suffering from any interruption of the electrospray process. Using a peptide sample solution, a shift toward higher charge states and increased signal-to-noise ratios was observed when the gap width was decreased. The limit of detection for the peptide insulin (chain B, oxidized) was approximately 4 nM. We also show a successful interfacing of the electrospray setup with capillary electrophoresis.
In this paper, we present a new concept for electrospray ionization mass spectrometry, where the sample is applied in a gap which is formed between the edges of two triangular-shaped tips. The size of the spray orifice can be changed by varying the gap width. The tips were fabricated from polyethylene terephthalate film with a thickness of 36 μm. To improve the wetting of the gap and sample confinement, the edges of the tips forming the gap were hydrophilized by means of silicon dioxide deposition. Electrospray was performed with gap widths between 1 and 36 μm and flow rates down to 75 nL/min. The gap width could be adjusted in situ during the mass spectrometry experiments and nozzle clogging could be managed by simply widening the gap. Using angiotensin I as analyte, the signal-to-noise ratio increased as the gap width was decreased, and a shift towards higher charge states was observed. The detection limit for angiotensin I was in the low nM range.
We describe a method for nanoelectrospray ionization mass spectrometry (nESI-MS) of very small sample volumes. Nanoliter-sized sample droplets were taken up by suction into a nanoelectrospray needle from a silicon microchip prior to ESI. To avoid a rapid evaporation of the small sample volumes, all manipulation steps were performed under a cover of fluorocarbon liquid. Sample volumes down to 1.5 nL were successfully analyzed, and an absolute limit of detection of 105 attomole of insulin (chain B, oxidized) was obtained. The open access to the sample droplets on the silicon chip provides the possibility to add reagents to the sample droplets and perform chemical reactions under an extended period of time. This was demonstrated in an example where we performed a tryptic digestion of cytochrome C in a nanoliter-sized sample volume for 2.5h, followed by monitoring the outcome of the reaction with nESI-MS. The technology was also utilized for tandem mass spectrometry (MS/MS) sequencing analysis of a 2 nL solution of angiotensin I.
In the present work, protein digest reactions in silicon-based microchips, coated with immobilized proteolytic enzymes, have been carried out. The performance of such vials, modified with trypsin or chymotrypsin, was tested with myoglobin as a substrate. Capillary electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry were utilized for analysis of the digests, and the influence of different instrumentation setups. immobilization procedures and reaction conditions are discussed.
In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.
A method was established using capillary electrophoresis with indirect UV detection for analysis of monosaccharides liberated from exopolysaccharides by acidic hydrolysis. Tangential flow filtration was used to isolate high molecular weight polysaccharides from seawater. The capillary electrophoresis method included the use of a background electrolyte consisting of 2,6-dimethoxyphenol and cetyltrimethylammonium bromide. Several neutral sugars commonly existing in marine polysaccharides were separated under optimized conditions. The relative standard deviations were between 1.3% and 2.3% for relative migration time and 1.3-2.5% for peak height. Detection limits (at S/N 3) were in the range of 27.2-47.8 mu M. The proposed approach was applied to the analysis of hydrolyzed colloidal polysaccharides in seawater collected from the Baltic Sea. Nanomolar levels of liberated monosaccharides in seawater samples can be detected by preconcentration up to 30,000 times.
In order to facilitate the determination of the primary and secondary origin of atmospheric organic aerosols, a novel method involving chiral capillary gas chromatography coupled with mass spectrometry has been developed and validated. The method was focused on the analysis of 2-methylerythritol and 2-methylthreitol, considered to be tracers of secondary organic aerosols from the oxidation of atmospheric isoprene. The method was validated by performing various tests using authentic standards, including pure enantiomeric standards. The result showed that the analytical method itself does not affect the enantiomeric composition of the samples analyzed. The method was applied on atmospheric aerosols from a boreal forest collected in Aspvreten, Sweden and on laboratory samples obtained from liquid phase oxidation of isoprene and smog chamber experiments. Aerosol samples contained one enantiomer of 2-methylerythritol in significantly larger quantities than the others. In contrast, the liquid-phase oxidation of isoprene and its gas-phase oxidation in the smog chamber produced all enantiomers in equal quantities. The results obtained where the enantiomer fraction, EF, is larger than 0.50 suggest that 2-methyltetrols in atmospheric aerosols may also have biological origin. Information about the differences between enantiomer fractions obtained using this method brings new insights in the area of atmospheric aerosols.
A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.
Protein microarray technology has an enormous potential for in vitro diagnostics (IVD)1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market. However, in order for protein microarrays to become broadly accepted tools in IVD, a number of criteria have to be fulfilled concerning robustness and automation. Robustness and automation are key demands to improve assay performance and reliability of multiplexed assays, and to minimize the time of analysis.
These key demands are addressed in this thesis and novel methods and techniques concerning assay automation, array fabrication as well as performance and detection strategies related to protein microarrays are presented and discussed. In the first paper an automated assay format, based on planar protein microarrays is described and evaluated by the detection of several auto-antibodies from human serum and by quantification of matrix metalloproteases present in plasma. Diffusion-rate limited solid phase reactions were enhanced by microagitation, using the surface acoustic wave technology, resulting in a slightly increased signal-to-noise ratio. In the second paper of the thesis, a novel multiplexed immunoassay system was developed by combining a direct immunoassay with a competitive system. This set-up allows quantification of analytes present in widely varying concentrations within a single multiplex assay. In the third paper, a new concept for sample deposition is introduced, addressing contemporary problems of contact or non-contact microarrayers in protein microarray fabrication.
In the fourth paper, a magnetic bead-based detection method for protein microarrays is described as a cost-effective alternative approach to the commonly used fluorescence-based confocal scanning systems. The magnetic bead-based detection could easily be performed by using an ordinary flatbed scanner. In addition, applying magnetic force to the magnetic bead-based detection approach enables to run the detection step more rapidly. Finally, in paper five, a microfluidic bead-based immunoassay for multiplexed detection of receptor tyrosine kinases in breast cancer tissue is presented. Since the assay is performed inside a capillary, the amounts of sample and reagent material could be reduced by a factor of 30 or more when compared with the current standard protein microarray assay.
Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.
BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.
Contemporary microarrayers of contact or noncontact format used in protein microarray fabrication still suffer from a number of problems, e. g. generation of satellite spots, inhomogeneous spots, misplaced or even absent spots, and sample carryover. In this paper, a new concept of non-contact sample deposition that reduces such problems is introduced. To show the potential and robustness of this pressure-assisted deposition technique, different sample solutions known to cause severe problems or to be even impossible to print with conventional microarrayers were accurately printed. The samples included 200 mg mL(-1) human serum albumin, highly concentrated sticky cell adhesion proteins, pure high-salt cell-lysis buffer, pure DMSO, and a suspension of 5-mu m polystyrene beads. Additionally, a water-immiscible liquid fluorocarbon, which was shown not to affect the functionality of the capture molecules, was employed as a lid to reduce evaporation during microarray printing. The fluorocarbon liquid lid was shown to circumvent hydrolysis of water-sensitive activated surfaces during long-term deposition procedures.
A methodology using stripping voltammetry has been elaborated to enable sensitive and reliable protein-chromium complexation measurements. Disturbing effects caused by adsorption of proteins on the mercury electrode were addressed. At low concentrations of proteins (< 60-85 nM), chromium-protein complexation measurements were possible. Chromium(VI) complexation was quantitatively determined using differently sized, charged, and structured proteins: serum albumin (human and bovine), lysozyme, and mucin. Generated results showed a strong relation between complexation and protein size, concentration, and the number of amino acids per protein mass. Complexation increased nonlinearly with increasing protein concentrations. The nature of this complexation was based on weak interactions judged from combined results with MALDI-TOF-MS and adsorptive cathodic stripping voltammetry.
In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated.
In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE.
A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal.
A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other.
In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work.
In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes.
A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein-hapten binding in the skin, is presented. Mass spectra of BSA-FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 m M. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined. 28 possible and 2 non-binding sites for FITC. (C) 2010 Elsevier B.V. All rights reserved.
In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged. Comparison was made between the EOF in a closed system (first capillary solely included in the electrical circuit) and in a closed-open system (first capillary and microcanal included in the electrical circuit). It was concluded that the experimental values of the EOF agreed with the theory. The influence of the capillary outer diameter on the peak dispersion was investigated using a closed-open-closed system (first capillary, microcanal and second capillary included in the electrical circuit). It was clearly seen that a capillary with 375 mu m od induced considerably higher peak dispersion than a 150 mu m od capillary, due to a larger liquid dead volume in the connection between the first capillary outlet and the microcanal. Mass spectrometric analysis has also been performed following CE separation runs in a closed-open-closed system with cytochrome c and lysozyme as model proteins. It was demonstrated that a signal distribution profile of the separated analytes could be recorded over a 30 mm long microcanal.
A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated.
In the present work a capillary electrophoretic method for the analysis of monosaccharides utilizing indirect UV-detection has been developed. Different probes for indirect detection have been assessed using model carbohydrate samples. Background electrolytes with or without addition of cetyltrimethylammonium bromide have also been evaluated regarding the separation power. Furthermore, a curve-fitting algorithm has been introduced to increase the separation resolution. The optimized method has been used for analysis of monosaccharides from an acidically hydrolyzed pulp sample.
An all-fiber capillary electrophoresis system is presented. It enables sensitive in-line electrophoresis separation and fluorescence detection. As a proof of concept, a biological sample (FITC-BSA) is electrokinetically separated and analyzed.
We model and simulate dielectrophoresis of microscale particles using the finite element method. A soft sphere system molecular dynamics model is presented, which solves a set of equations for the motion of every particle. The model couples most of the significant forces, i.e. the dielectrophoresis (DEP) forces, the particle-particle electrostatic forces, particle-particle interfacial repulsive forces, particle-wall repulsive forces and the hydrodynamic forces in Stokes flow. Since the system of equations is stiff, an implicit scheme is used. To obtain the particle trajectories, a constant time-step is applied. We present some numerical tests computing hydrodynamic force, electrostatic force and DEP force using our model, including simulated trapping of particles in a micro channel by dielectrophoresis. The results are in agreement with the theories and the experimental observations.
Isoprene, a compound emitted by vegetation, could be a major contributor to secondary organic aerosols (SOA) in the atmosphere. The main evidence for this contribution were the 2-methylbutane-1,2,3,4-tetraols, or 2-methyltetrols (2-methylerythritol and 2-methylthreitol) present in ambient aerosols. In this work, the four stereoisomers of these tetraols were analyzed in aerosols from Aspvreten, Sweden. 2-C-methyl-D-erythritol was found in excess over its enantiomer in the Spring/Summer, by up to 29% in July. This clearly indicated some biological origins for this enantiomer, consistent with its well-documented production by plants and other living organisms. In addition, a minimum of 20 to 60% of the mass of racemic tetraols appeared from biological origin. Thus, the SOA mass produced by isoprene in the atmosphere is less than what indicated by the 2-methyltetrols in aerosols. Our results also demonstrate that stereochemical speciation can distinguish primary and secondary organic material in atmospheric aerosols.
We suggest electro-osmotic driven flow (EOF) through closed-open-closed microchannels as a novel approach for spatial sample separation using capillary electrophoresis (CE) prior to matrix assisted laser desorption/ionization mass spectroscopy (MALDI-MS). For this purpose we built a system consisting of the series Coupling of a closed fused silica capillary for separation, a microfabricated open microcanal for future MS detection and a second closed fused silica capillary for downstream liquid collection. This work verifies the EOF transport of a peptide sample in such a system with low dispersion.
A new method has been developed for direct analysis of volatile polar trace compounds in aqueous samples by gas chromatography. Water samples are injected onto a short packed precolumn containing anhydrous lithium chloride. A capillary column is coupled in series with the prefractionation column for final separation of the analytes. The enrichment principle of the salt precolumn is reverse to the principles employed in conventional methods such as SPE or SPME in which a sorbent or adsorbent is utilized to trap or concentrate the analytes. Such methods are not efficient for highly polar compounds. In the LiCl pre-column concept, the water matrix is strongly retained on the hygroscopic salt, whereas polar as well as nonpolar volatile organic compounds show very low retention and are eluted ahead of the water. After transfer of the analytes to the capillary column, the retained bulk water is removed by backflushing the precolumn at elevated temperature. For direct injections of 120 μL of aqueous samples, the combined time for injection and preseparation is only 3.5 min. With this procedure, direct repetitive automated analyses of highly volatile polar compounds such as methanol or tetrahydrofuran can be performed, and a limit of quantification in the low parts-per-billion region utilizing a flame ionization detector is demonstrated.
A protocol for purification and analysis of chloroplast membrane proteins in the green macroalga Ulva lactuca has been developed, including reversed phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Five different solvents were evaluated for extraction of membrane proteins by three methods. The highest protein yield was achieved when proteins were extracted directly from the chloroplasts using the solvent hexafluoroisopropanol. A range of proteins of increasing hydrophobicity was separated by HPLC. Analysis of both HPLC fractions and non-separated samples by MALDI-TOF-MS revealed proteins with molecular weights spanning between 1 and 376 kDa.
Different sample handling methods for hydrophobic proteins and peptides were evaluated in association with the utilization of a structured matrix-assisted laser/desorption ionization (MALDI) target for increased sensitivity. The fluorinated organic solvent hexafluoroisopropanol (HFIP) was used for the solubilization of both the full-length protein bacteriorhodopsin (BR) and a cyanogen bromide digest thereof, and compared to the performance of the non-ionic detergents octyl-beta-D-glucopyranoside (OG), dodecyl-beta-D-maltoside (DM), and Triton X-100. A concentrating effect was seen when using the structured MALDI plate for BR dissolved in all the different detergents, of which OG generated the best-quality spectra for the full-length integral membrane protein as well as for the hydrophobic peptides. However, the uneven analyte distribution obtained with the detergent preparations required selective and thus time-consuming acquisition of spectra. When instead HFIP was used as sample solvent, a tenfold increase in sensitivity was achieved for full-length BR. Addition of acids to the HFIP-solubilized sample, or to the MALDI matrix solution, improved the signals for a few of the peptides, while degrading the spectra of others. Consequently, the addition of acid could be used as a complementary sample preparation method for hydrophobic peptides. On-target washing to remove contaminants (e.g., salt) was performed, and a recrystallization protocol for signal improvement specifically suited for hydrophobic peptides is described. Results from digestion and solubilization in different micro centrifuge tubes were examined to determine the influence of different materials on the possible sample loss due to wall adhesion. Studies of sample solution storage times suggest immediate analysis after solubilization to obtain best results.
A new prestructured target plate for matrix-assisted laser desorption/ionization (MALDI) was developed specifically for hydrophobic integral membrane proteins. This sample support contains predefined concentrating sample spots with a focusing effect on droplets with a high content of hexafluoroisopropanol (HFIP). This fluorinated organic solvent is advantageous for solubilizing hydrophobic proteins that are not soluble in water or the organic solvents normally used in sample preparation protocols for MALDI-MS. The prestructured plate was constructed by coating a regular steel plate with a thin layer of a silicone polymer, leaving sample spots of bare steel. Fabrication of the concentrating silicone structure was fast and very straightforward, without expensive or complicated equipment. Removing the layer, and thus regenerating the steel plate, was done by a simple washing procedure. The application and cleaning procedure are not constrained by a particular design of sample support or to any specific brand of mass spectrometer. When using the prestructured MALDI plate with HFIP as the sample solvent for 17 pmol of a cyanogen bromide digest of the highly hydrophobic membrane protein bacteriorhodopsin, an improved focusing effect and an increase of more than five-fold in average sensitivity were observed, compared with a regular steel target. Experimental results show a two-fold increase in average sensitivity when the new prestructured target plate was used, compared with a commercially available concentrating support
Fluorosurfactants have some unique properties that are advantageously used in a range of applications. Their solutions are commonly in contact with solid surfaces onto which the molecules adsorb. Despite this, the adsorption behavior of fluorosurfactants at solid/liquid interfaces is not sufficiently understood, and there is a need for more information. In this study we focus on cationic fluorosurfactant adsorption on negatively charged hydrophilic surfaces, especially with respect to the adsorbed layer structure, long-range interactions, and adhesion forces. To this end we combined results obtained from bimorph and interferometric surface force instruments and ellipsometry techniques. The initial adsorption to the oppositely charged surfaces occurs due to the electrostatic attraction between the charged headgroups and the surface. Further adsorption, driven by hydrophobic interactions, occurs readily as the surfactant concentration is increased. Surface force and ellipsometric experiments indicate that the surfactants self-assemble in the form of bilayer aggregates. The thickness of the bilayer aggregates was found to be consistent with the molecular structure. Further, ellipsometric measurements indicate that no complete bilayers were formed but rather that bilayer aggregates were present on the surface even at concentrations well above the cmc. Surface force data for low fluorosurfactant concentrations demonstrate that upon compression the bilayer aggregates assembled on the isolated surfaces are transformed, and as a result monolayer structures build up between the surfaces in contact. The force required to attain bilayer-bilayer contact increases with the surfactant bulk concentration due to an increase in the repulsive double-layer force. The force required to drive out surfactant molecules to achieve monolayer-monolayer contact also increases with surfactant concentration. Above the cmc some additional aggregates are present on top of the bilayer aggregates coating the surface. The adhesion found between the monolayer aggregates is an order of magnitude larger than between the bilayer aggregates. However, it is an order of magnitude lower than the corresponding value for Langmuir-Blodgett monolayer films of similar fluorosurfactants.
N-Methylformamide (NMF)-based matrices for capillary electrophoretic separation of nucleic acids have been developed. The use of an organic solvent as liquid base for the separation matrices allowed a hydrophobic polymer, C-16-derivatized 2-hydroxyethyl cellulose (HEC), to be employed as structural element in the sieving medium. With a matrix consisting of 5% w/v of this polymer dissolved in NMF containing 50 mM ammonium acetate, p(dA)(12-18) and p(dA)(40-60) oligonucleoticles were baseline separated. The addition of ammonium acetate to the buffer and separation matrix resulted in enhanced separation efficiency. Furthermore, it was possible to tailor the sieving performance of the separation medium by the use of a binary mixture of C16-derivatized HEC and PVP. Differences in sieving behavior of the various matrices evaluated are discussed.
A new sample target for matrix-assisted laser desorption/ionization mass spectrometry is described. The target consists of pairs of elevated hydrophilic anchor surfaces, positioned in proximity onto a microchip. The anchors are used to obtain separate preparations of sample and external standard, while both anchor surfaces are irradiated simultaneously by the laser pulse. Using a standard, based on six peptides, a 2-fold improvement in mass accuracy is observed. Also, ion suppression is significantly reduced. With a one peptide calibration standard, 22 tryptic fragments from a BSA digest are detected using the twin-anchor concept, whereas only 14 fragments are detected when the sample and standard are laser-ablated as a mixture from a conventional anchor target. A volume of similar to30 pL of sample solution of angiotensin I is transferred to the anchor surface, under a thin layer of a perfluorocarbon, to prevent a concentration bias due to evaporation. With this arrangement, a detection limit of 1.5 amol was achieved with a signal-to-noise ratio of 22:1.
Diode-pumped solid-state lasers (DPSSLs) have been integrated as light sources in confocal laser scanning microscopy (CLSM). The standard argon ion laser at 488 rim is compared with a DPSSL operating at 473 run in terms of noise and CLSM image characteristics. The equally high fluorescence image quality together with the many advantageous characteristics inherent to solid-state lasers suggest that excitation using a DPSSL is favored. The application of a dual-line DPSSL emitting at 491 and 532 nm for high resolution CLSM fluorescence imaging is shown for the first time.


text:
Call us+31(0)73-6106204
We like to share our experiences with you! With pleasure we will create a luxury individual TAILOR-MADE holiday itinerary. We are also very experienced in car driving in Latin America. We drove more than 50.000 km with our own car. Working with you, we will adjust until every detail fits your needs. CALL US and tell us what you're after.
Also see
Website; the official travel guide to Chile.
Travel inspiration
15-daagse rondreis Merendistrict Chili - Argentinië
Deze unieke reis neemt u mee naar het bijzondere Merendistrict aan de Chileense en Argentijnse zijde. Geniet hier van besneeuwde bergtoppen, diepblauwe meren, kristalheldere rivieren en uitgestrekte, dichte wouden terwijl u verblijft in bijzondere accommodaties.
19 daagse rondreis Chili - KLM Flying Dutchman
Tijdens deze reis ervaart u de vrijheid van het reizen met huurauto en geniet van al het moois dat de natuur te bieden heeft. U bezoekt zowel de droogste woestijn ter wereld, als de bruisende hoofdstad en de Carretera Austral, een van de mooiste autoroutes ter wereld. De ongerepte natuur waar u overdag doorheen rijdt zal u verrassen en in de avond kunt u heerlijk ontspannen in uitstekende en comfortabele accommodaties. Deze reis is verschenen in de december 2017 editie van het KLM Flying Dutchman magazine.
Johan van Rijswijcknextprev
My Unique Journey is driving with my own car through Latin America! I already drove over 40.000 miles in this continent. Soon more... A great experience, a huge enrichment!
Petra Horschnextprev
My Unique Journey is, with my boyfriend, driving through South America, to discover new places and enjoy the local culture. I grew up in Brazil and have traveled a lot by car. For me that's the best way to experience a country.
Pip keeps us company at the office. She has an affinity with travel, since she's born in Spain (oct.12). Along with her two brothers, then 8 weeks old, she was found in a box. Now we care for her and she is a very happy dog!
Sylvia Nederhoffnextprev
My Unique Journey is traveling together with Johan & Emma. Emma, only 9 years old, often accompanied us during our travels through Latin america. I am glad that we can make this possible for her!
Anke Hendriksnextprev
My Unique Journey is traveling through breathtaking landscapes, enjoying the colorful cultures and the great adventure. When I travelled through Latin America I saw many fantastic places, gained unforgettable experiences and added new places to my bucket list.
Suzanne Klerkxnextprev
Het ontdekken van de mooiste plekken ter wereld is wat ik het liefste doe. Een goede mix van cultuur en natuur is wat een reis voor mij uniek maakt. Latijns-Amerika heeft dit allemaal te bieden. Ik kan niet wachten om deze kleurrijke en bijzondere mooie landen te ontdekken en af te kunnen vinken van mijn bucketlist.
Luxury holiday Chile
Chile is a beautiful country with an enormous diversity. Chile curls around the Westside of the Andes like a long shoelace.
Why a long shoelace? Chile is 4275 kilometres long from North to South and at the narrowest point its only 60 kilometres wide.
Best travel period:
You can travel to Chile throughout the whole year. However, when you intend to visit the south of Chile, you must bear in mind that Chile is situated on the southern hemisphere so it has opposite seasons with the Netherlands. Best travel period in that case is their 'summer' : October to April.
Dry deserts, majestic volcanoes, vineyards, impenetrable woods and icy pampas. All in all Chile is characterized by its unprecedented diversity.
A paradise for adventurers, a must for nature lovers, but most of all a safe country with friendly inhabitants who will gladly welcome you to the country they're very proud of. The safety and good infrastructure also make it possible to discover this amazing country in a comfortable rental car.
A special place in the wine region at the end of a beautiful journey ...
Soon more...Read more
Hotel Altiplanico in the Atacama Desert...
... Is an oasis of peace! The hotel lies in the oasis town of San Pedro, in the Atacama Desert in Chile's north. This hotel complex is entirely made of adobe (a combination of clay and straw) …Read more
Chile, a country and people to love!
Thank you very much for the perfect organisation of my trip to CHILE. The three destinations Patagonia, Atacama and Rapa Nui were all 3 very special. The stops were well planned. All in all I can …Read more
- Load more...
Travelling through Chile individually is very doable, recommendable!
Pleasant people everywhere. From north to south, and thus a lot of variation and surprises. Deserts with coastal towns and villages, partly abandoned, inland as well. Inviting hot springs, good for …Read more
The 7 Lakes route and after that our visit to Torres del Paine were the highlights of our trip!
For years I wanted to learn Spanish in preparation of my trip to South America and lifelong I dreamed of Antarctica... After returning to the Netherlands Johan asked us to write a …Read more
Great planning and expertise of Johan and his team!
Soon more...Read more
It was a beautiful trip, with a lot of highlights and new experiences...
It was a beautiful trip with a lot of highlights and new experiences. Most of them planned, sometimes unexpected, such as the earthquake in San Pedro de Atacama or the boat trip to the Balamceda …Read more
Een onvergetelijke reis door Chili
Sapa Pana Travel heeft voor ons een onvergetelijke en mooie reis door Chili samengesteld.Read more
We also run in to sea lions and condors...
We are the only ones leaving the ship and we are brought to the Hosteria with a zodiac. A basic hostel, sealed off from the inhabited world by water and mountains. The bedroom has a wood-burning …Read more
Chile, Easter island, Lake district and Patagonia
A 19 day trip completely customized with all the highlights: Easter island with its impressive moa's, a trip in a jeep between the lakes and volcanoes such as the Osorno, and from the Last Hope …Read more
We enjoyed a carefree journey by car through beautiful South America!
Soon more...Read more
With our Volvo we drove 10.000 miles through South America.
Soon more.Read more
What a magnificent adventure this trip!
Soon more...Read more
Patagonia, you're so beautiful it's a waste to go to sleep.
We were the only guests in the hotel. Ripped from the group and raced to the hotel with a zodiac. No electricity, no warm water, our own wood-burning stove, friendly service and good food (including …Read more
Chili: een warm plekje in ons hart
Het was onze eerste ontmoeting met Zuid-Amerika en 'we loved it'!Read more
It was an unforgettable trip!
In autumn 2006 we made a trip to Chile which was organized by Sapa Pana Travel. In 23 days we successively visited the north, the middle and the south of Chile. We also visited Easter Island at the …Read more
Post academic UvT trip
We travelled to Chile with 20 colleagues in light of our post academic education as care manager at the UvT. Besides having a taste of this beautiful and varied country our aim was to learn more …Read more
We really enjoyed it and saw a lot
Hi Johan, Our compliments on the great organization of our trip. We really enjoyed it and we say a lot. The way you made up for our useless wait with a dinner in the Four Seasons was very much …Read more
Nearly 10.000 miles together in a car; laugh , cry and comfort each other, but mostly enjoying to the fullest!
Soon more...Read more
Our visit to stunning Chile was great!
Beautiful nature, varying climates, wonderful accommodations, overwhelming attractions. Everything had been taken care of WONDERFULLY, also thanks to the great cooperation with the Chilean agency …Read more
A fantastic trip, excellent carried out by Sapa Pana Travel and partners!
Soon moreRead more
Chile: from Santiago to Cape Horn
In the quiet lakes we see the reflection of the volcanoes' snowy tops. Churning and thundering the river races between the blocks of lava to become one with a lovely turquoise river. More to …Read more
Our trip through Chile and Argentina had many highlights...
...and one of them was the trip through Patagonia, where we took the boat along the Balmaceda glacier (see photo) and then (by recommendation of Sapa Pana Travel) we changed over to a Zodiac for a 2 …Read more
Chile and Easter Island combined with Argentina
Chile, Argentina and Easter Island had been on our holiday wish list for a long time. After looking through quite a number of brochures with group trips, some acquaintances told us about their good …Read more
Three months of travelling, mostly through Chile but also Argentina and Antarctica
After a long and very fun talk with Sapa Pana Travel, they planned the trip according to our wishes: car rental, flights and part of the accommodation. Everything was surprisingly well arranged. …Read more
Een waar paradijs voor de wijnliefhebber
Al langer hadden we het idee om een reis door Chili te maken. We wilden de wijncultuur ervaren en winery's bezoeken. Dit uiteraard in samenspel met het natuurschoon, de bevolking en de …Read more
Autorijden in Patagonië
Na kennis gemaakt te hebben met Sapa Pana Travel op de Beurs voor bijzondere reizen in Breda zaten we nog geen 2 weken later aan tafel bij Johan op kantoor. We hoefden er niet lang over na te denken, …Read more


text:
See what questions
a doctor would ask.
Karyotype disorders: Clinical conditions caused by an abnormal chromosome constitution in which there is extra or missing chromosome material (either a whole chromosome or a chromosome segment). (from Thompson et al., Genetics in Medicine, 5th ed, p429)
Source: Diseases Database
These medical condition or symptom topics may be relevant to medical information for Karyotype disorders:
Source: Diseases Database
Search to find out more about Karyotype disorders:
Tools & Services:
Medical Articles:
Search Specialists by State and City
By using this site you agree to our Terms of Use. Information provided on this site is for informational purposes only; it is not intended as a substitute for advice from your own medical team. The information on this site is not to be used for diagnosing or treating any health concerns you may have - please contact your physician or health care professional for all your medical needs. Please see our Terms of Use.
Copyright © 2014 Health Grades Inc. All rights reserved. Last Update: 13 August, 2015 (1:25)